SPLICEOSOME PERTURBATIONS AND USES THEREOF

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/383,244, filed November 10, 2022, U.S. Provisional Application No. 63/383,457, filed November 11, 2022, U.S. Provisional Application No. 63/387,930, filed December 16, 2022; which are all incorporated herein by reference in their entirety.

BACKGROUND OF THE DISCEOSURE

[0001] Spliceosome components are frequently mutated in cancer. Somatic mutations in the spliceosome and other splicing factors are prevalent in cancer. Less efficient splicing reveals new vulnerabilities.

INCORPORATION BY REFERENCE

[0002] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. Absent any indication otherwise, publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entireties.

SUMMARY OF THE DISCLOSURE

[0003] In some aspects, the present disclosure provides methods for treating a cancer in a subject. In some embodiments, said method comprises: selectively inhibiting an enzymatic component of a spliceosome in a cancer cell. In some embodiments, said inhibiting of said selected enzymatic component induces expression of one or more mis-spliced RNA that is different than that induced upon inhibition of a non-selected enzymatic component of said spliceosome. In some embodiments, said expression of said one or more different mis-spliced RNA results in an immune response to said cancer cell in said subject, thereby treating said subject, optionally wherein said immune response is different than that induced upon inhibition of a non-selected enzymatic component of said spliceosome.

[0004] In some aspects, the present disclosure provides methods for treating a cancer in a subject. In some embodiments, said method comprises: inhibiting an enzymatic component of a spliceosome that is a RNA helicase in a cancer cell in said subject. In some embodiments, said inhibiting induces expression of one or more mis-spliced RNA in said cancer cell. In some embodiments, said expression of said one or more mis-spliced RNA results in an anti-viral immune response to said cancer cell in said subject, thereby treating said subject.

[0005] In some aspects, the present disclosure provides methods for treating a cancer in a subject. In some embodiments, said method comprises: inhibiting an enzymatic component of a spliceosome in a cancer cell of said subject. In some embodiments, said inhibiting induces an immune response to said cancer cell in said subject, thereby treating said subject. [0006] In some aspects, the present disclosure provides methods for treating a cancer in a subject. In some embodiments, said method comprises: inducing degradation of an enzymatic component of a spliceosome in a cancer cell of said subject. In some embodiments, said degradation induces an immune response to said cancer cell in said subject, thereby treating said subject.

[0007] In some aspects, the present disclosure provides methods for inducing an immune response in a subject, said method comprising: inhibiting an enzymatic component of a spliceosome in a cell of said subject, wherein said inhibiting induces an immune response to said cell in said subject.

[0008] In some aspects, the present disclosure provides methods for inducing an immune response in a subject, said method comprising: inducing degradation of an enzymatic component of a spliceosome in a cell of said subject, wherein said degradation induces an immune response to said cell in said subject. [0009] In some embodiments, said cell is a cancer cell.

[0010] In some aspects, the present disclosure provides methods for treating a cancer in a subject, said method comprising: inhibiting an enzymatic component of a spliceosome in a cancer cell in said subject, wherein said inhibiting induces expression of one or more mis-spliced RNA in said cancer cell, and wherein said expression of said one or more mis-spliced RNA results in an anti-viral immune response to said cancer cell in said subject, thereby treating said subject.

[0011] In some aspect, the present disclosure provides methods for treating a cancer in a subject, said method comprising: inhibiting an enzymatic component of a spliceosome in a cancer cell in said subject, wherein said inhibiting induces expression of one or more mis-spliced RNA in said cancer cell, wherein said one or more mis-spliced RNA encode one or more neoantigens, wherein said one or more neoantigens induce an immune response to said cancer cell in said subject, thereby treating said subject. [0012] In some embodiments, said enzymatic component is a RNA helicase.

[0013] In some embodiments, said inhibiting comprises inducing degradation of the enzymatic component.

[0014] one or more enzymatic component.

[0015] In one aspect, provided herein is a method for treating a cancer in a subject, said method comprising: inhibiting splicing factor 3B subunit 1 (SF3B1) in a cancer cell in said subject, wherein said inhibiting induces an immune response to said cancer cell in said subject, thereby treating said subject.

[0016] In one aspect, provided herein is a method for inducing an immune response in a subject, said method comprising: inhibiting splicing factor 3B subunit 1 (SF3B1) in a cell in said subject, wherein said inhibiting induces an immune response to said cell in said subject.

[0017] In some embodiments, said inhibiting comprises inducing degradation of SF3B1.

[0018] In some embodiments, said inhibiting induces one or more mis-spliced RNA in said cancer cell. [0019] In some embodiments, said method further comprises inhibiting an enzymatic components of the spliceosome.

[0020] In some embodiments, the method further comprises inhibiting a RNA helicase.

[0021] In some embodiments, said inhibiting results in inhibition of a spliceosome activity. In some embodiments, said inhibiting results in inhibition of a spliceosome activity. In some embodiments, said inhibiting comprises degrading said enzymatic component. In some embodiments, said inhibiting comprises inhibiting post-translational modification of said enzymatic component. In some embodiments, said inhibiting comprises: administering an effective amount of one or more agents capable of inhibiting said enzymatic component.

[0022] In some embodiments, said one or more agents bind, degrade, and/or inhibit post-translational modification of said enzymatic component. In some embodiments, said one or more agents are a small molecule, protein, peptide, nucleic acid, carbohydrate, or a combination thereof. In some embodiments, said one or more agents are a siRNA, an antisense morphlino, an antisense oligonucleotide, a small molecule, or a combination thereof.

[0023] In some embodiments, said inhibiting induces secretion of interferon (e.g., via Pattern Recognition Receptors (PRRs including include TLRs, RIG-I-like receptors (RLRs), Nod-like receptors (NLRs) and C-type lectin receptors)). In some embodiments, the inhibiting induces a Jak-STAT signaling pathway. In some embodiments, the inhibiting results in activation of one or more interferon-stimulated genes (ISGs). In some embodiments, said inhibiting induces an increased expression or activity of a mitochondrial antiviral signaling protein. In some embodiments, said inhibiting induces an IFN signaling pathway in said cancer cell. In some embodiments, said inhibiting results in expression of one or more neoantigens, and/or an increase in level of said one or more neoantigens relative to that in a suitable control. In some embodiments, said inhibiting induces an increase in level and/or activity of a MHC class 1 polypeptide. In some embodiments, said inhibiting induces an increase in sensitivity of said cancer to a spliceosome-targeted therapy. In some embodiments, said inhibiting induces expression of one or more caspases in said cancer cell. In some embodiments, said inhibiting induces apoptosis of said cancer cell. In some embodiments, said inhibiting results in formation and/or increase in level of a R loop.

[0024] In some embodiments, said inhibiting and said formation and/or an increase in level of said R loop results in activation of a cGAS-STING pathway. In some embodiments, said inhibiting induces an increase in level and/or activity of a T cell of any kind, e.g., a cytotoxic T cell, e.g., a CD8+ T cell, a helper T cell, a memory T cell, an effector T cell. In some embodiments, said inhibiting comprises modifying a gene encoding the enzymatic component, wherein said modifying introduces a mutation in the enzymatic component. In some embodiments, said mutation is an amino acid substitution, deletion and/or insertion in said enzymatic component. [0025] In some embodiments, said modifying results in a decrease in the activity and/or level of said enzymatic component of the spliceosome. In some embodiments, said enzymatic component is DHX15, and said mutation is a R222G amino acid substitution in said DHX15 relative to a corresponding wild type DHX15; said enzymatic component is DDX46, and the mutation is a D529A amino acid substitution and/or D531 A amino acid substitution in said DDX46 relative to a corresponding wild type DDX46; and/or said enzymatic component is DDX23, and the mutation is a D549A amino acid substitution and/or D552A amino acid substitution in said DDX23 relative to a corresponding wild type DDX23. In some embodiments, said inhibiting comprises inhibiting the enzymatic activity of said enzymatic component of said spliceosome.

[0026] In some aspects, this disclosure provides methods for treating a cancer in a subject. In some embodiments, said method comprises: inducing degradation of an enzymatic component polypeptide of a spliceosome in a cancer cell in said subject. In some embodiments, said degradation induces expression of one or more mis-spliced RNA in said cancer cell. In some embodiments, said expression of said one or more mis-spliced RNA results in an immune response to said cancer cell in said subject, thereby treating said subject.

[0027] In some embodiments, said degradation results in inhibition of a spliceosome activity. In some embodiments, said degradation results in inhibition of a spliceosome activity. In some embodiments, said degradation comprises: administering an effective amount of an agent capable of degradation of said enzymatic component. In some embodiments, said agent binds, and/or degrades said enzymatic component. In some embodiments, said agent is a small molecule, protein, peptide, nucleic acid, carbohydrate, or a combination thereof. In some embodiments, said degradation induces secretion of interferon (e.g., via Pattern Recognition Receptors (PRRs including include TLRs, RIG-I-like receptors (RLRs), Nod-like receptors (NLRs) and C-type lectin receptors)). In some embodiments, the degradation induces a Jak-STAT signaling pathway. In some embodiments, the degradation results in activation of one or more interferon-stimulated genes (ISGs). In some embodiments, said degradation induces an increased expression or activity of a mitochondrial antiviral signaling protein. In some embodiments, said degradation induces an IFN signaling pathway in said cancer cell. In some embodiments, said degradation results in expression of one or more neoantigens, and/or an increase in level of said one or more neoantigens relative to that in a suitable control.

[0028] In some embodiments, said degradation induces an increase in level and/or activity of a MHC class 1 polypeptide. In some embodiments, said degradation induces an increase in sensitivity of said cancer to a spliceosome targeted therapy. In some embodiments, said degradation induces expression of one or more caspases in said cancer cell. In some embodiments, said degradation induces apoptosis of said cancer cell. In some embodiments, said degradation results in formation and/or increase in level of a R loop. In some embodiments, said degradation and said formation and/or an increase in level of said R loop results in activation of a cGAS-STING pathway. In some embodiments, said degradation induces an increase in level and/or activity of a T cell of any kind, e.g., a cytotoxic T cell, e.g., a CD8+ T cell, a helper T cell, a memory T cell, an effector T cell. In some embodiments, said degradation comprises inhibiting the enzymatic activity of said enzymatic component of said spliceosome. In some embodiments, said one or more mis-spliced RNA comprises one or more retained introns.

[0029] In some embodiments, said enzymatic component is a RNA helicase. In some embodiments, said enzymatic component is an ATP dependent RNA helicase. In some embodiments, said enzymatic component is a DEAD-box helicase. In some embodiments, said enzymatic component is a DEAH-box helicase. In some embodiments, said enzymatic component (e.g., a RNA helicase) is DHX8, DHX15, DHX16, DHX35, DHX33, DHX38, DHX40, DHX32, DHX34, DHX37, DHX36, DHX57, DHX29, DHX9, DHX30, UPF1, SMBP2, SETX, MOVIO, MOV10L1, DHX58, IFIH1, DDX58, AQR, DDX12, DDX11, HELZ2, ZNFX1, DICER, SUV3, ASCC3, Brr2, SKIV2, MTREX, DDX60, DDX28, DDX18, DDX10, DDX55, DDX31, DDX51, DDX24, DDX56, DDX19A, DDX19B, DDX25, eIF4Al, eIF4A2, eIF4A3, DDX39B, DDX39A, DDX20, DDX6, DDX50, DDX21, DDX1, DDX54, DDX5, DDX17, DDX53, DDX43, DDX23, DDX46, DDX42, DDX41, DDX3Y, DDX3X, DDX4, DDX52, DDX59, DDX47, DDX49, DDX39B, PRPF8, or DDX27. In some embodiments, said enzymatic component is selected from the group consisting of DHX8, DHX15, DHX38, DHX8, DHX16, DDX46, DDX23, DDX41, DDX47, AQR, and DDX21. In some embodiments, said enzymatic component is DHX15, DHX38, DHX46, or DHX23. In some embodiments, said enzymatic component is DHX46, or DHX23. In some embodiments, said enzymatic component is DHX15, or DHX38. In some embodiments, said enzymatic component is DHX46. In some embodiments, said enzymatic component is DHX15, or DHX38. In some embodiments, said enzymatic component is DHX23. In some embodiments, said enzymatic component is DHX15. In some embodiments, said enzymatic component is a Ski-21ike helicase. In some embodiments, said enzymatic component is Prp5, Sub2, Prp28, Prpl9, Brr2, Prpl6, Prp22, or Prp43.

[0030] In some embodiments, said one or more mis-spliced RNA forms a dsRNA in said cancer cell. In some embodiments, said method comprises inducing expression of one or more mis-spliced RNA that forms a dsRNA in said cancer cell. In some embodiments, said dsRNA is located in cytoplasm of said cancer cell. In some embodiments, said dsRNA induces interferon signaling pathway. In some embodiments, said dsRNA induces an immune response. In some embodiments, the dsRNA induces an innate immune response. In some embodiments, said immune response is an anti-viral immune response. In some embodiments, the dsRNA induces interferon secretion. In some embodiments, the dsRNA induces interferon activation. In some embodiments, the dsRNA are detected by MDA5, RIG1, PKR, TLR3, or a combination thereof. In some embodiments, the dsRNA induces expression and/or activation of MDA5, RIG1, PKR, TLR3, or a combination thereof. [0031] In some embodiments, said immune response is an antiviral immune response. In some embodiments, said method comprises inducing expression of one or more mis-spliced RNA that encodes one or more neoantigens. In some embodiments, said one or more mis-spliced RNA encode said one or more neoantigens. In some embodiments, said one or more neoantigen comprises a neoepitope that binds to a HLA protein of said subject. In some embodiments, the method further comprises binding of a neoepitope of said one or more neoantigen to a HLA protein. In some embodiments, said binding to said HLA protein induced a T cell response (e.g., a cytotoxic T cell response or a helper T cell response). In some embodiments, said immune response comprises induction of a T cell response (e.g., a cytotoxic T cell response or a helper T cell response). In some embodiments, said immune response is a T-cell immune response. In some embodiments, said immune response is a memory immune response. In some embodiments, said immune response comprises an increase in level of one or more cytokine and/or chemokines in said subject.

[0032] In some embodiments, the cancer is selected from the group consisting of melanoma, ovarian cancer, lung cancer, prostate cancer, breast cancer, colorectal cancer, endometrial cancer, lymphoma, and a leukemia. In some embodiments, said cancer is a solid tumor. In some embodiments, the cancer is a hematological tumor.

[0033] In some embodiments, the method further comprises administering one or more additional cancer therapeutic agent, such as an agent targeting an oncogene (e.g., a kinase, a RAS downstream effector pathway). In some embodiments, the one or more additional cancer therapeutic agent comprises a chemotherapeutic agent, radiation, or immunotherapy.

[0034] In some embodiments, the method further comprises administering one or more anti immunosuppressive/immunostimulatory agents. In some embodiments, the one or more anti- immunosuppressive/immunostimulatory agent provides a CTLA4, a PD-1, a PD-L1 blockade, or a combination thereof. In some embodiments, the anti-immunosuppressive/immunostimulatory agent provides a CTLA4, a PD-1, a PD-L1, a TIM3, a LAG-3, a TIGIT, or a OX40L blockade. In some embodiments, the one or more anti -immunosuppressive/immunostimulatory agents comprises an anti- CTLA4 antibody, an anti-PD 1 antibody, an anti-PD-Ll antibody, or a combination thereof.

[0035] In some embodiments, the one or more additional cancer therapeutic agents are capable of binding to and/or inhibiting programmed cell death 1 (PDCD1, PD1, PD-1), CD274 (CD274, PDL1, PD-L1), PD- L2, cytotoxic T-lymphocyte associated protein 4 (CTLA4, CD 152), CD276 (B7H3); V-set domain containing T cell activation inhibitor 1 (VTCN1, B7H4), CD272 (B and T lymphocyte associated (BTLA)), killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1), lymphocyte activating 3 (LAG3, CD223), hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3), V-set immunoregulatory receptor (VSIR, B7H5, VISTA), T cell immunoreceptor with Ig and ITIM domains (TIGIT), programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273), immunoglobulin superfamily member 11 (IGSF11, VSIG3), TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML), PVR related immunoglobulin domain containing (PVRIG, CD112R), galectin 9 (LGALS9), killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); and killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1), killer cell lectin like receptor Cl (KLRC1, NKG2A, CD159A), killer cell lectin like receptor DI (KLRD1, CD94), killer cell lectin like receptor G1 (KLRG1, CLEC15A, MAP A, 2F1), sialic acid binding Ig like lectin 7 (SIGLEC7), SIGLEC, sialic acid binding Ig like lectin 9 (SIGLEC9), CEACAM (e.g., CEACAM-1, CEACAM-3 and/or CEACAM-5), VISTA, LAIR1, CD160, 2B4, CD80, CD86, B7-H1, B7-H3 (CD276), B7-H4 (VTCN1), CD134 (OX40L), KIR, A2AR, A2BR, MHC class I, MHC class II, GAL9, adenosine, TGFR (e g., TGFRbeta) , CD94/NKG2A, IDO, TDO, CD39, CD73, GARP, CD47, SIRP alpha, SIRP beta, PVRIG, CSF1R, orNOX. In some embodiments, said additional cancer therapeutic agent is a PD-1 modulator.

[0036] In some embodiments, said PD-1 modulator is Pembrolizumab (humanized antibody), Pidilizumab (CT-011, monoclonal antibody, binds DLL1 and PD-1), Spartalizumab (PDR001, monoclonal antibody), Nivolumab (BMS-936558, MDX-1106, human IgG4 monoclonal antibody), MEDI0680 (AMP-514, monoclonal antibody), Cemiplimab (REGN2810, monoclonal antibody), Dostarlimab (TSR-042, monoclonal antibody), Sasanlimab (PF- 06801591, monoclonal antibody), Tislelizumab (BGB-A317, monoclonal antibody), BGB-108 (antibody), Tislelizumab (BGB-A317, antibody), Camrelizumab (INCSHR1210, SHR-1210), AMP-224, Zimberelimab (AB 122, GLS-010, WBP-3055, monoclonal antibody), AK-103 (HX- 008, monoclonal antibody), AK-105 (anti-PD-1 antibody), CS1003 (monoclonal antibody), HLX10 (monoclonal antibody), Retifanlimab (MGA-012, anti-PD-1 monoclonal antibody), BI- 754091 (antibody), Balstilimab (AGEN2034, PD-1 antibody), toripalimab (JS-001, antibody), cetrelimab (JNJ-63723283, anti-PD-1 antibody), genolimzumab (CBT- 501, anti-PD-1 antibody), LZM009 (anti-PD-1 monoclonal antibody), Prolgolimab (BCD- 100, anti-PD-1 monoclonal antibody), Sym021 (antibody), ABBV-181 (antibody), BAT- 1306 (antibody), JTX-4014, sintilimab (IBI-308), Tebotelimab (MGD013, PD-l/LAG-3 bispecific), MGD-019 (PD- 1/CTLA4 bispecific antibody), KN-046 (PD-1/CTLA4 bispecific antibody), MEDI-5752 (CTLA4/PD-1 bispecific antibody), RO7121661 (PD-l/TIM-3 bispecific antibody), XmAb20717 (PD-1/CTLA4 bispecific antibody), or AK-104 (CTLA4/PD-1 bispecific antibody).

[0037] In some embodiments, said one or more additional cancer therapeutic agent is a CTLA4 modulator. In some embodiments, said one or more additional cancer therapeutic agent is a CD40L antibody, an OX-40 antibody, a CD28 antibody, or a combination thereof. [0038] In some embodiments, the subject has a breast cancer that is resistant to a therapy (e.g., an antiestrogen therapy), is an MSI breast cancer, is a metastatic breast cancer, is a Her2 negative breast cancer, is a Her2 positive breast cancer, is an ER negative breast cancer, is an ER positive breast cancer or any combination thereof. In some embodiments, the breast cancer expresses an estrogen receptor with a mutation.

[0039] In some embodiments, the method further comprises administering at least one additional therapeutic agent or modality.

[0040] In some embodiments, said cancer cell comprises increased expression or activation of an oncogene (e.g., MYC) relative to that in a corresponding normal cell. In some embodiments, said cancer is a myc dependent cancer. In some embodiments, said subject is a mammal, such as a human. In some embodiments, said cancer is a human cancer. In some embodiments, said immune response is long-term immune response.

[0041] In some aspects, the present disclosure provides a method of enhancing sensitivity of a cancer to one or more spliceosome targeted therapies (STT). In some embodiments, the method comprises: inhibiting a selected enzymatic component of a spliceosome in a cancer cell, wherein said inhibiting of said selected enzymatic component induces expression of one or more mis-spliced RNA that is different than that induced upon inhibition of a non-selected enzymatic component associated with a spliceosome. [0042] In some aspects, the present disclosure provides a method of predicting responsiveness of a subject to a spliceosome targeted therapy (STT) comprising; (a) inhibiting an enzymatic component of a spliceosome in said subject; (b) determining a RNA expression profde in a biological sample of said subject; (c) comparing said RNA expression profde with a known mis-spliced RNA expression profde associated with sensitivity to said STT; and (d) identifying said subject as a responsive subject or a non- responsive subject based on the comparison of step (b). In some embodiments, said subject is identified as responsive if said RNA expression profde matches said known mis-spliced RNA expression profde. In some embodiments, said subject is identified as non-responsive if said RNA expression profde is different than said known mis-spliced RNA expression profde.

[0043] In some aspects, the present disclosure provides a method for inducing an immune response to a cancer cell in a subject, comprising: inhibiting an enzymatic component of a spliceosome that is a RNA helicase in a cancer cell in said subject. In some embodiments, said inhibiting induces expression of one or more mis-spliced RNA in said cancer cell. In some embodiments, said expression of said one or more mis-spliced RNA results in an anti-viral immune response to said cancer cell in said subject.

[0044] In some aspects, the present disclosure provides a method for inducing an immune response to a cancer cell in a subject, comprising: inducing degradation of an enzymatic component polypeptide of a spliceosome in a cancer cell in said subject. In some embodiments, said degradation induces expression of one or more mis-spliced RNA in said cancer cell. In some embodiments, said expression of said one or more mis-spliced RNA results in an immune response to said cancer cell in said subject, thereby treating said subject.

[0045] In some aspects, the present disclosure provides a method for treating a cancer in a subject, comprising: inhibiting DDX46, DDX23, DHX15 or a combination thereof in a said subject. In some embodiments, said inhibiting induces expression of one or more mis-spliced RNA in said cancer cell. In some embodiments, said expression of said one or more mis-spliced RNA results in an immune response to said cancer cell, thereby treating said subject.

[0046] In some aspects, the present disclosure provides a method for inducing an immune response to a cancer cell in a subject, comprising: inhibiting DDX46, DDX23, DHX15 or a combination thereof in said subject. In some embodiments, said inhibiting induces expression of one or more mis-spliced RNA in said cancer cell. In some embodiments, said expression of said one or more mis-spliced RNA results in an immune response to said cancer cell.

[0047] In some embodiments of aspects, and embodiments herein, the dsRNA induces JAK/STAT signaling pathway. In some embodiments, the dsRNA induces expression of one or more IRF-family transcription factors (e g., IRF-1, IRF-2, IRF-3, IRF-4, IRF-5, IRF-6, IRF-7, IRF, 8, and IRF-9). In some embodiments, the dsRNA induces expression of one or more NF-KB transcription factors. In some embodiments, the dsRNA induces Jak/Stat signaling pathway. In some embodiments, the dsRNA induces protein-kinase R-signaling pathway. In some embodiments, the dsRNA induces 2', 5 '-oligoadenylate synthetase (OAS)-RNase L pathway. In some embodiments, the one or more mis-spliced RNA induces JAK/STAT signaling pathway. In some embodiments, the one or more mis-spliced RNA induce expression of one or more IRF-family transcription factors (e.g., IRF-1, IRF-2, IRF-3, IRF-4, IRF-5, IRF- 6, IRF-7, IRF, 8, and IRF-9). In some embodiments, the one or more mis-spliced RNA induces expression of one or more NF-KB transcription factors. In some embodiments, the one or more mis-spliced RNA induces Jak/Stat signaling pathway. In some embodiments, the one or more mis-spliced RNA induces protein-kinase R-signaling pathway. In some embodiments, the one or more mis-spliced RNA induces 2', 5 '-oligoadenylate synthetase (OAS)-RNase L pathway.

[0048] In some embodiments, the immune response comprises activation JAK/STAT signaling pathway. In some embodiments, the immune response comprises expression of one or more IRF-family transcription factors (e g., IRF-1, IRF-2, IRF-3, IRF-4, IRF-5, IRF-6, IRF-7, IRF, 8, and IRF-9). In some embodiments, the immune response comprises expression of one or more NF-KB transcription factors. In some embodiments, the immune response comprises activation of Jak/Stat signaling pathway. In some embodiments, the immune response comprises activation of protein-kinase R-signaling pathway. In some embodiments, the immune response comprises activation of 2', 5 '-oligoadenylate synthetase (OAS)-RNase L pathway. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure can be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:

[0050] FIG. 1 shows exemplary enzymatic components of spliceosome and their role in splicing cycle. [0051] FIG. 2 shows RNA misprocessing signatures upon inhibition or degradation of exemplary enzymatic components of the spliceosome. The data was generated in two cell lines; MDA-MB231-LM2, and SUM 159. Splicing perturbations of different exemplary enzymatic components show different RNA mis-processing signatures. Splicing perturbations of select enzymatic components shows expression of one or more mis-spliced RNA that is different than that of others.

[0052] FIG. 3 shows RNA processing signatures upon splicing perturbations (e.g., by inhibition or degradation of exemplary enzymatic components e.g., helicases of a spliceosome). Selective perturbations of exemplary helicases result in distinct mis-splicing signatures.

[0053] FIG. 4 shows fingerprints of inhibition (e.g., selective inhibition) of helicases.

[0054] FIG. 5 shows DHX15 perturbation (e.g., inhibition or degradation) results in robust expression of mis-spliced RNA. The mis-spliced RNA comprises intron retention. The data shows that selective inhibition of DHX15 results in mis-splicing events (i.e., expression of one or more mis-spliced RNA) that is different than those obtained upon perturbations (e.g., inhibition or degradation) of other helicases (e.g., DHX38, DHX16, DHX46).

[0055] FIG. 6 shows perturbation (e.g., inhibition or degradation) of enzymatic components of a spliceosome modulates expression of OAS1, and results in activation of an antiviral immune signaling. Perturbations of different exemplary enzymatic components (e.g., DDX46, DHX8, DHX16, DDX23) results in differential effects on expression of OAS1, demonstrating a differential ability to activate an immune response (e.g., an antiviral immune). Perturbations of different exemplary enzymatic components (e.g., DDX46, DHX8, DHX16, DDX23) results in a different immune response.

[0056] FIG. 7 shows perturbation (e.g., inhibition or degradation) of enzymatic components of a spliceosome modulates expression of CXCL10, and results in activation of an antiviral immune signaling. Perturbations of different exemplary enzymatic components (e.g, DDX46, DHX8, DHX16, DDX23) results in differential effects on expression of CXCL10, demonstrating a differential ability to activate an immune response (e.g., an antiviral immune). Perturbations of different exemplary enzymatic components (e.g., DDX46, DHX8, DHX16, DDX23) results in a different immune response.

[0057] FIG. 8 shows perturbation (e.g., inhibition or degradation) of spliceosome RBP-X in cancer cells induced anti-tumor activity. [0058] FIG. 9 shows perturbation (e.g., inhibition or degradation) of RBP-X results in durable (i.e., a long term) immune response. In some embodiments, RBP-X is an enzymatic component of the spliceosome.

[0059] FIG. 10A and 10B shows perturbation (e.g., inhibition or degradation) of RBP-X-degradation results in immune memory response to a cancer. In some embodiments, RBP-X is an enzymatic component of the spliceosome. FIG. 10A shows results in murine TNBC cells ((PyMT-M; mouse mammary tumor virus-polyoma middle tumor-antigen) mouse model of breast cancer model). FIG. 10B shows results in EO771 model.

[0060] FIG. 11 shows panel of cell line models with inducible degradation of spliceosome components were used to determine unique function in splicing fidelity. SUM159-Cas9 cells were transduced with FKBP-tagged spliceosome components and spliceosome-targeted sgRNAs to generate cell lines with endogenous knockout and expression of degradable exogenous alleles. Targets were chosen to represent core spliceosome components with recurrent spliceosome mutations and helicases characterized to play a role in splicing quality control. Schematic illustrates the splicing cycle and where each component modeled functions within that process.

[0061] FIGs. 12A-12K, FIG13, FIG 14A, 14B, 15, 16, and 17 demonstrate rapid degradation of individual spliceosome components causes distinct patterns of RNA mis-splicing. FIG. 12A-12H shows target cDNA fused to FKBP12 ^F36V were expressed in SUM159 cells in which the endogenous loci of the corresponding spliceosome component was knocked out via CRISPR/Cas9. Cells were treated with dTagVl for 6, 9, and 12 hrs, and the endogenous protein level of the spliceosome component were measure using western blot. FIG. 12A shows degradation of spliceosome component U2AF2. FIG. 12B shows degradation of spliceosome component DDX46. FIG. 12C shows degradation of spliceosome component SF3B1. FIG. 12D shows degradation of spliceosome component PRPF8. FIG. 12E shows degradation of spliceosome component DHX16. FIG. 12F shows degradation of spliceosome component AQR. FIG. 12G shows degradation of spliceosome component DHX38. FIG. 12H shows degradation of spliceosome component DHX15.

[0062] FIGs. 12I-12K shows measured changes in RNA splicing induced by maximum target degradation using paired-end poly(A)+ RNAseq followed by classification of misprocessed fragments across annotated introns. FIG. 121 shows Model of mis-splicing algorithm using to quantify RNA mis- splicing upon spliceosome component degradation. Briefly, exon-level annotations from ENSEMBL were collapsed across isoforms to generate a “multi-gene” annotation. Read-pairs were then classified as “misspliced” if one or more of the read mates mapped into intronic regions. All other read-pairs were defined as “spliced” (includes exonic sequence only). Global mis-splicing summarizes the proportion of misspliced read-pairs across all mapped read-pairs. For each intron, a generalized binomial model was applied to assess significance of the proportion of mis-spliced to spliced read-pairs between two conditions (e.g., DMSO vs dTag treatment).

[0063] FIGs. 12J and 12K show degradation of spliceosome targets results in increased RNA missplicing. Swarm plot represents the fold change in global mis-splicing (proportion of total mis-spliced (or misprocessed) fragments versus properly spliced fragments) with target degradation at (FIG. 12 J) 6 hours and (FIG. 12K) 12 hours compared to vehicle (mean +/- SEM, n=3 biological replicates/condition, two- tailed unpaired Student’s t-test).

[0064] FIG. 13 shows spliceosome component degradation leads to increased RNA mis-splicing. RNA mis-splicing analysis was performed on RNA from SUM159-FKBP cell lines treated with dTAG13 or dTAGVl for 9hrs. RNA mis-splicing ratio was calculated on a single intron basis as the difference in proportion of mis-spliced fragments versus properly spliced fragments. Volcano plot represents the change in mis-splicing ratio between DMSO and dTAG-treated samples. Color indicates number of introns.

[0065] FIG. 14A shows degradation of specific spliceosome targets results in differential RNA mis- splicing. Swarm plot represents the fold change in number of mis-processed introns with maximum target degradation at 9 hours compared to OnM dose (mean +/- SEM, n=3 biological replicates/condition, two- tailed unpaired Student’s t-test).

[0066] FIG. 14B shows spliceosome target degradation induces misprocessing of a distinct set of introns. Sashimi plot of representative introns selectively misprocessed with SF3B1 degradation, left, and AQR degradation, right.

[0067] FIG. 15 shows DHX15 has a unique misprocessing signature compared to other targets. Dimension reduction analysis of misprocessing scores was used to identify target clusters, suggesting similar signatures of misprocessing. Three clusters were identified: U2-complex (SF3B1, U2AF2, and DDX46), catalytic spliceosome (DHX38, DHX16, AQR, and PRPF8), and DHX15 in a distinct cluster. [0068] FIG. 16 shows degradation of targets within the same cluster induces misprocessing of a unique set of introns. Cluster-specific misprocessing was identified using linear regressions. Data are shown as intron-level misprocessing (n=3 biological replicates/condition).

[0069] FIG. 17 shows degradation of targets within a functional cluster leads to retention of distinct intron sets. RNA-seq read coverage of introns identified as specifically misprocessed by U2-complex (left), catalytic spliceosome (middle), and DHX15 (right) are shown. Data are representative of biological triplicates for each condition. *p<0.05, **p<0.0I, ***p<0.00I, ****p<0.0001

[0070] FIGs 18A-18L demonstrate degradation of DHX15 leads to widespread 5’ and 3’ cryptic splicing. FIG. 18A and FIG. 18B show DHX15 degradation induces splicing at cryptic splice sites. FIG. 18A is a sashimi plot of reads mapping to the SNAPCI intron and flanking exons. Data are representative of biological triplicates for each condition. FIG. 18B is a box plot quantifies number of reads mapping to cryptic splice junctions upon DMSO or dTAG13 treatment (median (line), QI to Q3 quartile values (boundaries of the box), and range (whiskers), n=3 biological replicates per condition).

[0071] FIG. 18C and FIG. 18D show increased levels of DHX15 degradation results in dose-dependent cryptic splice junction accumulation. FIG. 18C is a sashimi plot of reads mapping to the RBM17 intron and flanking exons. FIG. 18D is a box plot quantifies number of cryptic splice junctions upon DMSO and dTAG13 treatment (median (line), QI to Q3 quartile values (boundaries of the box), and range (whiskers), n=3 biological replicates per condition).

[0072] FIG. 18E shows degradation of DHX15 leads to use of multiple cryptic splice sites within a single intron. Bar plot of number of introns with 1-5, and 6+ cryptic splice junctions in DMSO and dTAG13 treated states (mean and SEM, n=3 biological replicate s/treatment, two-tailed unpaired Student’s t-test). [0073] FIG. 18F shows Cryptic splice junctions can be classified by the cryptic splice site utilized. Depicted are three bins of cryptic splice junctions: Cryptic 3’ss (spliced to canonical 5’ss), Cryptic 5’ss (spliced to canonical 3’ss), and Dual cryptic (splicing between cryptic 5’ and 3’ss within an intron). Canonical splicing events are used for normalization as a proxy for sequencing depth.

[0074] FIG. 18G shows Degradation of DHX15 uniquely induces all classifications of cryptic splicing. Magnitude of cryptic splicing is quantified as the total number of unique cryptic splice junctions normalized to the number of known intron splicing events. Bar plot depicts fold change in cryptic splicing magnitude compared to DMSO treatment after 9hrs of target degradation (mean +/- SEM, n=3 biological replicates/condition, two-tailed unpaired Student’s t-test).

[0075] FIG. 18H shows DHX15 degradation similarly induces cryptic splicing in an additional TNBC cell line, MDA-MB-231-LM2. Magnitude of cryptic splicing is quantified as the number of unique cryptic splice junctions normalized to the number of known intron splicing events. Bar plot depicts fold change in cryptic splicing magnitude compared to DMSO treatment after 9hrs of target degradation (mean +/- SEM, n=3 biological replicates/condition, two-tailed unpaired Student’s t-test).

[0076] FIG. 181 shows similar cryptic splice junctions are utilized in both SUM 159 and LM2 cells upon DHX15 degradation. Sashimi plot of reads mapping to the SNAPCI intron and flanking exons. Data are representative of biological triplicates for each condition. Splice junctions with >= 3 reads are shown. [0077] FIG. 18J shows Usage of cryptic splice sites is similarly increased in both SUM159 and LM2 cells with DHX15 degradation. Frequency of cryptic splicing was quantified using a generalized binomial model comparing the splicing frequency of cryptic versus canonical junctions. Scatter plot of differential frequency of cryptic splicing -/+ DHX15 degradation in both SUMI 59 and LM2 cells. Differential cryptic splicing frequency is significantly correlated between the two cell lines (p = 6.2e-100).

[0078] FIG. 18K and FIG. 18L shows cryptic splice junction usage is increased in both SUM159 and LM2 cells upon DHX15 degradation. FIG. 18K is a box plot depicts fold change in cryptic junction usage between DMSO and dTAG13 treated states induces increased cryptic junction usage in both SUM159 and LM2 cells (median (line), QI to Q3 quartile values (boundaries of the box), and range (whiskers), n=3 biological replicates per condition). FIG. 18L is a sashimi plot of reads mapping to the cryptic splice junction and flanking exons. Data are representative of biological triplicates for each condition. *p<0.05, **p<0.01, ***p<0.001. See also FIGs 19A-19J.

[0079] FIGs. 19A-19J demonstrate effects of degradation of DHX15. FIG. 19A shows DHX15 degradation induces cryptic splicing in SNAPCI. SUM159 FKBP-DHX15 cells were treated with dTag for 9hrs and cryptic splicing was measured using RT-qPCR. Data are shown as cryptic splicing vs canonical splicing relative to DMSO (mean -/+ SEM, n=3 biological replicates).

[0080] FIG. 19B shows increasing dose of dTag results in dose-dependent decrease in DHX15 protein levels. SUM 159 FKBP-DHX15 cells were treated with dTag for 9 hours and DHX15 protein levels were measured using Western blot. Tubulin was probed as a load control.

[0081] FIG. 19C shows cryptic splicing of RBM17 is exacerbated by increased DHX15 degradation. SUMI 59 FKBP-DHX15 cells were treated with dTag for 9hrs and cryptic splicing was measured using RT-qPCR. Data are shown as cryptic splicing vs canonical splicing relative to DMSO (mean -/+ SEM, n=3 biological replicates).

[0082] FIG. 19D shows cryptic splice junctions are detected in long read sequencing. Cryptic splicing events were identified in short read sequencing (read density shown at top) of LM2 FBKP-DHX15 dTag treated cells. Long reads from cells treated with DMSO (bottom) or dTag (middle) mapping to RBM25 are shown. Within long reads, gray boxes represent read coverage, blue lines represent skipped coverage due to splicing, and novel read coverage due to cryptic splicing is represented by maroon boxes.

[0083] FIG. 19E shows circular RNAseq reads support intron lariat formation from cryptic splicing events. Circular RNAseq reads from the dTag treated state suggest intron lariat formation from splicing at both the canonical and cryptic 3’ splice sites in LM2 FKBP-DHX15 cells. Sashimi plot of standard RNAseq reads mapping to representative introns and flanking exons with cryptic junctions indicated with red lines shown top. Circular RNAseq read density from cells treated with DMSO (middle) or dTag (bottom). Black lines under circular RNAseq read density indicate region included that supports canonical junction usage and the red line indicate region included that supports cryptic junction usage.

[0084] FIG. 19F shows LM2 FKBP-DHX15 cell line expresses degradable DHX15 with knockout of endogenous DHX15. Cells were treated with dTag 13 for 9 hours and DHX15 protein levels were measured using Western blot. Probing with DHX15 antibody detects both endogenous and FKBP- DHX15allele, probing with HA antibody detects FKBP-DHX15 allele alone. Tubulin was probed as a load control.

[0085] FIG. 19G shows DHX15 degradation induces cryptic splicing in SNAPCI. LM2 FKBP-DHX15 cells were treated with dTag for 9 hours and cryptic splicing was measured using RT-qPCR. Data are shown as cryptic splicing vs canonical splicing relative to DMSO (mean -/+ SEM, n=3 biological replicates).

[0086] FIG. 19H-FIG. 191 shows DHX15 degradation induces cryptic splicing at conserved sites in independent TNBC cell lines. FIG. 19H shows number of reads mapping to cryptic (x-axis) and canonical (y-axis) splice junctions in SUMI 59 cells. FIG. 191 shows number of reads mapping to cryptic (x-axis) and canonical (y-axis) splice junctions in LM2 cells are shown. Reads numbers from each DMSO treated replicate are shown with black dots and each dTag treated replicate with red dots. The proportion of these reads were compared using a generalized binomial model to determine relative frequency of splicing at a given cryptic junction.

[0087] FIG. 19J shows DHX15 degradation induces cryptic splicing

[0088] FIGs. 20A-20B show sequence proximal to cryptic splice sites is similar to canonical splice site. FIG. 20A shows motif information content for 20-mers past the 5’ss. FIG. 20B shows motif information content for 20-mers preceding the 3’ss are shown. Image generated with the Bio.motifs package in Python. [0089] FIG. 20C shows sequence surrounding the predicted branch point of cryptic 3’ss is similar to that of canonical splice sites. Motif information content for 7-mers centered on the branchpoint predicted by Branch Point Prediction algorithm is shown. Image generated with the Bio.motifs package in Python.

[0090] FIG. 20D shows predicted branch point strength is similar between cryptic and canonical splice sites. Empirical cumulative distribution curves of predicted branch point strength are plotted.

[0091] FIGs. 20E and 20F show cryptic splice sites used upon DHX15 degradation are weaker than canonical splice sites. FIG. 20E shows empirical cumulative distribution curves of MaxENT predicted splice site strength of both cryptic and canonical 5’ss. FIG. 20F shows that of 3’ss. A leftward shift in the blue curve indicates decreased splice site strength of cryptic splice junctions.

[0092] FIG. 20G shows cryptic splice sites used upon DHX15 degradation are predicted splice sites. Read density plot of reads mapping to the SNAPCI intron and flanking exons. Data are representative of biological triplicates for each condition. Splice junctions with >= 3 reads are shown. Acceptor splice site probability predicted by spliceAI are shown on the right y-axis.

[0093] FIG. 20H shows cryptic splice sites used upon DHX15 degradation have higher splice prediction scores compared to surrounding nucleotides. SpliceAI analysis was used to predict the splice site probability of each nucleotide in a 200bp window centered on a cryptic splice site. Shown is the mean SpliceAI probability score for 100 randomly selected cryptic splice sites. Splice donor and acceptor probability were calculated for canonical 3’ss and canonical 5’ss, respectively, as a negative control [0094] FIGs. 201 - 20J show cryptic splice sites used upon DHX15 degradation are weaker than canonical splice sites. FIG. 201 shows empirical cumulative distribution curves of SpliceAI predicted splice site strength of both cryptic and canonical 5’ss. FIG. 20 J shows that of 3’ss. A leftward shift in the red curve indicates decreased splice site strength of cryptic splice junctions. [0095] FIGs. 21A-21C show effects of degradation of DHX15. FIG. 21A shows intron length.

[0096] FIG. 2 IB shows cryptic splice sites are distributed throughout intronic space, with modestly increased frequency near the canonical splice junction. Bar plot represents the frequency of cryptic 5’ splice sites (upper panel) and cryptic 3 ’ splice sites (bottom panel) across normalized intron space (0 = canonical 5’ splice site, 1 = canonical 3’ splice site) for SUM159 (blue bars) and LM2 FKBP-DHX15 (green bars) cells treated with dTag. Lines represent the kernel distribution estimation plot for SUM159 (blue) and LM2 (green).

[0097] FIG. 21C shows the distance between putative branch point and splice site is similar between cryptic and canonical 3’splice sites. Empirical cumulative distribution curves of distance between putative branch point and 3’ splice site are plotted for canonical (black line) and cryptic (red line) 3’ splice sites. [0098] FIG. 22A shows meta-view analysis used to assess RBP binding in the proximity of canonical and cryptic splice sites. Meta-views represent 75 “exonic” and 250 “intronic” nucleotides surrounding canonical and cryptic splice sites as illustrated in schematic identified in RNAseq of SUM159 FKBP- DHXI5 cells treated with dTAG13. RBP peak calls from the ENCODE eCLIP experiments in K562 and HepG2 cells were used.

[0099] FIG. 22B shows cryptic 5’ and 3’ss are lacking binding of key spliceosome components. Lines show binding frequency of PRPF8 at the 5 ’ss (left) and SF3B4 at the 3’ss (right) with 5th to 95th percentile shaded.

[0100] FIG. 22C shows cryptic 3’ss maintain U2AF recognition while lacking SF3 complex binding. Cryptic 5’ss lack binding of canonical factors such as AQR and PRPF8. Heatmap indicates peak coverage (requiring >= 8-fold and p value <= 10 ^A -3) in immunoprecipitations versus paired size-matched input in published ENCODE data. Color indicates average peak density across 500 splice junctions.

[0101] FIG. 22D shows cryptic 3’ss are bound by the U2AF complex similarly to canonical junctions. Lines show binding frequency of U2AF2 (left) and U2AF2 (right) at the 3’ss with 5th to 95th percentile shaded.

[0102] FIG. 22E shows SF3B4 binds to canonical splice sites with a higher frequency than U2AF2. Bar plot depicts fraction of total SF3B4 and U2AF2 peaks that overlap with canonical splice sites used in SUMI 59 FKBP-DHX15 cells treated with dTAG13.

[0103] FIG. 22F shows DHX15 degradation leads to increased splicing of U2AF2-bound sites. Bar plot depicts fraction of SF3B4 and U2AF2 peaks that overlap with cryptic splice sites compared to peaks that do not overlap with canonical splice sites.

[0104] FIG. 22G shows U2AF2 binding motif is similar between peaks overlapping cryptic and canonical splice sites. Scatter plot shows 6-mer frequency of pyrimidine -rich and 3’ splice site containing sequences, blue and red respectively, in U2AF2 peaks overlapping cryptic and canonical splice sites. [0105] FIGs. 22H, and 221 show U2AF2 binding motif at sites that neither overlap cryptic or canonical splice sites have increased pyrimidine content but lack 3’ splice site sequence. FIG. 22H shows a scatter plot shows 6-mer frequency of pyrimidine-rich and 3’ splice site containing sequences, blue and red respectively, in U2AF2 peaks overlapping canonical splice sites and sequences with neither canonical or cryptic splicing. FIG. 221 shows a bar plot showing 6-mer frequency in U2AF2 peaks.

[0106] FIG. 22J shows Sequences bound by U2AF2 that are neither cryptic or canonical splice sites are predicted to be significantly weaker splice sites. Empirical cumulative distribution curves of MaxENT predicted splice site strength of both cryptic, canonical, and neither sequences are plotted. A leftward shift in the green curve indicates decreased splice site strength of U2AF2-bound sequences that do not overlap cryptic or canonical splice junctions.

[0107] FIGs. 22K, and 22L show degradation of DHX15 increases SF3B4 binding at cryptic 3’ splice sites. Lines show binding frequency of SF3B4 in SUM159 cells at baseline (FIG. 22K) and upon DHX15 (FIG. 22L) degradation with 5th to 95th percentile shaded. Canonical and cryptic splice sites are defined based on RNAseq of SUMI 59 FKBP-DHX15 cells treated with dTAG13.

[0108] FIG. 23A-23K shows Cancer hotspot mutation compromises DHX15 quality control function and results in increased cryptic splice site usage.

[0109] FIG. 23 A shows Recurrent hotspot mutations in DHX15 have been identified in AML patient tumors. RUNX1-RUNX1T1 AML samples have recurrent R222G mutations in the RNA binding domain. Amino acids are colored based on their predicted importance to protein function as calculated by Evolutionary Trace analysis. Protein domains of DHX15 are shown.

[0110] FIG. 23B shows R222G mutation impacts interaction between DHX15 and ssRNA substrate by decreasing contact with the RNA base. Decreased interaction with RNA by the R222G mutant can be seen by increased exposure of the RNA base. RNA binding by DHX15 has been modeled using prp43 structure. DHX15 is colored by domains in ribbon format with R222 residue shown in red in space fill. The R222G mutation has been modeled into the published DHX15 structure.

[OHl] FIG. 23C shows LM2 FKBP-DHX15 cells were engineered to express GFP, DHX15WT, and DHX15R222G cDNA. Treatment with dTAG13 results in degradation of the FKBP-DHX15 allele alone and maintained expression of the protein encoded by the rescue cDNA alone.

[0112] FIG. 23D shows DHX15R222G does not rescue RNA misprocessing induced by DHX15 degradation. RNA misprocessing analysis was performed on RNA from LM2-FKBP cell lines expressing GFP, DHX15WT or DHX15R222G and treated with DMSO or dTAG13. RNA misprocessing ratio is calculated on a single intron basis as the difference in proportion of mis-spliced fragments versus properly spliced fragments. Volcano plot represents the misprocessing ratio between DMSO and dTAG-treated samples. Color indicates number of introns. [0113] FIG. 23E and FIG. 23F show DHX15R222G does not rescue cryptic splicing induced by DHX15 degradation. FIG. 23E shows a sashimi plot of reads mapping to the SNAPCI intron and flanking exons. Data are representative of biological triplicates for each condition. FIG. 23F shows a cryptic splice junctions were quantified in LM2 FKBP-DHX15 cells expressing GFP, DHX15WT, and DHX15R222G upon degradation of the FKBP-DHX15 allele. Expression of DHX15WT suppresses accumulation of cryptic splice junctions while GFP and DHX15R222G do not. Bar plot depicts fold change in cryptic splicing magnitude compared to DMSO treatment after 6hrs of target degradation (mean +/- SEM, n=3 biological replicate s/condition, two-tailed unpaired Student’s t-test).

[0114] FIG. 23G shows model of inducible DHX15 R222G allele. DHX15 exon 3 harboring the c.664C>G mutation encoding the R222G allele flanked by Lox sites was inserted into the endogenous DHX15 locus. Generating a DHX15 R222Gflox mouse in a Rosa26CreER background allows for tamoxifen-inducible allele recombination and expression of DHX15R222G.

[0115] FIG. 23H shows AML-ETO9a transformation of hematopoietic stem cells from DHX15R222G allows for analysis of the impact of this allele in AML. Hematopoietic stem cells from fetal liver were harvested and transduced with AML-ETO9a. GFP+ transformed cells were sorted and treated -/+ tamoxifen to induce R222G allele recombination before collection for RNA sequencing.

[0116] FIG. 231 shows introduction of heterozygous and homozygous DHX15R222G mutation in AML- ETO cell lines induces misprocessing of similar introns. Principle component analysis was used to identify the most important features that distinguish heterozygous DHX15R222G mutation from DHX15WT. Heat map depicts misprocessing ratio of introns significantly increased with heterozygous DHX15 mutation.

[0117] FIG. 23J shows DHX15R222G mutation in AML-ETO cell lines induces cryptic splicing. Sashimi plot of representative intron misprocessed in both heterozygous and homozygous DHX15R222G cell lines.

[0118] FIG. 23K shows Introduction of DHX15R222G mutation increases cryptic splicing. Number of unique cryptic splice junctions was quantified in introns misprocessed in heterozygous and homozygous DHX15R222G mutation in AML-ETO cell lines. Bar plot depicts the fold change in number of cryptic splice junctions in tamoxifen vs vehicle treated cells (mean +/- SEM, n=3 biological replicates/condition, two-tailed unpaired Student’s t-test). *p<0.05, ***p<0.001, ****p<0.0001.

[0119] FIGs. 24A-24D show effects of DHX15 degradation. FIG. 24B shows canonical 5’ and 3’ splice sites. FIG. 24B shows cryptic 5’ and 3’ splice sites broadly lack key spliceosome-associated RBP binding. Binding frequency (calculated from ENCODE eCLIP data) of 37 additional RBPs indicate most spliceosome-associated RBPs do not bind near cryptic 5’ or 3’ splice sites identified in SUM 159 FKBP- DHX15 cells treated with dTag for 9hrs as compared to canonical splice sites. U2AF1/2 bind at both canonical and cryptic 3’ splice sites. Heatmap indicates peak coverage (requiring >= 8-fold and p value <= 10 ^A -3) in immunoprecipitations versus paired size-matched input in published ENCODE data. Color indicates average peak density across 500 splice junctions.

[0120] FIG. 24C-24D show expression of Dhxl5R222G induces RNA mis-splicing. FIG. 24A shows Tamoxifen treatment induces recombination and expression of Dhxl5 c.664C>G, encoding the R222G mutation. Plot represents the nucleotide frequency at Dhxl5 c.664 as quantified by RNA sequencing in samples as indicated. FIG. 24D shows homozygous and heterozygous Dhxl5R222G mutation results a distinct pattern of RNA mis-splicing. Principal component analysis of mis-splicing events was used to identify clusters, suggesting similar signatures of mis-splicing. PC2 separates out the Dhxl5R222G mutant lines treated with tamoxifen from Vehicle treated and Dhxl5WT cells.

[0121] FIGs. 25A and 25B show DHX15 signature cryptic splice sites are spliced at increased proportion upon DHX15 degradation in both SUM 159 and LM2 FKBP-DHX15 cells. FIG. 25 A shows a heat map depicts proportion of cryptic splicing in SUM159 and LM2 FKBP-DHX15 cells with DMSO or dTAG13 treatment. Average proportion of cryptic junction usage across the 122 signature junctions was calculated as the “DHX15 signature CSJ score”. FIG. 25B shows a sashimi plot of reads mapping to a DHX15 signature cryptic splice junction and flanking exons. Data are representative of biological triplicates for each condition.

[0122] FIG. 25C shows increased levels of DHX15 degradation results in dose-dependent increase in DHX15 signature CSJ score. Box plot shows CSJ score upon DMSO and increasing dTAG13 treatment (median (line), QI to Q3 quartile values (boundaries of the box), and range (whiskers), n=3 biological replicates per condition).

[0123] FIG. 25D shows degradation of DHX15 uniquely increases DHX15 signature CSJ score. Box plot represents CSJ score after 9hrs of target degradation (median (line), QI to Q3 quartile values (boundaries of the box), and range (whiskers), n=3 biological replicates per condition).

[0124] FIG. 25E shows DHX15R222G does not suppress increased DHX15 signature CSJ score induced by DHX15 degradation. Box plot represents CSJ score after 6hrs of dTAG treatment (median (line), QI to Q3 quartile values (boundaries of the box), and range (whiskers), n=3 biological replicates per condition). [0125] FIG. 25F shows DHX15 signature is upregulated in cancer cells vs normal. Box plot depicts DHX15 signature CSJ score in panel of tumor and normal cell lines.

[0126] FIG. 25G shows DHX15 signature CSJ score correlates with dependency on DHX15 pan-cancer. Box plot of DHXI5 dependency (measured by Demeter2 score of shRNA screen) for cell lines in the bottom and top quartile of CSJ score.

[0127] FIG. 25H shows DHX15 signature CSJ score correlates with dependency on DHX15 in breast cancer cell lines. Box plot of DHX15 dependency (measured by Demeter2 score of shRNA screen) for cell lines in the bottom and top tertile of CSJ score.

[0128] FIG 251 shows DHX15 signature. [0129] FIGs. 26A-26D show deletion of DHX15 allele correlates with increased DHX15 signature CSJ score.

[0130] FIG. 26A shows a Manhatan plot of allele association with DHX15 CSJ score in BRCA TCGA and Loss of CSJ score associated alleles occurs in -26% of BRCA patients.

[0131] FIG. 26B shows loss of DHX15 correlates with increased CSJ score in TCGA BRCA cohort. [0132] FIG. 26C shows loss of SUGP1 correlates with increased CSJ score in TCGA BRCA cohort. [0133] FIG. 27 shows engineering degradable SF3B1 in syngeneic (murine) TNBC models. The FKBP12F36V fragment (Nabet et al., 2018) was fused to the C-terminus of SF3B1 cDNA and cloned into a pHAGE-PGK backbone. PyMT-M cells were transduced with the SF3B1- FKBP12F36V lentivirus and then selected with puromycin. To knock out the endogenous SF3B1 locus, Cas9 protein was electroporated with Edit-R tracrRNA (Dharmacon) and crRNA targeting the first intron-exon junction of SF3B 1. A single clone was then selected, and Western bloting was used to confirm knockout of the endogenous protein and expression of SF3B1- FKBP12F36V.

[0134] FIG. 28A shows degradation leads to dose-dependent cell death in murine TNBC cells (PyMT-M model). SF3B1 degradation in PyMT-M FKBP-SF3B1 clones was assayed by Western blot after treatment with the indicated dose of dTAG13. dTAG13 dose curve assays were performed by treatment of FKBP12F36V -SF3B1 for 48 hours at indicated concentrations. Cell numbers were determined by Hoechst 33342 staining, followed by nuclei counting using the Celigo Imaging Cell Cytometer (Brooks). Cell number is normalized to the count of cells treated with Vehicle, DMSO. PI positive cells were assessed by incubating cells with 1: 100 dilution of Propidium Iodide (Sigma Aldrich, P4864) for 15 minutes before counting using the Celigo Imaging Cell Cytometer (Brooks). Number of PI positive foci was normalized to cell number determined by Hoechst 33342 staining, followed by nuclei counting using Celigo Imaging Cell Cytometer (Brooks). FIG. 28B is a negative control that shows no effect on cell number in the parental PyMT-M.

[0135] FIG. 29 shows spliceosome SF3B1 degradation leads to dose-dependent tumor regression. Tumor cells (500,000 cells in 25uL PBS) were transplanted into cleared mammary fat pad of 3-4 week old female C57/BL/6J female mice. Tumors were randomized onto vehicle or dTAG-13 (indicated dose in 5% DMSO + 20% solutol in saline via daily IP injection) at 250-400mm ^A 3. Tumor volume was measured using calipers three times per week. Ploted is the mean tumor volume -/+ SEM.

[0136] FIGs. 30A and 30B shows an increased infiltration of CD8a+ T cell upon SF3B1 degradation in tumor cells. FIG. 30A shows PyMT-M FKBP12 ^F36V -SF3B1 Tumor CD8a quantification. FIG. 30A shows increased number of CD8+ T cells upon degradation of SF3B1. FIG. 30A shows increased staining for CD8+ T cells compared to control vehicle (left panel) upon degradation of SF3B1 (right panel). Tumor chunks were fixed in 10% formalin overnight at 4°C overnight, and subsequently transferred into 70% ethanol, embedded in paraffin, and sectioned at regular intervals. Slides were deparaffinized and hydrated using xylene, graded ethyl alcohol, and dH2O. After antigen retrieval, 15 minutes of steaming at 90°C with pressure in 0. IM Tris-HCl, pH 9.0, sections were treated with 3% hydrogen peroxide solution for 5 minutes. Sections were incubated with primary antibody (CD8a, Cell Signaling 98941 diluted 1: 100) for Ihr at RT. Sections were then incubated with Envision Labelled Polymer-HRP (Dako) for 30 minutes at RT. DAB+ solution (DakoCytomation) was then added to section, incubated for 15 minutes, followed by application of DAB Sparkle Enhancer (Biocare). Sections were counterstained with Harris Hematoxylin. Counting of CD8a+ stained cells was done using the Count Tool in Adobe Photoshop. FIG. 30B shows the corresponding staining.

[0137] FIG. 31 shows degradation of spliceosome SF3B1 (within tumor cells) triggers anti-tumor immunity and immune memory. FIG. 31 Spliceosome SF3B1 -degradation leads to dose-dep. tumor regression. Tumor cells were transplanted into cleared mammary fat pad of 5-6 week old female C57/BL/6J female mice. Tumors were randomized onto vehicle or dTAG-13 (indicated dose in 5% DMSO + 20% solutol in saline via daily IP injection). Tumor volume was measured using calipers three times per week. Plotted is the mean tumor volume -/+ SEM. Animals with durable tumor regression for >100 days, along with age-matched naive mice, were transplanted with tumor cells (500,000 cells in 25uL) into the cleared mammary fat pad contralateral to the primary tumor. Animals were monitored for tumor growth three times per week.

[0138] FIG. 32 shows Spliceosome SF3B1 degradation leads to anti-tumor immune memory. Tumor cells were transplanted into cleared mammary fat pad of 5-6 week old female C57/BL/6J female mice. Tumors were randomized onto vehicle or dTAG-13 (indicated dose in 5% DMSO + 20% solutol in saline via daily IP injection). Tumor volume was measured using calipers three times per week. Plotted is the mean tumor volume -/+ SEM. Animals with durable tumor regression for >100 days, along with age-matched naive mice, were transplanted with tumor cells (500,000 cells in 25uL) into the cleared mammary fat pad contralateral to the primary tumor. Animals were monitored for tumor growth three times per week.

[0139] FIG. 33 shows method of tumor rechallenge after SF3B1 degradation. Tumor cells were transplanted into cleared mammary fat pad of 5-6 week old female C57/BL/6J female mice. Tumors were randomized onto vehicle or dTAG-13 (indicated dose in 5% DMSO + 20% solutol in saline via daily IP injection) at 250-400mm ^A 3. Tumor volume was measured using calipers three times per week. Plotted is the mean tumor volume -/+ SEM. Animals with durable tumor regression for >90 days were injected with antiCD8 or IgG antibodies (clone YTS 169.4 or vehicle control rat IgG2B via IP injection every 3 days). Four days after initial injection, animals were transplanted with additional tumor cells into the cleared mammary fat pad on the opposite side. Animals were monitored for tumor growth three times per week. Growth curves of individual tumors is shown.

[0140] FIG. 34 shows SF3B1 perturbation (within tumor cells) triggers immune memory in a CD8+ T- cell dependent manner. The FIG. shows spliceosome SF3B1 -degradation leads to anti -tumor immune memory that is dependent on CD8+ T-cells. Tumor cells were transplanted into cleared mammary fat pad of 5-6 week old female C57/BL/6J female mice. Tumors were randomized onto vehicle or dTAG-13 (indicated dose in 5% DMSO + 20% solutol in saline via daily IP injection) at 250-400mm ^A 3. Tumor volume was measured using calipers three times per week. Plotted is the mean tumor volume -/+ SEM. Animals with durable tumor regression for >90 days were injected with antiCD8 or IgG antibodies (clone YTS 169.4 or vehicle control rat IgG2B via IP injection every 3 days). Four days after initial injection, animals were transplanted with additional tumor cells into the cleared mammary fat pad on the opposite side. Animals were monitored for tumor growth three times per week. Growth curves of individual tumors is shown.

[0141] FIG. 35 shows method of Tumor rechallenge with independent TNBC tumor model. Tumor cells were transplanted into cleared mammary fat pad of 5-6 week old female C57/BL/6J female mice. Tumors were randomized onto vehicle or dTAG-13 (indicated dose in 5% DMSO + 20% solutol in saline via daily IP injection). Tumor volume was measured using calipers three times per week. Plotted is the mean tumor volume -/+ SEM. Animals with durable tumor regression for >100 days, along with age-matched naive mice, were transplanted with tumor cells (500,000 cells in 25uL) into the cleared mammary fat pad contralateral to the primary tumor. Animals were monitored for tumor growth three times per week.

[0142] FIG. 36 shows SF3B1 perturbation (within tumor cells) triggers immune memory against an independent TNBC model. FIG. 36 shows Spliceosome SF3B1 -degradation leads to immune memory against independent TNBC model. The data shows immune response to spliceosome degradation is not limited to mis-splicing derived neoantigens. Tumor cells were transplanted into cleared mammary fat pad of 5-6 week old female C57/BL/6J female mice. Tumors were randomized onto vehicle or dTAG-13 (indicated dose in 5% DMSO + 20% solutol in saline via daily IP injection). Tumor volume was measured using calipers three times per week. Plotted is the mean tumor volume -/+ SEM. Animals with durable tumor regression for >100 days, along with age-matched naive mice, were transplanted with tumor cells (500,000 cells in 25uL) into the cleared mammary fat pad contralateral to the primary tumor. Animals were monitored for tumor growth three times per week.

[0143] FIG. 37 illustrates methods to evaluate if spliceosome degradation induce the elimination of bystander tumor cells. Single cell suspensions of the PyMT-M FKBP12F36V - SF3B 1 cell line and parental PyMT-M cell line were mixed to generate mixed suspensions consisting of the following ratios: 100% FKBP12F36V - SF3B1: 0% parental; 99.8% FKBP12F36V - SF3B1: 0.2% parental; 99% FKBP12F36V - SF3B1: 1% parental, 90% FKBP12F36V - SF3B1: 10% parental; 0% FKBP12F36V - SF3B1: 100% parental. 500,000 cells per mixed suspension were transplanted into 4-5 week old female C57BL6/J mice. Mice in each group were randomized to receive vehicle or dTAG-13 (30 mg/kg) by daily IP injection at tumor size 150-300 mm3. Tumors reaching 1000 mm3 were harvested at endpoint. [0144] FIG. 38 shows SF3B1 degradation enables clearance of bystander tumor cells that do not harbor SF3B1 degradation (and do not harbor acute RNA mis-splicing). Single cell suspensions of the PyMT-M FKBP12F36V - SF3B1 cell line and parental PyMT-M cell line were mixed to generate mixed suspensions consisting of the following ratios: 100% FKBP12F36V - SF3B1: 0% parental; 99.8% FKBP12F36V - SF3B1: 0.2% parental; 99% FKBP12F36V - SF3B1: 1% parental, 90% FKBP12F36V - SF3B1: 10% parental; 0% FKBP12F36V - SF3B1: 100% parental. 500,000 cells per mixed suspension were transplanted into 4-5 week old female C57BL6/J mice. Mice in each group were randomized to receive vehicle or dTAG-13 (30 mg/kg) by daily IP injection at tumor size 150-300 mm3. Tumors reaching 1000 mm3 were harvested at endpoint.

[0145] FIGs. 39A-39B shows degradation of spliceosome RNA helicases leads to distinct patterns of RNA mis-splicing. FIG. 39A shows target clusters with similar and differential signatures of misprocessing. Dimension reduction analysis of misprocessing scores was used to identify target clusters. Four clusters were identified: U2-associated (SF3B1, U2AF2, DDX46 and DDX23), catalytic spliceosome (DHX38, DHX16 and DHX8), no splicing function (DDX21 and DDX47) and DHX15 in a distinct cluster. The targets of different cluster show differential missprocessing signatures. FIG. 39B shows degradation of targets within the same cluster induces misprocessing of a unique set of introns. Cluster-specific misprocessing was identified using linear regressions. Data are shown as intron-level misprocessing (n=3 biological replicate s/condition). The data shows that select (but not all) spliceosome RBPs (including RNA helicases) leads to distinct patterns of mis-splicing that are similar to SF3B 1- associated splicing patterns, suggesting that perturbation for example, by degradation of these RNA helicases (e.g., DDX46, and DDX23) can lead to an anti-viral immune response and immune memory phenotypes against tumor.

[0146] FIG. 40 shows activation of antiviral programs by degradation of select spliceosome helicases. The data shows that spliceosome proteins that clustered with SF3B1 shows similar immune response as degradation of SF3B1. The data indicates that degradation of DDX46 modulates expression of OAS1 & IL1B and results in activation of an antiviral immune signaling. SUM159 FKBP-DDX46 cells were treated with dTagl3 for 72hrs and then RNA was isolated. qPCR was performed and expression was calculated as fold change relative to control data using the AACt method.

[0147] FIG. 41 shows perturbations, for example, by degradation, of different exemplary enzymatic components (e.g., DDX46, DHX38, DHX16, DDX23) results in differential effects on expression of OAS1, demonstrating a differential ability to activate an immune response (e.g., an antiviral immune). Perturbations of different exemplary enzymatic components (e.g., DDX46, DHX8, DHX16, DDX23) results in a different immune response.

[0148] FIGs. 42A-42C show degradation of spliceosome RNA helicases leads to distinct RNA mis- splicing signatures and activation of antiviral signaling. FIG. 42A shows perturbations of different exemplary enzymatic components (e.g, DDX46, DHX38, DHX16, DDX23) results in differential effects on expression of OAS1. FIG. 42B shows perturbations of different exemplary enzymatic components (e.g., DDX46, DHX38, DHX16, DDX23) results in differential effects on expression of IL1B. FIG. 42C shows perturbations of different exemplary enzymatic components (e.g., DDX46, DHX38, DHX16, DDX23) results in differential effects on expression of CD80, demonstrating a differential ability to activate an immune response (e.g., an antiviral immune). The data demonstrates perturbations of different exemplary enzymatic components (e.g., DDX46, DHX8, DHX16, DDX23) results in a different immune response.

[0149] FIG. 43 A shows perturbations of different exemplary enzymatic components (e.g, DDX46, DHX38, DHX16, DDX23) results in differential effects on expression of OAS1, CD80, IL1B, CCL5, CXCL10 and CXCL11, demonstrating a differential ability to activate an immune response (e.g., an antiviral immune). Perturbations of different exemplary enzymatic components (e.g, DDX46, DHX8, DHX16, DDX23) results in a different immune response.

[0150] FIG. 43B shows cell number relative to untreated at the 72 hour time point in which RNA was harvested to assess antiviral signaling and expression changes.

[0151] FIG. 43C shows protein abundance relative to untreated at the 72 hour time point in which RNA was harvested to assess antiviral signaling and expression changes.

[0152] FIGs. 44A-44D show that immune response e.g., antiviral signaling, induced by DDX46 degradation is rescued by non-degradable WT but not DEAD-box mutant DDX46. The data shows that the enzymatic activity of DDX46 is required to suppress antiviral signaling. Antiviral signaling induced by DDX46 degradation is rescued by non-degradable WT but not DEAD-box mutant DDX46 (enzymatic activity is required for antiviral suppression). Expression of non-degradable DDX46 rescues the increase in antiviral signaling seen upon DDX46 degradation (WT vs GFP upon dTagl3 treatment) while expression of ATP binding mutants of DDX46 do not rescue antiviral signaling. Cells were treated with dTagl3 for 72hrs and then RNA was isolated. qPCR was performed and expression was calculated as fold change relative to control data using the AACt method. FIG. 44A shows expression of IL6. FIG. 44B shows expression of IL1B. FIG. 44C shows expression of CD80. FIG. 44D shows expression of OAS1. [0153] FIGs. 45A-45E show degradation of DDX46 leads to dose-dependent cell death (dependent on enzymatic activity)

[0154] FIG. 45A shows dose dependent degradation of DDX46 after dTagl3 treatment in SUM159 cells. [0155] FIG. 45B shows DDX46 degradation impairs cell growth DDX46 degradation impairs SUM159 breast cancer cell growth. Cells were fixed and nuclei were counted 72 hours post-treatment with dTagl3. [0156] FIG. 45C shows DDX46 degradation causes apoptotic cell death. DDX46 degradation results in apoptotic cell death. Caspase 3/7 activity was measured at the indicated time after dTagl3 treatment. [0157] FIG. 45D shows DDX46 degradation causes intron retention (DYNCH1). DDX46 degradation results in intron retention. RNA was isolated from SUM159 cells treated with dTagl3 for the indicated time and qPCR was performed to measure mis-spliced and properly spliced DYNCH1. Expression of the mis-spliced transcript was normalized to expression of the proper spliced transcript and then to untreated within each time point.

[0158] FIG. 45E shows DDX46 ATP binding is required to support growth. DDX46 enzymatic activity (ATPase) is required to support SUM 159 breast cancer cell growth. Expression of non-degradable DDX46 rescues the growth defect seen upon DDX46 degradation (WT vs GFP upon dTagl3 treatment) while expression of ATP binding mutants of DDX46 do not rescue growth.

[0159] FIGs. 46A-46E show degradation of DDX23 leads to dose-dependent cell death that is dependent on enzymatic activity.

[0160] FIG. 46A shows dose dependent degradation of DDX23 after dTagl3 treatment in SUM 159 cells. [0161] FIG. 46B shows DDX23 degradation impairs SUM159 breast cancer cell growth. Cells were fixed and nuclei were counted 72 hours post-treatment with dTagl3.

[0162] FIG. 46C shows DDX23 degradation results in apoptotic cell death. Caspase 3/7 activity was measured at the indicated time after dTagl3 treatment.

[0163] FIG. 46D shows DDX23 degradation results in intron retention. RNA was isolated from SUM159 cells treated with dTagl3 for the indicated time and qPCR was performed to measure mis-spliced and properly spliced MAEA. Expression of the mis-spliced transcript was normalized to expression of the proper spliced transcript and then to untreated within each time point.

[0164] FIG. 46E shows DDX23 enzymatic activity (ATPase) is required to support SUMI 59 breast cancer cell growth. Expression of non-degradable DDX23 rescues the growth defect seen upon DDX23 degradation (WT vs GFP upon dTagl3 treatment) while expression of ATP binding mutants of DDX23 do not rescue growth.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0165] The following discussion of the present disclosure has been presented for purposes of illustration and description. The following is not intended to limit the invention to the form or forms disclosed herein. Although the description of the present disclosure has included description of one or more embodiments and certain variations and modifications, other variations and modifications are within the scope of the present disclosure, e.g. , as may be within the skill and knowledge of those in the art, after understanding the present disclosure. It is intended to obtain rights which include alternative embodiments to the extent permitted, including alternate, interchangeable and/or equivalent structures, functions, ranges or steps to those claimed, whether or not such alternate, interchangeable and/or equivalent structures, functions, ranges or steps are disclosed herein, and without intending to publicly dedicate any patentable subject matter. [0166] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0167] Although various features of the disclosure may be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the disclosure may be described herein in the context of separate embodiments for clarity, various aspects and embodiments can be implemented in a single embodiment.

[0168] The practice of some embodiments disclosed herein employ, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See for example Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (R.I. Freshney, ed. (2010)).

EXAMPLES OF DEFINITIONS

[0169] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g., to any commonly owned patent or application. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present disclosure, the particular materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0170] In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting.

[0171] The terms “and/or” and “any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and/or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.”

[0172] The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use. [0173] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which can depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0174] As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the invention, and vice versa. Furthermore, compositions of the invention can be used to achieve methods of the invention.

[0175] Reference in the specification to “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the inventions.

[0176] As used herein, a "subject", "patient", "individual" and like terms are used interchangeably and refers to a vertebrate, for example, in some embodiments, a subject refers to a mammal. In some embodiments, a subject is a primate. In some embodiments, a subject is a human. Mammals include, without limitation, humans, primates, rodents, wild or domesticated animals, including feral animals, farm animals, sport animals, and pets. Primates include, for example, chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include, for example, mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include, for example, cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog, fox, wolf, avian species, e.g., chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon. The terms, "individual," "patient" and "subject" are used interchangeably herein. A subject can be male or female.

[0177] In some embodiments, the subject is a mammal. The mammal can be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can be advantageously used as subjects that represent animal models of conditions, disorders or their associated symptoms disclosed herein (e.g., cancer, or one or more symptoms thereof. In addition, the compositions and methods described herein can be used to treat domesticated animals and/or pets. Accordingly, in some embodiments, the subject can be a domesticated pet (e.g., a cat, or a dog).

[0178] A subject can be one who has been previously diagnosed with or identified as suffering from or under medical supervision for a cancer or a symptom thereof. A subject can be one who is diagnosed and currently being treated for, or seeking treatment, monitoring, adjustment or modification of an existing therapeutic treatment, or is at a risk of developing a cancer.

[0179] “Improving,” “enhancing,” “bettering,” and its grammatical equivalents as used herein can mean any improvement recognized by one of skill in the art. For example, improving can encompass a decrease, lessening, or diminishing of an undesirable effect or symptom. In some embodiments, the subject is suffering from, is at risk of suffering from a cancer or showing one or more symptoms of a cancer.

[0180] The terms "increased" /'increase", "increasing" or "enhance" are all used herein to generally mean an increase by a statically significant amount; for the avoidance of doubt, the terms "increased", "increase", or "enhance", can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3 -fold, or at least about a 4-fold, or at least about a 5 -fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. The increase can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is in some embodiments to a level accepted as within the range of normal for an individual without a given disease.

[0181] The terms, "decrease", "reduce", "reduction", "lower" or "lowering," or "inhibit" are all used herein generally to mean a decrease by a statistically significant amount. For example, "decrease", "reduce", "reduction", or "inhibit" can mean a decrease by at least 10% as compared to a reference level, for example a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level or non-detectable level as compared to a reference level), or any decrease between 10-100% as compared to a reference level. In the context of a marker or symptom, by these terms is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is in some embodiments down to a level accepted as within the range of normal for an individual without a given disease.

[0182] Some numerical values disclosed throughout are referred to as, for example, “X is at least or at least about 100; or 200 [or any numerical number].” This numerical value includes the number itself and all of the following: i) X is at least 100; ii) X is at least 200; iii) X is at least about 100; and iv) X is at least about 200.

[0183] All these different combinations are contemplated by the numerical values disclosed throughout. All disclosed numerical values should be interpreted in this manner, whether it refers to an administration of a therapeutic agent or referring to days, months, years, weight, dosage amounts, etc., unless otherwise specifically indicated to the contrary.

[0184] The ranges disclosed throughout are sometimes referred to as, for example, “X is administered on or on about day 1 to 2; or 2 to 3 [or any numerical range].” This range includes the numbers themselves (e.g., the endpoints of the range) and all of the following: i) X being administered on between day 1 and day 2; ii) X being administered on between day 2 and day 3; iii) X being administered on between about day 1 and day 2; iv) X being administered on between about day 2 and day 3; v) X being administered on between day 1 and about day 2; vi) X being administered on between day 2 and about day 3; vii) X being administered on between about day 1 and about day 2; and viii) X being administered on between about day 2 and about day 3.

[0185] All these different combinations are contemplated by the ranges disclosed throughout. All disclosed ranges should be interpreted in this manner, whether it refers to an administration of a therapeutic agent or referring to days, months, years, weight, dosage amounts, etc., unless otherwise specifically indicated to the contrary.

[0186] As used herein, the terms "polypeptide", "protein" and "peptide" are used interchangeably and refer to a polymer of amino acid residues linked via peptide bonds and which may be composed of two or more polypeptide chains. The terms "polypeptide", "protein" and "peptide" refer to a polymer of at least two amino acid monomers joined together through amide bonds. An amino acid may be the L-optical isomer or the D-optical isomer. More specifically, the terms "polypeptide", "protein" and "peptide" refer to a molecule composed of two or more amino acids in a specific order; for example, the order as determined by the base sequence of nucleotides in the gene or RNA coding for the protein. Proteins are essential for the structure, function, and regulation of the body's cells, tissues, and organs, and each protein has unique functions. Examples are hormones, enzymes, antibodies, and any fragments thereof. In some cases, a protein can be a portion of the protein, for example, a domain, a subdomain, or a motif of the protein. In some cases, a protein can be a variant (or mutation) of the protein, wherein one or more amino acid residues are inserted into, deleted from, and/or substituted into the naturally occurring (or at least a known) amino acid sequence of the protein. A protein or a variant thereof can be naturally occurring or recombinant.

[0187] Methods for detection and/or measurement of polypeptides in biological material are well known in the art and include, but are not limited to, Western-blotting, flow cytometry, ELISAs, RIAs, and various proteomics techniques. An exemplary method to measure or detect a polypeptide is an immunoassay, such as an ELISA. This type of protein quantitation can be based on an antibody capable of capturing a specific antigen, and a second antibody capable of detecting the captured antigen. Exemplary assays for detection and/or measurement of polypeptides are described in Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, (1988), Cold Spring Harbor Laboratory Press. Methods for detection and/or measurement of RNA in biological material are well known in the art and include, but are not limited to, Northern-blotting, RNA protection assay, RT PCR. Suitable methods are described in Molecular Cloning: A Laboratory Manual (fourth Edition) By Michael R. Green, Joseph Sambrook, Peter MacCallum 2012, 2,028 pp, ISBN 978-1-9361 13-42-2.

Enzymatic component of a spliceosome

[0188] The term “spliceosome” as used herein refers to the macromolecular complex responsible for RNA splicing. As used herein, the term “RNA splicing” refers to processing of RNA in which a newly made precursor messenger RNA transcript (often referred to as a “pre-mRNA”) is converted into a mature messenger RNA (mRNA). Such splicing includes removal of introns (non-coding regions) and linking of exons (coding regions). For many eukaryotic introns, a series of reactions catalyzed by the spliceosome produces the spliced mRNA. The spliceosome is composed of five small nuclear ribonucleoproteins (snRNPs), known as Ul, U2, U3, U4, U5 and U6, and more than 100 additional proteins. The term "intron" refers to both the DNA sequence within a gene and the corresponding sequence in the unprocessed RNA transcript. As part of the RNA processing pathway, introns can be removed by RNA splicing either shortly after or concurrent with transcription. Introns are found in the genes of most organisms and many viruses. They can be located in a wide range of genes, including those that generate proteins, ribosomal RNA (rRNA), and transfer RNA (tRNA). An "exon" can be any part of a gene that encodes a part of the final mature RNA produced by that gene after introns have been removed by RNA splicing. The term "exon" refers to both the DNA sequence within a gene and to the corresponding sequence in RNA transcripts.

[0189] In some embodiments, methods of the disclosure comprise inhibiting an enzymatic component of a spliceosome. In some embodiments, methods of the disclosure comprise inducing degradation of an enzymatic component of a spliceosome. As used herein the term “enzymatic component of a spliceosome” refers to any protein that comprises an enzymatic activity that is directly or indirectly associated with a spliceosome. In some embodiments, an enzymatic component catalyzes conformational changes in the spliceosome during RNA splicing. In some embodiments, an enzymatic component catalyzes unwinding of RNA structures. In some embodiments, an enzymatic component catalyzes remodeling of RNA-protein complexes during RNA splicing. In some embodiments, an enzymatic component catalyzes displacement of a RNA binding protein from the RNA. In some embodiments, an enzymatic component is an RNA helicase. There are at least seven configurations of the spliceosome complex during splicing: the pre-catalytic complex (B), the activated complex (Bact), the catalytically activated complex (B*), the catalytic step I spliceosome (C), the step II catalytically activated complex (C*), the post-catalytic complex (P), and the intron lariat spliceosome (ILS). In some embodiments, the enzymatic component catalytically activates a complex. In some embodiments, the enzymatic component catalyzes conversion between complexes.

RNA helicases

[0190] In some embodiments, an enzymatic component is an RNA helicase. RNA helicases catalyze unwinding of RNA duplexes, RNA strand separation, displace proteins from RNA molecules, annealing of RNA strands, act as RNA clamps or placeholders, and stabilize on-pathway folding intermediates. In some embodiments, the RNA helicase is a SF2 RNA helicase. In some embodiments, the RNA helicase is a SF1 RNA helicase. In some embodiments, the RNA helicase is a SF2 RNA helicase. SF2 RNA helicases include the DEAD box, DEAH box, and Ski2-like proteins, generally referred to as DExD/H box RNA helicases, named after one of the consensus amino acid sequence motifs (Caruthers and McKay, 2002, incorporated herein by reference in its entirety. In some embodiments, the RNA helicase is an ATP dependent RNA helicase. In some embodiments, the RNA helicase is a DEAD-box helicase. In some embodiments, the RNA helicase is a DEAH-box helicase. In some embodiments, said enzymatic component (e.g., a RNA helicase) is DHX8, DHX15, DHX16, DHX35, DHX33, DHX38, DHX40, DHX32, DHX34, DHX37, DHX36, DHX57, DHX29, DHX9, DHX30, UPF1, SMBP2, SETX, MOVIO, MOVIOLI, DHX58, IFIH1, DDX58, AQR, DDX12, DDX11, HELZ2, ZNFX1, DICER, SUV3, ASCC3, Brr2, SKIV2, MTREX, DDX60, DDX28, DDX18, DDX10, DDX55, DDX31, DDX51, DDX24, DDX56, DDX19A, DDX19B, DDX25, eIF4Al, eIF4A2, eIF4A3, DDX39B, DDX39A, DDX20, DDX6, DDX50, DDX21, DDX1, DDX54, DDX5, DDX17, DDX53, DDX43, DDX23, DDX46, DDX42, DDX41, DDX3Y, DDX3X, DDX4, DDX52, DDX59, DDX47, DDX49, DDX39B, PRPF8, or DDX27. In some embodiments, the RNA helicase is selected from the group consisting of DHX8, DHX15, DHX38, DHX8, DHX16, DDX46, DDX23, DDX41, DDX47, AQR, and DDX21. In some embodiments, the RNA helicase is DHX15, or DHX38. In some embodiments, the RNA helicase is a Ski-21ike helicase. In some embodiments, the RNA helicase is Prp5, Sub2, Prp28, Prpl9, Brr2, Prpl6, Prp22, or Prp43. Various methods can be used to measure helicase activity e.g., a strand displacement assay; e.g., Stephanie S. et al, RNA Unwinding Assay for DExD/H-Box RNA Helicases Springer Protocols (2004), the contents of which are incorporated herein by reference in its entirety. In some embodiments, the enzymatic component is a human enzymatic component. In some embodiments, the RNA helicase is a human RNA helicase. The protein and nucleic acid sequences of the enzymatic component, e.g., human enzymatic component e.g., RNA helicases are known and available publicly, for example in Pubmed. Polypeptide sequences of exemplary enzymatic components are provided below:

[0191] Polypeptide sequence of human DHX15 protein

[0192] MSKRHRLDLGEDYPSGKKRAGTDGKDRDRDRDREDRSKDRDRERDRGDREREREKEKE KELRASTNAMLISAGLPPLKASHSAHSTHSAHSTHSTHSAHSTHAGHAGHTSLPQCINPF TNLPHT PRYYDILKKRLQLPVWEYKDRFTDILVRHQSFVLVGETGSGKTTQIPQWCVEYMRSLPGP KRGV ACTQPRRVAAMSVAQRVADEMDVMLGQEVGYSIRFEDCSSAKTILKYMTDGMLLREAMND PL LERYGVIILDEAHERTLATDILMGVLKEVVRQRSDLKVIVMSATLDAGKFQIYFDNCPLL TIPGRT HPVEIFYTPEPERDYLEAAIRTVIQIHMCEEEEGDLLLFLTGQEEIDEACKRIKREVDDL GPEVGDI KIIPLYSTLPPQQQQRIFEPPPPKKQNGAIGRKVVVSTNIAETSLTIDGVVFVIDPGFAK QKVYNPRI RVESLLVTAISKASAQQRAGRAGRTRPGKCFRLYTEKAYKTEMQDNTYPEILRSNLGSVV LQLK

KLGIDDLVHFDFMDPPAPETLMRALELLNYLAALNDDGDLTELGSMMAEFPLDPQLA KMVIASC DYNCSNEVLSITAMLSVPQCFVRPTEAKKAADEAKMRFAHIDGDHLTLLNVYHAFKQNHE SVQ WCYDNFINYRSLMSADNVRQQLSRIMDRFNLPRRSTDFTSRDYYINIRKALVTGYFMQVA HLER TGHYLTVKDNQVVQLHPSTVLDHKPEWVLYNEFVLTTKNYIRTCTDIKPEWLVKIAPQYY DMSN FPQCEAKRQLDRIIAKLQSKEYSQY (SEQ ID NO: 1)

[0193] Polypeptide sequence of human DDX23

[0194] MAGELADKKDRDASPSKEERKRSRTPDRERDRDRDRKSSPSKDRKRHRSRDRRRGGSRS RSRSRSKSAERERRHKERERDKERDRNKKDRDRDKDGHRRDKDRKRSSLSPGRGKDFKSR KDR

DSKKDEEDEHGDKKPKAQPLSLEELLAKKKAEEEAEAKPKFLSKAEREAEALKRRQQ EVEERQR MLEEERKKRKQFQDLGRKMLEDPQERERRERRERMERETNGNEDEEGRQKIREEKDKSKE LHAI KERYLGGIKKRRRTRHLNDRKFVFEWDASEDTSIDYNPLYKERHQVQLLGRGFIAGIDLK QQKR EQSRFYGDLMEKRRTLEEKEQEEARLRKLRKKEAKQRWDDRHWSQKKLDEMTDRDWRIFR ED YSITTKGGKIPNPIRSWKDSSLPPHILEVIDKCGYKEPTPIQRQAIPIGLQNRDIIGVAE TGSGKTAAF LIPLLVWITTLPKIDRIEESDQGPYAIILAPTRELAQQIEEETIKFGKPLGIRTVAVIGG ISREDQGFRL RMGCEIVIATPGRLIDVLENRYLVLSRCTYVVLDEADRMIDMGFEPDVQKILEHMPVSNQ KPDTD EAEDPEKMLANFESGKHKYRQTVMFTATMPPAVERLARSYLRRPAVVYIGSAGKPHERVE QKV FLMSESEKRKKLLAILEQGFDPPIIIFVNQKKGCDVLAKSLEKMGYNACTLHGGKGQEQR EFALS NLKAGAKDILVATDVAGRGIDIQDVSMVVNYDMAKNIEDYIHRIGRTGRAGKSGVAITFL TKED SAVFYELKQAILESPVSSCPPELANHPDAQHKPGTILTKKRREETIFA* (SEQ ID NO:2) [0195] Polypeptide sequence of human DDX46

[0196] MGRESRHYRKRSASRGRSGSRSRSRSPSDKRSKRGDDRRSRSRDRDRRRERSRSRDKRRS RSRDRKRLRRSRSRERDRSRERRRSRSRDRRRSRSRSRGRRSRSSSPGNKSKKTENRSRS KEKTDG GESSKEKKKDKDDKEDEKEKDAGNFDQNKLEEEMRKRKERVEKWREEQRKKAMENIGELK KEI EEMKQGKKWSLEDDDDDEDDPAEAEKEGNEMEGEELDPLDAYMEEVKEEVKKFNMRSVKG G GGNEKKSGPTVTKVVTVVTTKKAVVDSDKKKGELMENDQDAMEYSSEEEEVDLQTALTGY QT KQRKLLEPVDHGKIEYEPFRKNFYVEVPELAKMSQEEVNVFRLEMEGITVKGKGCPKPIK SWVQ CGISMKILNSLKKHGYEKPTPIQTQAIPAIMSGRDLIGIAKTGSGKTIAFLLPMFRHIMD QRSLEEG EGPIAVIMTPTRELALQITKECKKFSKTLGLRVVCVYGGTGISEQIAELKRGAEIIVCTP GRMIDML AANSGRVTNLRRVTYVVLDEADRMFDMGFEPQVMRIVDNVRPDRQTVMFSATFPRAMEAL AR RILSKPIEVQVGGRSVVCSDVEQQVIVIEEEKKFLKLLELLGHYQESGSVIIFVDKQEHA DGLLKD LMRASYPCMSLHGGIDQYDRDSIINDFKNGTCKLLVATSVAARGLDVKHLILVVNYSCPN HYED YVHRAGRTGRAGNKGYAYTFITEDQARYAGDIIKALELSGTAVPPDLEKLWSDFKDQQKA EGKI IKKSSGFSGKGFKFDETEQALANERKKLQKAALGLQDSDDEDAAVDIDEQIESMFNSKKR VKDM AAPGTSSVPAPTAGNAEKLEIAKRLALRINAQKNLGIESQVDVMQQATNAILRGGTILAP TVSAK TIAEQLAEKINAKLNYVPLEKQEEERQDGGQNESFKRYEEELEINDFPQTARWKVTSKEA LQRISE YSEAAITIRGTYFPPGKEPKEGERKIYLAIESANELAVQKAKAEITRLIKEELIRLQNSY QPTNKGR YKVL* (SEQ ID N0:3)

Perturbation of enzymatic component of a spliceosome

[0197] In some embodiments, the methods of the disclosure comprises perturbation of an enzymatic component of a spliceosome in a cancer cell. In some embodiments, the perturbation comprises inhibiting the enzymatic component of the spliceosome. In some embodiments, the methods of the disclosure comprises inhibiting the enzymatic component of the spliceosome. In some embodiments, the perturbation comprises degradation of the enzymatic component of the spliceosome. In some embodiments, the methods of the disclosure comprises inducing degradation of an enzymatic component of the spliceosome. In some embodiments, the inhibiting or degradation of the enzymatic component results in inhibition of a spliceosome activity. The term “inhibition of a spliceosomal activity” refers to decreasing splicing activity in a cell (e.g., a cancer cell) by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% as compared to a corresponding control cell.

Inhibiting an enzymatic component of a spliceosome

[0198] In some embodiments, the inhibiting of an enzymatic component in a cell (e.g., a cancer cell) comprises inhibiting the activity, expression and/or levels of an RNA (e.g., mRNA) that encodes the enzymatic component protein relative to that in a corresponding control cell and/or inhibiting the activity, expression and/or levels of the enzymatic component protein relative to that in a corresponding control cell. In some embodiments, a corresponding control cell is a cell that lacks or is not subject to perturbation (e.g., inhibition or degradation of the enzymatic component). In some embodiments, a corresponding control cell is a corresponding healthy cell, e.g., a corresponding non-cancerous cell. In some embodiments, the corresponding control cell is a corresponding cell in which no change in the activity or level of the enzymatic component has been affected. In some embodiments, the corresponding control cell is the same cell type as the cell in which the inhibiting or degradation is induced. In some embodiments, the corresponding control cell is from the same species or a different species. In some embodiments, the corresponding control cell is from the same subject as the cell in which the enzymatic component is inhibited or degraded (e.g., a cancer cell). In some embodiments, the corresponding control cell is the same cell type (e.g., cancer cell in which the enzymatic component is inhibited or degraded), but is one that is not subject to the inhibition or degradation.

[0199] In some embodiments, the inhibiting can be effected at the genomic level (e.g. by homologous recombination and site specific endonucleases) and/or the transcript level using a variety of molecules which interfere with transcription and/or translation (e.g., RNA silencing agents) and/or on the protein level (e.g., aptamers, small molecules and inhibitory and inhibitory peptides, antagonists, enzymes that cleave the polypeptide, antibodies and the like). In some embodiments, the inhibiting is transient. In some embodiments, the inhibiting is permanent. In some embodiments, the inhibiting is constitutive. In some embodiments, the inhibiting is inducible. In some embodiments, the inhibiting refers to a decrease in level of mRNA that encodes the enzymatic component e.g., as determined by RT-PCR. In some embodiments, the inhibiting refers to a decrease in level of an enzymatic component protein, e.g., as determined by Western blot or ELISA assay.

[0200] In some embodiments, inhibiting of an enzymatic component comprises inhibiting a level (e.g., expression level) of an RNA e.g., an mRNA that encodes the enzymatic component relative to that in a corresponding control cell. In some embodiments, inhibiting of an enzymatic component comprises inhibiting a level (e.g., expression level) of the enzymatic component protein relative to that in a corresponding control cell. In some embodiments, the level of said RNA and/or said enzymatic component protein is inhibited by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% as compared to that in a corresponding control cell. In some embodiments, inhibiting of an enzymatic component comprises inhibiting an activity of the enzymatic component relative to that in a corresponding control cell. In some embodiments, said activity of the enzymatic component is inhibited by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% as compared to that in a corresponding control cell. [0201] In some embodiments, the inhibiting comprises administering an effective amount of an agent capable of inhibiting the enzymatic component. In some embodiments, the inducing degradation comprises administering an effective amount of an agent capable of inducing degradation of the enzymatic component. In some embodiments, the agent binds, degrades, or inhibits post-translational modification of said enzymatic component. In some embodiments, is a small molecule, protein, peptide, nucleic acid, carbohydrate, or a combination thereof. In some embodiments, the agent is a siRNA, an antisense morphlino, an antisense oligonucleotide, a small molecule, or a combination thereof. In some embodiments, the agent directly inhibits or degrades the enzymatic component. The term “directly” means that the agent acts upon and/or directly interacts with the nucleic acid sequence (e.g., DNA or RNA) that encodes the enzymatic component or with the enzymatic component polypeptide, and not on a co-factor, an upstream activator or downstream effector of the enzymatic component.

[0202] In some embodiments, the agent indirectly inhibits or degrades an enzymatic component. The term “indirectly” means that the agent acts upon a co-factor, an upstream activator or downstream effector of the enzymatic component. In some embodiments, an agent capable of inhibiting and/or degradation of the enzymatic component polypeptide binds to and/or cleaves the enzymatic component. Such exemplary agents can be small molecules, antagonists, or inhibitory peptides. In some embodiments, a non-functional analogue of at least a catalytic or binding portion of an enzymatic component can be also used as an agent which inhibits the enzymatic component. In some embodiments, an agent e.g., a small molecule or peptides can interfere with an activity of the enzymatic component. In some embodiments, the activity is a catalytic activity. In some embodiments, the activity comprises binding and/or interacting with a downstream cofactor, subunit component, another spliceosomal factor, or RNA. In some embodiments, an agent prevents the activation or substrate binding of the enzymatic component. In some embodiments, an agent e.g., an agent that can inhibit or degrade the enzymatic component includes an antibody, an antibody fragment, or an aptamers. In some embodiments, the antibody or an antibody fragment binds an epitope of the enzymatic component. Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference). As used herein, the term “aptamer” refers to double stranded or single stranded RNA molecule that binds to specific molecular target, such as a protein. Various methods are known in the art which can be used to design protein specific aptamers. The skilled artisan can employ SELEX (Systematic Evolution of Ligands by Exponential Enrichment) for efficient selection as described in Stoltenburg R, Reinemann C, and Strehlitz B (Biomolecular engineering (2007) 24(4):381-403).

[0203] In some embodiments, an agent is capable of inhibiting a nucleic acid sequence (e.g., DNA or RNA; e.g., mRNA) that encodes the enzymatic component. Exemplary agents that inhibit a nucleic acid sequence comprises a nucleic acid agent, having a nucleic acid backbone, DNA, RNA, mimetics thereof or a combination of same. The nucleic acid agent may be encoded from a DNA molecule or provided to the cell per se. In some embodiments, the agent comprises an RNA silencing agent e.g., shRNA, siRNA, and miRNAs. In some embodiments, the agent is an antisense oligonucleotide e.g., that hybridizes to the RNA that encodes the enzymatic component.

[0204] In some embodiments, the inhibiting comprises modifying a gene that encodes the enzymatic component. In some embodiments, the modifying introduces a mutation in the enzymatic component. In some embodiments, the mutation comprises an amino acid substitution, deletion and/or insertion. In some embodiments, the mutation is a loss of function mutation. In some embodiments, the modifying results in a decrease in the activity e.g., enzymatic, or catalytic activity of the enzymatic component.

[0205] In some embodiments, the enzymatic component is DHX15, and the mutation is R222G amino acid substitution in said DHX15 relative to a corresponding wild type DHX15. In some embodiments, the enzymatic component is DDX46, and the mutation is D529A and/or D531 A in said DDX46 relative to a corresponding wild type DDX46. In some embodiments, the enzymatic component is DDX23, and the mutation is D549A and/or D552A in said DDX23 relative to a corresponding wild type DDX23.

[0206] In some embodiments, the modifying results in inhibition of the level and/or activity of the expressed product, i.e., the RNA that encodes the enzymatic component and/or the translated enzymatic component protein. Non-limiting examples of mutations include a missense mutation, i.e., a mutation which changes an amino acid residue in the protein with another amino acid residue and thereby abolishes the enzymatic activity of the protein; a nonsense mutation, i.e., a mutation which introduces a stop codon in a protein, e.g., an early stop codon which results in a shorter protein devoid of the enzymatic activity; a frame-shift mutation, i.e., a mutation, usually, deletion or insertion of nucleic acid(s) which changes the reading frame of the protein, and may result in an early termination by introducing a stop codon into a reading frame (e.g., a truncated protein, devoid of the enzymatic activity), or in a longer amino acid sequence (e.g., a readthrough protein) which affects the secondary or tertiary structure of the protein and results in a non-functional protein, devoid of the enzymatic activity of the non-mutated polypeptide; a readthrough mutation due to a frame-shift mutation or a modified stop codon mutation (i.e., when the stop codon is mutated into an amino acid codon), with an abolished enzymatic activity; a promoter mutation, i.e., a mutation in a promoter sequence, usually 5' to the transcription start site of a gene, which results in down-regulation of a specific gene product; a regulatory mutation, i.e., a mutation in a region upstream or downstream, or within a gene, which affects the expression of the gene product; a deletion mutation, i.e., a mutation which deletes coding nucleic acids in a gene sequence and which may result in a frame-shift mutation or an in-frame mutation (within the coding sequence, deletion of one or more amino acid codons); an insertion mutation, i.e., a mutation which inserts coding or non-coding nucleic acids into a gene sequence, and which may result in a frame-shift mutation or an in-frame insertion of one or more amino acid codons; an inversion, i.e., a mutation which results in an inverted coding or non-coding sequence; a splice mutation i.e., a mutation which results in abnormal splicing or poor splicing; and a duplication mutation, i.e., a mutation which results in a duplicated coding or non-coding sequence, which can be in-frame or can cause a frame-shift. In some embodiments, the modifying comprises modifying at least one allele of the gene. In some embodiments, the modifying comprises modifying both alleles of the gene. In such instances the e.g., enzymatic component may be in a homozygous form or in a heterozygous form. Methods of introducing nucleic acid alterations to a gene of interest are well known in the art [see for example Menke D. Genesis (2013) 51 : -618; Capecchi, Science (1989) 244: 1288-1292; Santiago et al. Proc Natl Acad Sci USA (2008) 105:5809-5814; International Patent Application Nos. WO 2014085593, WO 2009071334 and WO 2011146121; U.S. Pat. Nos. 8,771,945, 8,586,526, 6,774,279 and UP Patent Application Publication Nos. 20030232410, 20050026157, US20060014264; the contents of which are incorporated by reference in their entireties] and include targeted homologous recombination, site specific recombinases, PB transposases and genome editing by engineered nucleases such as CRISPR/Cas9, talens, zinc finger nucleases. Agents for introducing nucleic acid alterations to a gene of interest can be designed publically available sources or obtained commercially from Transposagen, Addgene and Sangamo Biosciences.

[0207] Methods for detecting sequence alteration are well known in the art and include, but not limited to, DNA sequencing, electrophoresis, an enzyme-based mismatch detection assay and a hybridization assay such as PCR, RT-PCR, RNase protection, in-situ hybridization, primer extension, Southern blot, Northern Blot and dot blot analysis. Sequence alterations in a specific gene can also be determined at the protein level using e.g., chromatography, electrophoretic methods, immunodetection assays such as EUISA and western blot analysis and immunohistochemistry.

[0208] Inhibition of spliceosomal activity can be assessed using any method known in the art, such as assays measuring splicing of select endogenous gene transcripts can be carried out. Such methods are discussed in Kerstin A. Effenberger, Veronica K. Urabe, and Melissa S. Jurica, “Modulating splicing with small molecular inhibitors of the spliceosome”, Wiley Interdiscip Rev RNA. Author manuscript; PMC 2018 Mar. 1, incorporated herein by reference in its entirety. In some embodiments, the agent can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

Degradation of an enzymatic component of a spliceosome

[0209] In some embodiments, methods of the disclosure comprise degradation of the enzymatic component polypeptide. In some embodiments, the degradation comprises proteasomal degradation. In some embodiments, inducing degradation comprises ubiquitination of the enzymatic component. In some embodiments, the degradation results in a decrease in the level of the enzymatic component protein relative to that in a corresponding control cell. In some embodiments, inducing degradation comprises administering an effective amount of an agent capable of inducing degradation of the enzymatic component protein. In some embodiments, the inducing degradation comprises inducing transient degradation. In some embodiments, the inducing degradation comprises inducing permanent degradation. In some embodiments, the inducing degradation comprises inducing constitutive degradation. In some embodiments, the inducing degradation comprises inducing inducible degradation. In some embodiments, degradation is induced by a small molecule degrader (such as proteolysis targeting chimera (PROTAC) or molecular glue). In some embodiments, degradation is induced by a degron tagging of the enzymatic component. Methods for inducing targeted degradation of a protein is known in the art, for example, see Bekes, M., Langley, D.R. & Crews, C.M. PROTAC targeted protein degraders: the past is prologue. Nat Rev Drug Discov 21, 181-200 (2022); Fang et al. Targeted protein degrader development for cancer: advances, challenges, and opportunities, VOLUME 44, ISSUE 5, P303-317, May 2023; Zhang T, Liu C, Li W, Kuang J, Qiu XY, Min L, Zhu L. Targeted protein degradation in mammalian cells: A promising avenue toward future. Comput Struct Biotechnol J. 2022 Sep 28;20:5477-5489. doi: 10.1016/j.csbj.2022.09.038. PMID: 36249565; PMCID: PMC9535385; the contents of each are incorporated herein by reference in its entirety.

Selectively inhibiting

[0210] In some embodiments, the methods of the disclosure comprise selectively inhibiting an enzymatic component of a spliceosome in a cancer cell. As used herein, the term “selectively inhibiting” refers to specifically inhibiting a particular, desired enzymatic component of a spliceosome in comparison to inhibiting other non-selected enzymatic components of a spliceosome. In some embodiments, selectively inhibiting refers to absence of inhibition of the non-selected enzymatic components of a spliceosome. In some embodiments, the agent can selectively bind, inhibit and/or degrade a particular enzymatic component. Selectively inhibiting can be achieved, for example, by administering an agent that selectively binds, inhibits and/or degrades a particular enzymatic component over the other non-selected enzymatic components and/or selectively targeting an agent capable of inhibiting and/or degrading an enzymatic component to a particular enzymatic component. In some embodiments, selectively inhibiting comprises selectively degrading a particular enzymatic component, for example using a small molecule degrader (such as proteolysis targeting chimera (PROTAC) or molecular glue). In some embodiments, selectively inhibiting an enzymatic component induces expression of one or more mis-spliced RNA that is different than that induced upon inhibition of the excluded non-selected enzymatic components. In some embodiments, the selectively inhibited enzymatic component is selected from the group consisting of DDX23, DDX46, DHX15, SF3B1, or a combination thereof. In some embodiments, the selectively inhibited enzymatic component is selected from the group consisting of DDX23, DDX46, SF3B1, or a combination thereof. In some embodiments, the selectively inhibited enzymatic component is selected from the group consisting of DDX23, DDX46, or a combination thereof. In some embodiments, the selectively inhibited enzymatic component is selected from the group consisting of DDX23. In some embodiments, the selectively inhibited enzymatic component is selected from the group consisting of DDX46.

Effects of the perturbation of enzymatic component of a spliceosome

Mis-spliced RNA expression

[0211] In some embodiments, inhibiting an enzymatic component induces expression of one or more mis-spliced RNA. As used herein “mis-spliced RNA” or “mis-processed RNA” refers to an altered spliced RNA upon processing of a pre-mRNA transcript such that it contains either a different combination of exons as a result of exon skipping or exon inclusion, a deletion in one or more exons, or additional sequence not normally found in the spliced RNA (e.g., intron sequence) upon processing of that pre- mRNA transcript in a cell (e.g., a corresponding control cell), “mis-spliced RNA” can also refer to an increase or decrease in a spliced RNA than that is normally found in a cell (e.g., a corresponding control cell). In some embodiments, the methods of the disclosure expresses the one or more mis-spliced RNA relative to the spliced RNA expressed in a corresponding control cell. In some embodiments, the expression of a mis-spliced RNA results in an immune response to the cell. In some embodiments, the expression of a mis-spliced RNA results in induction of an antiviral immune response. In some embodiments, the mis-spliced RNA forms a dsRNA. In some embodiments, the mis-spliced RNA encodes one or more neoantigens.

[0212] In some embodiments, one or more mis-spliced RNA are expressed due to intron retention upon inhibition or degradation of the enzymatic component of the spliceosome. In some embodiments, the mis- spliced RNA comprises one or more retained introns of a pre-mRNA transcript. In some embodiments, the mis-spliced RNA is expressed due to splicing at a cryptic splice site sequence of a pre-mRNA transcript. A “cryptic splice site” is a splice site that is not normally used in the splicing event for a given pre-mRNA transcript.

[0213] In some embodiments, the one or more mis-spliced RNA expressed upon selectively inhibiting or degrading an enzymatic component is different than that expressed upon inhibiting or degrading a nonselected enzymatic component. As used herein “different than that expressed” refers to a presence or absence of a mis-spliced RNA upon selectively inhibiting or degrading an enzymatic component relative to that upon inhibiting or degrading a non-selected enzymatic component, “different than that expressed” also refers to increased or decreased expression of a mis-spliced RNA upon selectively inhibiting or degrading an enzymatic component relative to that upon inhibiting or degrading a non-selected enzymatic component. In some embodiments, an increased expression of a mis-spliced RNA is at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% relative to that upon inhibiting or degrading a nonselected enzymatic component. In some embodiments, a decreased expression of a mis-spliced RNA is at least aboutl%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% relative to that upon inhibiting or degrading a non-selected enzymatic component. In some embodiments, the one or more mis-spliced RNA induces JAK/STAT signaling pathway.

[0214] In some embodiments, the one or more mis-spliced RNA induce expression of one or more IRF- family transcription factors (e g., IRF-1, IRF-2, IRF-3, IRF-4, IRF-5, IRF-6, IRF-7, IRF, 8, and IRF-9) In some embodiments, the one or more mis-spliced RNA induces expression of one or more NF-KB transcription factors. In some embodiments, the one or more mis-spliced RNA induces Jak/Stat signaling pathway. In some embodiments, the one or more mis-spliced RNA induces protein-kinase R-signaling pathway. In some embodiments, the one or more mis-spliced RNA induces 2', 5 '-oligoadenylate synthetase (OAS)-RNase L pathway. In some embodiments, the one or more mis-spliced RNA comprises one or more cryptic junction.

[0215] Methods for determining mis-spliced RNA expression and level are known in the art. For example, a total RNA extraction can be performed on cells or a cell lysate and the resulting extracted total RNA can be purified (e.g., on a column comprising oligo-dT beads) to obtain extracted mRNA.For example, the mis-spliced RNA transcripts can be detected and measured using deep sequencing, such as ILLUMINA® RNASeq, ILLUMINA® next generation sequencing (NGS), ION TORRENT™ RNA next generation sequencing, 454™ pyrosequencing, or Sequencing by Oligo Ligation Detection (SOLID™). The mis-spliced RNA transcripts can be measured using an exon array, such as the GENECHIP® human exon array, RT-PCR, RT-qPCR. Techniques for conducting these assays are known to one skilled in the art. In some embodiments, a statistical analysis or other analysis is performed on data from the assay utilized to measure an mis-spliced RNA transcript. In some embodiments, a student t-test statistical analysis is performed on data from the assay utilized to measure an RNA transcript to determine those RNA transcripts that have an alternation in amount relative to the amount in a corresponding control cell. In some embodiments, the stability of mis-spliced RNA transcripts can be determined by serial analysis of gene expression (SAGE), differential display analysis (DD), RNA arbitrarily primer (RAP)- PCR, restriction endonuclease-lytic analysis of differentially expressed sequences (READS), amplified restriction fragment-length polymorphism (ALFP), total gene expression analysis (TOGA), RT-PCR, RT- qPCR, high-density cDNA filter hybridization analysis (HDFCA), suppression subtractive hybridization (SSH), differential screening (DS), cDNA arrays, oligonucleotide chips, or tissue microarrays. In other embodiments, the stability of one or more RNA transcripts can be determined by Northern blots, RNase protection, or slot blots.

Induction of an immune response

[0216] In some embodiments, the inhibiting of an enzymatic component of a spliceosome in a cell (e.g., a cancer cell) results in an immune response to the cell. In some embodiments, the inhibiting of an enzymatic component of a spliceosome in a cell (e.g., a cancer cell) induces expression of one or more mis-spliced RNA in said cancer cell, and the expression of the one or more mis-spliced RNA results in an immune response to the cell (e.g., cancer cell). In some embodiments, the degradation of an enzymatic component protein of a spliceosome in a cell (e.g., a cancer cell) results in an immune response to the cell. In some embodiments, the degradation of an enzymatic component of a spliceosome in a cell (e.g., a cancer cell) induces expression of one or more mis-spliced RNA in said cancer cell, and the expression of the one or more mis-spliced RNA results in an immune response to the cell (e.g., cancer cell). [0217] “Immune response” generally refers to innate and acquired immune responses including, but not limited to, both humoral immune responses (e.g., mediated by B lymphocytes) and cellular immune responses (e.g., mediated by an immune cell). A cellular immune response can be mediated by an immune cell, such as a B cell, T cell (CD4+ or CD8+), regulatory T cell, antigen-presenting cell, dendritic cell, monocyte, macrophage, NKT cell, NK cell, basophil, eosinophil, or neutrophil. A cellular immune response can be a T cell mediated response. As used herein, the term "T cell-mediated response" refers to a response mediated by T cells, including effector T cells (e.g., CD8+ cells) and helper T cells (e.g., CD4+ cells). A T cell mediated responses include, for example, T cell cytotoxicity and/or proliferation. A cellular immune response can be a cytotoxic T lymphocyte (CTL) response. As used herein, the term "cytotoxic T lymphocyte (CTL) response" refers to an immune response induced by cytotoxic T cells. CTL responses are mediated primarily by CD8+ T cells. A T cell, can include e.g., an effector T cell or a Th cell, such as a CD4+ or CD8+ T cell, or the inhibition or depletion of a Treg cell. "T effector" ("Teff ') cells refers to T cells (e.g., CD4+ and CD8+ T cells) with cytolytic activities as well as T helper (Th) cells, which secrete cytokines and activate and direct other immune cells. In some embodiments, an immune response is a T cell response, such as a CD4+ response or a CD8+ response. In some embodiments, an immune response can include, for example, 1) cytotoxicity of the cell (e.g., a cancer cell) in which an enzymatic component of a spliceosome has been inhibited or degraded, 2) cytotoxicity of cells in proximity of cells (e.g., a cancer cell) in which an enzymatic component of a spliceosome has been inhibited or degraded, 3) cytokine production and/or increase in level of cytokine, 4) chemokine production and/or increase in level of chemokine, 5) interferon production and/or increase in level of interferon, 4) proliferation and/or an increase in level of the immune cell, 6) trafficking of the immune cell to a cell (e.g., cancer cell) in which the enzymatic component of a spliceosome has been inhibited and/or degraded, 7) infiltration by an immune cell into a particular site (e.g., a tumor site, or a tumor microenvironment) that harbors the cell (e.g., a cancer cell) in which an enzymatic component of a spliceosome has been inhibited and/or degraded, 8) expression of one or more caspases in the cell (e.g., cancer cell) in which the enzymatic component of a spliceosome has been inhibited and/or degraded, 9) apoptosis of the cell (e.g., cancer cell) in which the enzymatic component of a spliceosome has been inhibited and/or degraded, 10) activation of one or more immune signaling pathways, and/or 11) phagocytosis of a cell (e.g., cancer cell) in which the enzymatic component of a spliceosome has been inhibited and/or degraded.

[0218] In some embodiments, the immune response is a memory immune response. Accordingly, in one aspect provided herein is a method of generating a memory immune response to a cancer cell comprising inhibiting or degrading an enzymatic component of a spliceosome of the cancer cell. A "memory immune response" results when the provided treatment for cancer (e.g., inhibiting or degrading an enzymatic component of a spliceosome in a cancer cell) facilitates the adaptation of the immune system and the immune response of the subject or patient in its ability to slow, reduce or prevent the return or the recurrence, e.g., lengthening the time of remission, of the tumor or cancer being treated in the subject or patient. In some embodiments, the memory immune response may slow, reduce or prevent the development of tumors or cancers that are different than the cancer being treated, e.g., through epitope spreading. In some embodiments, the methods of the disclosure induces memory B cells, and/or memory T cells. In some embodiments, the immune response comprises inductions of memory B cells and/or memory T cells. The memory B cells and memory T cells remains in a resting state until re-exposure to an antigenic cancer cell. Accordingly, in some embodiments, upon recurrence or remission of the treated cancer, the memory B cells undergo activation, proliferation and induction of humoral immune response to the cancer. In some embodiments, upon recurrence or remission of the treated cancer, the memory T cells, undergo activation, proliferation, and a T cell response.

[0219] In some embodiments, the induced immune response upon selectively inhibiting or degrading an enzymatic component is different than that upon inhibiting or degrading a non-selected enzymatic component. In some embodiments, a different immune response comprises a presence or absence of an immune response upon selectively inhibiting or degrading an enzymatic component relative to that upon inhibiting or degrading a non-selected enzymatic component. In some embodiments, a different immune response comprises an increased or decreased immune response upon selectively inhibiting or degrading an enzymatic component relative to that upon inhibiting or degrading a non-selected enzymatic component. In some embodiments, an increased immune response is at least aboutl%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% relative to that upon inhibiting or degrading a non-selected enzymatic component. In some embodiments, a decreased immune response is at least aboutl%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% relative to that upon inhibiting or degrading a non-selected enzymatic component. In some embodiments, an increased or decreased immune response comprises an increased or decreased level of an immune cell, activity of an immune cell and/or level of a cytokine, chemokine, and/or interferon.

Anti-viral immune response

[0220] In some embodiments, the immune response (e.g., an innate immune response) is an antiviral immune response. In some embodiments, the one or more mis-spliced RNA forms a double stranded RNA (dsRNA). In some embodiments, the methods of the disclosure comprise inducing one or more misspliced RNA that forms a dsRNA. In some embodiments, the dsRNA is located in the cytoplasm of the cancer cell. In some embodiments, the dsRNA induces the immune response. Viral double stranded RNA (dsRNA) induces an IFN response, a molecule that occurs during viral infection as a result of viral genomic replication and viral RNAs with extensive secondary structure (review in (Jacobs et al., Virology 219:339-49 (1996)). In some embodiments, a dsRNA induces an interferon (IFN) response. An interferon response can comprise, for example, secretion of an interferon, increase in level of an interferon, and/or activation of interferon (IFN) signaling pathway. An interferon signaling pathway can be, for example, a signaling pathway activated or induced upon binding of an interferon to an interferon receptor. In some embodiments, said dsRNA induces interferon signaling pathway. In some embodiments, said dsRNA induces an immune response. In some embodiments, the dsRNA induces an innate immune response. In some embodiments, said immune response is an anti-viral immune response. In some embodiments, the dsRNA induces interferon secretion. In some embodiments, the dsRNA induces interferon activation. In some embodiments, the dsRNA are detected by MDA5, RIG1, PKR, TLR3, or a combination thereof. In some embodiments, the dsRNA induces expression and/or activation of MDA5, RIG1, PKR, TLR3, or a combination thereof. In some embodiments, the dsRNA induces JAK/STAT signaling pathway.In some embodiments, the dsRNA induces expression of one or more IRF-family transcription factors (e.g., IRF-1, IRF-2, IRF-3, IRF-4, IRF-5, IRF-6, IRF-7, IRF, 8, and IRF-9). In some embodiments, the dsRNA induces expression of one or more NF-KB transcription factors. In some embodiments, the dsRNA induces Jak/Stat signaling pathway. In some embodiments, the dsRNA induces protein-kinase R-signaling pathway. In some embodiments, the dsRNA induces 2', 5 '-oligoadenylate synthetase (OAS)-RNase L pathway.

[0221] In some embodiments, the immune response (e.g., an antiviral immune response) comprises production and/or an increase in level of an interferon and/or complement proteins. In some embodiments, the inhibiting and/or degradation induces production and/or an increase in level of an interferon and/or complement proteins. In some embodiments, the immune response (e.g., an antiviral immune response) comprises activation of IFN signaling pathway. In some embodiments, the inhibiting and/or degradation induces an IFN signaling pathway. In some embodiments, the interferon is a type I Interferon (e.g., IFN-a, IFN-P, IFN-a, IFN-K or IFN-co). In some embodiments, the interferon is an IFN-a, and/or IFN- . In some embodiments, the IFN signaling pathway is an IFN-a signaling pathway, and/or IFN-P signaling pathway. In some embodiments, the dsRNA is recognized by the TLR3 receptor, which in some embodiments, activates myeloid differentiation factor 88 (Myd88)-dependent and independent signal transduction cascades, leading to the expression of IFNp.

[0222] Interferons (IFN) comprise a family of cytokines which are expressed in response to viral infection and other insults, and regulate a myriad of cellular and systematic responses directed to control viral propagation (see Levy et al., Cytokine Growth Factor Rev. 12: 143-156 (2001) and Goodbum et al., J. Gen. Virol. 81:2341-2364 (2000) for a review). During a viral infection, IFN induces signal transduction through the Janus kinase/Signal Transducer and Activator of Transcription (JAK/STAT) system resulting in the induction of hundreds of IFN inducible genes (de Veer et al., L. Leuc. Proc. Biol. 69:;912-20 (2001); Ehrt et al., J. Exp. Med. 194: 1123-40 (2001)). Mechanisms of interferon signaling are described in Platanias, L. Mechanisms of type-I- and type-II-interferon-mediated signalling. Nat Rev Immunol 5, 375- 386 (2005), the contents of which is incorporated herein by reference in its entirety.

[0223] In some embodiments, the IFN induces activation of Jak-STAT signaling pathway. In some embodiments, the IFN induces expression of one or more interferon inducible genes. In some embodiments, the immune response (e.g., an antiviral immune response) comprises activation of Jak- STAT signaling pathway. In some embodiments, the inhibiting and/or degradation induces Jak-STAT signaling pathway. In some embodiments, the immune response (e.g., an antiviral immune response) comprises expression of one or more interferon stimulated genes. In some embodiments, the inhibiting and/or degradation induces expression (activation) of one or more interferon stimulated genes. IFN stimulated genes include, for example, those encoding RNA-dependent protein kinase (PKR), the MXlprotein, oligoadenylate synthetase (OAS), and IFNs themselves. In some embodiments, the immune response (e.g., an antiviral immune response) comprises expression of one or more nuclear factor KB (NF- KB)-responsive genes (e.g., TNF, IL1B). In some embodiments, the inhibiting and/or degradation induces expression (activation) of one or more nuclear factor KB (NF-KB)-responsive genes (e.g., TNF, IL1B).

[0224] In some embodiments, the immune response (e.g., an antiviral immune response) comprises an increased expression and/or activation of a mitochondrial antiviral signaling protein. In some embodiments, the inhibiting and/or degradation induces an increased expression and/or activation of a mitochondrial antiviral signaling protein. In some embodiments, the immune response (e.g., an antiviral immune response) comprises an activation of a cGas-STING pathway. In some embodiments, the inhibiting and/or degradation induces activation cGas-STING pathway.

[0225] In some embodiments, the immune response comprises activation JAK/STAT signaling pathway. In some embodiments, the immune response comprises expression of one or more IRF-family transcription factors (e g., IRF-1, IRF-2, IRF-3, IRF-4, IRF-5, IRF-6, IRF-7, IRF, 8, and IRF-9). In some embodiments, the immune response comprises expression of one or more NF-KB transcription factors. In some embodiments, the immune response comprises activation of Jak/Stat signaling pathway. In some embodiments, the immune response comprises activation of protein-kinase R-signaling pathway. In some embodiments, the immune response comprises activation of 2', 5 '-oligoadenylate synthetase (OAS)-RNase L pathway.

[0226] Methods to determine presence and level of a protein (e.g., an interferon, expression products of interferon stimulated genes e.g., an OAS, or expression products of NF-KB-responsive genes) are known in the art, for example by western blot, ELISA, and RT-PCR, and described in the specification. Methods to determine induction and activation of a signaling pathway are also known in the art. For example, induction and/or activation of a signaling pathway can be determined by detection and/or measurement of expression of a signaling protein component of the pathway, or detection and/or measurement of a phosphorylation status of a signaling protein component of the pathway. Methods to measuring cellular signaling pathways are known in the art, for example, reviewed in Svoboda KK, Reenstra WR. Approaches to studying cellular signaling: a primer for morphologists. Anat Rec. 2002 Apr 15;269(2): 123-39. doi: 10.1002/ar.10074. PMID: 12001220; PMCID: PMC2862383, the contents of which are incorporated herein by reference in its entirety.

Neoantigenic immune response

[0227] In some embodiments, the one or more mis-spliced RNA encodes one or more neoantigen. In some embodiments, the methods of the disclosure comprises induction of one or more mis-spliced RNA that encodes one or more neoantigens in the cell (e.g., a cancer cell).

[0228] “Neoantigen” means a class of antigens which when expressed by a cell are identified as foreign by the immune system, thereby inducing an immune response to the neoantigen. Neoantigens can arise, for example, by mutations in proteins, or translation of mis-spliced RNA into proteins which are identified as foreign. Neoantigens encompass, but are not limited to, antigens which arise from, for example, substitution in the protein sequence, frame shift mutation, fusion polypeptide, in-frame deletion, and insertion.

[0229] The term “neoepitope” as used herein refers to an antigenic determinant region within the neoantigenic peptide. A neoepitope may comprise at least one “anchor residue” and at least one “anchor residue flanking region.” A neoepitope may further comprise a “separation region.” The term “anchor residue” refers to an amino acid residue that binds to specific pockets on HLAs, resulting in specificity of interactions with HLAs. Neoepitopes may bind to HLA molecules through primary and secondary anchor residues protruding into the pockets in the peptide -binding grooves. In the peptide -binding grooves, specific amino acids compose pockets that accommodate the corresponding side chains of the anchor residues of the presented neoepitopes.

[0230] In some embodiments, the immune response comprises an immune response to one or more neoantigen expressed by the cell. In some embodiments, the immune response to one or more neoantigen is a cellular immune response. In some embodiments, the inhibiting results in expression of one or more neoantigens, and/or an increase in level of said one or more neoantigens relative to that in a corresponding control cell. In some embodiments, the degradation results in expression of one or more neoantigens, and/or an increase in level of said one or more neoantigens relative to that in a corresponding control cell. In some embodiments, a level of a neoantigen is increased by at least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or 100% relative to that in a corresponding control cell. In some embodiments, the one or more neoantigen comprises a neoepitope that binds to a HLA protein. In some embodiments, the methods of the disclosure further comprises binding of a neoepitope of said one or more neoantigen to a HLA protein. In some embodiments, binding to said HLA protein induced a T cell response (e.g., a cytotoxic T cell response or a helper T cell response). Methods of the disclosure

[0231] In some aspects, provided herein are methods of predicting responsiveness of a subject to a spliceosome targeted therapy comprising: (a) inhibiting an enzymatic component of a spliceosome in said subject; (b) determining a RNA expression profile in a biological sample of said subject; (c) comparing said RNA expression profile with a known mis-spliced RNA expression profile associated with sensitivity to said STT; and (d) identifying said subject as a responsive subject or a non-responsive subject based on the comparison of step (b), wherein said subject is identified as responsive if said RNA expression profile matches said known mis-spliced RNA expression profile, and wherein said subject is identified as non- responsive if said RNA expression profile is different than said known mis-spliced RNA expression profile. In some embodiments, the RNA expression profile refers to the expression of total RNA or a select candidate RNA sequences. In some aspects, provided herein is a method of enhancing sensitivity of a cancer to a spliceosome targeted therapy (STT), the method comprising:

[0232] inhibiting a selected enzymatic component of a spliceosome in a cancer cell, wherein said inhibiting of said selected enzymatic component induces expression of one or more mis-spliced RNA that is different than that induced upon inhibition of a non-selected enzymatic component associated with a spliceosome, wherein said expression of one or more mis-spliced RNA results in enhancing susceptibility of the cancer cell to a STT. The term "enhancing the susceptibility to a STT" refers to increasing the likelihood cancer cell inhibition or killing by, or decreasing resistance of the cancer cell to, the STT. The phrase encompasses direct and indirect activity of a STT agent on the cancer cell. In some embodiments, an STT can be a small molecule inhibitor of a spliceosome. A number of STT are known and reviewed for example in Bonner EA, Lee SC. Therapeutic Targeting of RNA Splicing in Cancer. Genes. 2023; 14(7): 1378, the contents of which are incorporated herein by reference in its entirety.

[0233] In some aspect, provided herein are methods of inducing an immune response in a subject. In some aspect, provided herein is a method of treating a cancer in a subject. In some embodiments, the methods of the disclosure comprise inhibiting an enzymatic component of a spliceosome in a cancer cell. In some embodiments, the methods of the disclosure comprise inducing degradation of an enzymatic component of a spliceosome in a cancer cell. In some embodiments, the methods of the disclosure comprise inhibiting a DDX46, DDX23, DHX15, or a combination thereof in a subject. In some embodiments, the inhibiting of DDX46, DDX23, DHX15, or a combination thereof is in a cancer cell in a subject. In some embodiments, the methods of the disclosure comprise inducing degradation of a DDX46, DDX23, DHX15, or a combination thereof in a subject. In some embodiments, the inducing degradation of DDX46, DDX23, DHX15, or a combination thereof is in a cancer cell in a subject. In some embodiments, the methods of the disclosure comprise selectively inhibiting an enzymatic component of a spliceosome in a cell. [0234] In some embodiments, the methods of the disclosure comprise inducing expression of one or more mis-spliced RNA in a cell (e.g., a cancer cell) of a subject (e.g., via inhibition of an enzymatic component of a spliceosome in the cell). In some embodiments, the methods of the disclosure comprise inhibiting an enzymatic component of a spliceosome in a cell (e.g., a cancer cell) of a subject, and inducing one or more mis-spliced RNA in the cell. In some embodiments, the methods of the disclosure comprise inhibiting an enzymatic component of a spliceosome in a cell (e.g., a cancer cell) of a subject, and inducing an immune response to the cell. In some embodiments, the methods of the disclosure comprise inhibiting an enzymatic component of a spliceosome in a cell (e.g., a cancer cell) of a subject, inducing one or more mis-spliced RNA in the cell and inducing an immune response to the cell. In some embodiments, the methods of the disclosure comprise inducing degradation of an enzymatic component of a spliceosome in a cell (e.g., a cancer cell) of a subject, and inducing one or more mis-spliced RNA in the cell. In some embodiments, the methods of the disclosure comprise inducing degradation of an enzymatic component of a spliceosome in a cell (e.g., a cancer cell) of a subject, and inducing an immune response to the cell. In some embodiments, the methods of the disclosure comprise inducing degradation of an enzymatic component of a spliceosome in a cell (e.g., a cancer cell) of a subject, inducing one or more mis-spliced RNA in the cell and inducing an immune response to the cell.

[0235] In some embodiments, the methods of the disclosure comprise inducing IFN signaling pathway. In some embodiments, the methods of the disclosure comprise inducing IFN expression and/or an increase in interferon expression. In some embodiments, the methods of the disclosure comprises inducing Jak- STAT signaling pathway. In some embodiments, the methods of the disclosure comprises inducing expression of one or more interferon stimulated genes. In some embodiments, the methods of the disclosure comprises inducing cGas-STING pathway.

[0236] In some embodiments, the methods disclosed herein induce a change (e.g., an increase) in level and/or activity of an immune cell (e.g., a T cell), a change in level of an immunomodulatory molecule (e.g., inflammatory cytokines, chemokines), or a combination thereof in a subject. As used herein, the terms level, number, count and concentration can be used interchangeably. It will be appreciated by those skilled in the art that both a cell culture system and the immune system of a subject comprise basal levels of immune cells and immunomodulatory molecules. The phrases basal level and normal level can be used interchangeably. As used herein, the basal level of a type of immune cell, or a immunomodulatory molecule, refers to the average number of that cell type, or immunomodulatory molecule, present in a population of individuals considered healthy (i.e., free of cancer) or the basal level of a type of immune cell, or an immunomodulatory molecule, refers to the average level of that cell type, or immunomodulatory molecule, present in a population of cells that is not-activated. Those skilled in the art are capable of determining if an immune cell, or a population of such cells, is activated. For example, the expression of CD69, CD25 and/or CD 154 proteins by a T-cell indicates that the cell has been activated. For example, the expression of MHC-class II, B220 and CD3 proteins by B-cell indicates that the B-cell has been activated. For example, the expression of IL-12, iNOS, Arg-1, or IL-1 proteins by macrophage indicates the macrophage has been activated.

[0237] Methods to measure immune cells are well known in the art including methods based on identifying expression of specific surface marker proteins e.g., by flow cytometry. Level of immune cell can be measure, for example, by measuring proliferation by 3H-Thymidine Uptake, Bromodeoxyuridine Uptake (BrdU), ATP Luminescence, Fluorescent Dye Reduction (carboxyfluorescein succinimidyl ester (CFSE)-like dyes); cytokine measurement, for example, using Multi-Analyte ELISArray Kits, bead-based multiplex assay; measuring surface antigen expression, for example, by flow cytometry; measuring cell cytotoxicity, for example, by Two-Label Flow Cytometry, Calcein AM Dye Release, Luciferase Transduced Targets, or Annexin V. Methods to measure T-cell responses and B-cell responses are well known in the art, for example see Expert Rev. Vaccines 9(6), 595-600 (2010), mBio. 2015 Jul-Aug; 6(4). Surface antigen or surface markers of CD8+ T cells are known in the art. For example, effector T cells may express CD25, CD69, KLRG1, CD30, 0X40, ICOS, TIM3; effector memory T cells may express CD44, CD45RO, CD62LlowCD127, CCR71ow, KLRG1, central memory T cells may express CD45RO, CD62LlowCD127++, CCR71ow, CD27, CD28. Anergic and regulatory T cells may express CD57, CD28-, KLRG1++, Lag-3, PD-1, HLADR.

[0238] The reference level or basal level of a cell or molecule can be a specific amount (e.g., a specific concentration) or it can encompass a range of amounts. Basal levels, or ranges, of immune cells and immunoregulatory molecules are known to those in the art. For example, in a healthy individual, the normal level of CD4+ T-cells present in human blood is 500-1500 cells/ml. Basal levels of cells can also be reported as a percentage of a total cell population.

[0239] Immune cell number and function, for example may be monitored by assays that detect immune cells by an activity such as cytokine production, proliferation, or cytotoxicity. For example, Lymphoproliferation Assay, which assays the ability of T cells to proliferate in response to an antigen can be used as an indicator of the presence of cancer cell specific CD4+ helper T cells. For example, flow cytometric detection of intracellular perforin and granzyme B or degranulation assay, ELISPOT assay or ELISA for the detection of IL-2 and IFNy can be performed to determine activity of CD8+ T cells. For example, expression kinetics for CD25, flow cytometric detection of intracellular perforin and granzyme B can be performed for memory T cells. Assays for degranulation evaluate the cytotoxic potential of CD8+ T cells. Activated effector CD8+ T cells release cytolytic granules perforin and granzyme B, which induces killing of cancer cells. Mobilization of CD107/LAMP-1 is a measure of cytotoxic potential of killer cells. CD 107a glycoproteins line the luminal surface of resting T cells. Upon activation, lytic granules get localized to the site of interaction with the target cell and merge with the plasma membrane. During this process, granzymes and perforin get exocytosed and CD 107 expression appears on the cell surface. Mobilization of CD107/LAMP-1 can be detected using flow cytometry. Chromium (51Cr) release cytotoxicity assay, and Annexin V binding cytotoxicity assay can be performed to measure tumor specific cytotoxicity of T cells. Typically, the specimen of purified T cells or PBMCs is mixed with various dilutions of antigen or antigen in the presence of stimulator cells (irradiated autologous or HLA matched antigen-presenting cells). After 72-120 h, [3H] thymidine is added, and DNA synthesis (as a measure of proliferation) is quantified by using a gamma counter to measure the amount of radiolabeled thymidine incorporated into the DNA. A stimulation index can be calculated by dividing the number of cpm for the specimen by the number of counts per minute in a control. A proliferation assay can be used to compare T-cell responses before and after treatment according to methods of the present disclosure. Another example of assay that can be employed for detection of proliferation of immune cells (e.g., T-cell, B-cells) include use of intracellular fluorescent dye, 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) in mixed lymphocyte reaction (CFSE-MLR) and determination of proliferating cells using flow cytometry. Another example of an assay that can be employed is detection of secreted cytokines by ELISA and ELISPOT Assays.

[0240] Cytokine secretion by immune cells in a subject may be detected by measuring either bulk cytokine production (by an ELISA) or enumerating individual cytokine producing immune cells (by an ELISPOT assay). Generally, in an ELISA assay, PBMC specimens are incubated with antigen (with or without antigen-presenting cells), and after a defined period of time, the supernatant from the culture is harvested and added to microtiter plates coated with antibody for cytokines of interest such as IFN-y, TNF-a, or IL-2. Antibodies ultimately linked to a detectable label or reporter molecule are added, and the plates are washed and read. In a modification of the assay, cytokine secretions can be measured in samples (e.g., serum or other body fluids) obtained from a subject (e.g., a subject suffering from cancer) using ELISA or ELISPOT before and after treatment with the peptides disclosed herein or pharmaceutical compositions disclosed herein. Other useful assays include, measurement of detection of intracellular cytokine assay by flow cytometry, measurement of cytokine mRNA levels by RT-PCR and direct cytotoxicity assays of T-cell (See: Clay et al., 2001). Macrophage activation can be determined, for example, by measuring levels of chemokines such as IL-8/CXCL8, IP-10/CXCL10, MIP-1 alpha/CCL3, MIP-1 beta/CCL4, and RANTES/CCL5, which are released as chemoattractants for neutrophils, immature dendritic cells, natural killer cells, and activated T cells. Levels of pro-inflammatory cytokines are released including IL-1 beta/IL-lF2, IL-6, and TNF -alpha can also be measured by assays well known in the art. Levels of proteolytic enzymes, MMP-1, -2, -7, -9, and -12, which degrade Collagen, Elastin, Fibronectin, and other ECM components can also be measured to determine macrophage activation. Leukocytes are attracted by the macrophage via its release of chemokines including MDC/CCL22, PARC/CCL18, and TARC/CCL17. Levels of activated B-cell can be determined, for example, by measuring antigen specific antibody secretion or detecting activated B-cell specific surface markers such as CD27, CD19, CD20, CD25, CD30, CD69, CD80, CD86, CD135, by assays such as flow cytometry. [0241] In some embodiments, the methods disclosed herein can increase the level of an immune cell by at least about 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, of the total immune cell population. Methods of measuring different types of T-cells in the T-cell population are known to those skilled in the art. In some embodiments, methods of the present disclosure increase the number of immune cells by at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95%. In another embodiment, methods of the present disclosure increase the number of immune cells by a factor of at least 10, at least 100, at least 1,000, at least 10,000. In some embodiments, the level of immune cells is increased so that immune cells comprise at least about 0.5%, 1%, 5%, 10%, 15%, 20%, 25%, 30, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, of the total immune cell population.

[0242] Provided herein are methods of treating a cancer. The term “cancer” refers to any of the various malignant neoplasms characterized by the proliferation of cells that have the capability to invade surrounding tissue and/or metastasize to new colonization sites, including but not limited to leukemias, lymphomas, carcinomas, melanomas, sarcomas, germ cell tumors and blastomas. Exemplary cancers include cancers of the brain, bladder, breast, cervix, colon, head and neck, kidney, lung, non-small cell lung, mesothelioma, ovary, prostate, stomach and uterus, leukemia and medulloblastoma.

[0243] Neoplastic tissues can originate from any cell type or tissue found in a mammal, including, but not limited to hepatic, skin, breast, prostate, neural, optic, intestinal, cardiac, vasculature, lymph, spleen, renal, bladder, lung, muscle, connective, tissue, pancreatic, pituitary, endocrine, reproductive organs, bone, and blood. The neoplastic tissue for analysis may include any type of solid tumor or hematological cancer. In some embodiments, the neoplastic tissue is a breast cancer tissue. In other embodiments, the neoplastic tissue is a breast tissue with atypical hyperplasia.

[0244] The term “leukemia” refers to broadly progressive, malignant diseases of the blood-forming organs and is generally characterized by a distorted proliferation and development of leukocytes and their precursors in the blood and bone marrow. Leukemia diseases include, for example, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, aleukemic leukemia, a leukocythemic leukemia, basophylic leukemia, blast cell leukemia, bovine leukemia, chronic myelocytic leukemia, leukemia cutis, embryonal leukemia, eosinophilic leukemia, Gross' leukemia, hairy-cell leukemia, hemoblastic leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myelocytic leukemia, myeloid granulocytic leukemia, myelomonocytic leukemia, Naegeli leukemia, plasma cell leukemia, plasmacytic leukemia, promyelocytic leukemia, Rieder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemic leukemia, and undifferentiated cell leukemia.

[0245] The term “carcinoma” refers to a malignant new growth made up of epithelial cells tending to infdtrate the surrounding tissues and give rise to metastases. Exemplary carcinomas include, for example, acinar carcinoma, acinous carcinoma, adenocystic carcinoma, adenoid cystic carcinoma, carcinoma adenomatosum, carcinoma of adrenal cortex, alveolar carcinoma, alveolar cell carcinoma, basal cell carcinoma, carcinoma basocellulare, basaloid carcinoma, basosquamous cell carcinoma, bronchioalveolar carcinoma, bronchiolar carcinoma, bronchogenic carcinoma, cerebriform carcinoma, cholangiocellular carcinoma, chorionic carcinoma, colloid carcinoma, comedo carcinoma, corpus carcinoma, cribriform carcinoma, carcinoma en cuirasse, carcinoma cutaneum, cylindrical carcinoma, cylindrical cell carcinoma, duct carcinoma, carcinoma durum, embryonal carcinoma, encephaloid carcinoma, epiennoid carcinoma, carcinoma epitheliale adenoides, exophytic carcinoma, carcinoma ex ulcere, carcinoma fibrosum, gelatiniform carcinoma, gelatinous carcinoma, giant cell carcinoma, carcinoma gigantocellulare, glandular carcinoma, granulosa cell carcinoma, hair-matrix carcinoma, hematoid carcinoma, hepatocellular carcinoma, Hurthle cell carcinoma, hyaline carcinoma, hypemephroid carcinoma, infantile embryonal carcinoma, carcinoma in situ, intraepidermal carcinoma, intraepithelial carcinoma, Krompecher's carcinoma, Kulchitzky-cell carcinoma, large-cell carcinoma, lenticular carcinoma, carcinoma lenticulare, lipomatous carcinoma, lymphoepithelial carcinoma, carcinoma medullare, medullary carcinoma, melanotic carcinoma, carcinoma molle, mucinous carcinoma, carcinoma muciparum, carcinoma mucocellulare, mucoepidermoid carcinoma, carcinoma mucosum, mucous carcinoma, carcinoma myxomatodes, naspharyngeal carcinoma, oat cell carcinoma, carcinoma ossificans, osteoid carcinoma, papillary carcinoma, periportal carcinoma, preinvasive carcinoma, prickle cell carcinoma, pultaceous carcinoma, renal cell carcinoma of kidney, reserve cell carcinoma, carcinoma sarcomatodes, Schneiderian carcinoma, scirrhous carcinoma, carcinoma scroti, signet-ring cell carcinoma, carcinoma simplex, smallcell carcinoma, solanoid carcinoma, spheroidal cell carcinoma, spindle cell carcinoma, carcinoma spongiosum, squamous carcinoma, squamous cell carcinoma, string carcinoma, carcinoma telangiectaticum, carcinoma telangiectodes, transitional cell carcinoma, carcinoma tuberosum, tuberous carcinoma, verrucous carcinoma, and carcinoma villosum.

[0246] The term “sarcoma” generally refers to a tumor which arises from transformed cells of mesenchymal origin. Sarcomas are malignant tumors of the connective tissue and are generally composed of closely packed cells embedded in a fibrillar or homogeneous substance. Sarcomas include, for example, chondrosarcoma, fibrosarcoma, lymphosarcoma, melanosarcoma, myxosarcoma, osteosarcoma, Abernethy's sarcoma, adipose sarcoma, liposarcoma, alveolar soft part sarcoma, ameloblastic sarcoma, botryoid sarcoma, chloroma sarcoma, chorio carcinoma, embryonal sarcoma, Wilms' tumor sarcoma, endometrial sarcoma, stromal sarcoma, Ewing's sarcoma, fascial sarcoma, fibroblastic sarcoma, giant cell sarcoma, granulocytic sarcoma, Hodgkin's sarcoma, idiopathic multiple pigmented hemorrhagic sarcoma, immunoblastic sarcoma of B cells, lymphomas (e.g., Non-Hodgkin Lymphoma), immunoblastic sarcoma of T-cells, Jensen's sarcoma, Kaposi's sarcoma, Kupffer cell sarcoma, angiosarcoma, leukosarcoma, malignant mesenchymoma sarcoma, parosteal sarcoma, reticulocytic sarcoma, Rous sarcoma, serocystic sarcoma, synovial sarcoma, and telangiectaltic sarcoma.

[0247] The term “melanoma” is taken to mean a tumor arising from the melanocytic system of the skin and other organs. Melanomas include, for example, acral-lentiginous melanoma, amelanotic melanoma, benign juvenile melanoma, Cloudman's melanoma, S91 melanoma, Harding-Passey melanoma juvenile melanoma, lentigo maligna melanoma, malignant melanoma, nodular melanoma subungal melanoma, and superficial spreading melanoma.

[0248] The cancer may be of any type or grade or tissue of origin. It may or may not be metastatic. The cancer may include any malignant cell type, such as those found in a solid tumor or a hematological tumor. Specific cancers for which the inhibitors identified through methods disclosed herein are useful include non-small cell lung cancer adenocarcinoma, ovarian cancer, esophageal cancer, HCC, head and neck cancer, non-small cell lung squamous cancer, breast cancer (including at least triple -negative), gastric cancer, pancreatic cancer, bladder cancer, colon cancer, cecum cancer, stomach cancer, brain cancer, kidney cancer, larynx cancer, sarcoma, lung cancer, melanoma, prostate cancer, and so on. Exemplary hematological tumors include tumors of the bone marrow, T or B cell malignancies, leukemias, lymphomas, blastomas, myelomas, and the like. Further examples of cancers that may be treated using the methods provided herein include, but are not limited to, lung cancer (including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung), cancer of the peritoneum, gastric or stomach cancer (including gastrointestinal cancer and gastrointestinal stromal cancer), pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, various types of head and neck cancer, and melanoma. The cancer may specifically be of the following histological type, though it is not limited to these: neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma; small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma; hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma; trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp; adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant; branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma; acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma; granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma; nonencapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometroid carcinoma; skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminous adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infdtrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; pagef s disease, mammary; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; thymoma, malignant; ovarian stromal tumor, malignant; thecoma, malignant; granulosa cell tumor, malignant; androblastoma, malignant; sertoli cell carcinoma; leydig cell tumor, malignant; lipid cell tumor, malignant; paraganglioma, malignant; extra- mammary paraganglioma, malignant; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; lentigo malignant melanoma; acral lentiginous melanomas; nodular melanomas; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; blue nevus, malignant; sarcoma; fibrosarcoma; fibrous histiocytoma, malignant; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; mixed tumor, malignant; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; mesenchymoma, malignant; brenner tumor, malignant; phyllodes tumor, malignant; synovial sarcoma; mesothelioma, malignant; dysgerminoma; embryonal carcinoma; teratoma, malignant; struma ovarii, malignant; choriocarcinoma; mesonephroma, malignant; hemangiosarcoma; hemangioendothelioma, malignant; kaposi's sarcoma; hemangiopericytoma, malignant; lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; chondroblastoma, malignant; mesenchymal chondrosarcoma; giant cell tumor of bone; ewing's sarcoma; odontogenic tumor, malignant; ameloblastic odontosarcoma; ameloblastoma, malignant; ameloblastic fibrosarcoma; pinealoma, malignant; chordoma; glioma, malignant; ependymoma; astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma; oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma; ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; meningioma, malignant; neurofibrosarcoma; neurilemmoma, malignant; granular cell tumor, malignant; malignant lymphoma; hodgkin's disease; hodgkin's; paragranuloma; malignant lymphoma, small lymphocytic; malignant lymphoma, large cell, diffuse; malignant lymphoma, follicular; mycosis fungoides; other specified non-hodgkin's lymphomas; B-cell lymphoma; low grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate grade/follicular NHL; intermediate grade diffuse NHL; high grade immunoblastic NHL; high grade lymphoblastic NHL; high grade small non-cleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; Waldenstrom's macroglobulinemia; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia; mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; hairy cell leukemia; chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); acute myeloid leukemia (AML); and chronic myeloblastic leukemia. [0249] As used herein, the terms "treat," "treatment," "treating," or "amelioration" refer to therapeutic treatments, wherein the object is to reverse, alleviate, ameliorate, inhibit, slow down or stop the progression or severity of a condition associated with, a disease or disorder (e.g., a cancer). The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with a cancer. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of a disease is reduced or halted. That is, "treatment" includes not just the improvement of symptoms or markers, but also a cessation of at least slowing of progress or worsening of symptoms that would be expected in absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptom(s), diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. The term "treatment" of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0250] The efficacy of the treatment methods for cancer of the present disclosure can be measured by various endpoints commonly used in evaluating cancer treatments, including but not limited to, tumor regression, tumor weight or size shrinkage, time to progression, duration of survival, progression free survival, overall response rate, duration of response, and quality of life. The methods disclosed herein can, for example, reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and/or stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and/or stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the disorder. The methods disclosed herein can result in a cytostatic and/or cytotoxic effect to a cancer. For cancer therapy, efficacy in vivo can, for example, be measured by assessing the duration of survival, duration of progression free survival (PFS), the response rates (RR), duration of response, and/or quality of life. The therapy may delay the onset of cancer, reduce the severity of one or more symptoms of cancer, or both.

[0251] In other embodiments, described herein are methods for increasing progression free survival of a human subject susceptible to or diagnosed with a cancer. Time to disease progression is defined as the time from treatment until disease progression or death. In some embodiments, the methods disclosed herein alone, or in combination with one or more additional therapeutic agents (e.g., an immunosuppressive agent, an immunomodulatory agent, an immunotherapeutic agent) may significantly increase progression free survival by at least about 1 month, 1.2 months, 2 months, 2.4 months, 2.9 months, 3.5 months, such as by about 1 to about 5 months, when compared to a treatment with said additional therapeutic alone. In another embodiment, the methods described herein may significantly increase response rates in a group of human subjects susceptible to or diagnosed with a cancer that are treated with various therapeutics. Response rate is defined as the percentage of treated subjects who responded to the treatment. In some embodiments, the methods described herein alone or in a combination treatment with one or more additional therapeutic agents significantly increases response rate in the treated subject group compared to the group treated with said one or more additional therapeutic agents. Accordingly, in some embodiments, the methods disclosed herein further comprise administering to a subject one or more additional therapeutic agents.

[0252] For example, in some embodiments, the methods described herein alleviate a symptom of a cancer. As used herein, "alleviating a symptom of a cancer" is ameliorating or reducing any condition or symptom associated with the cancer. As compared with an equivalent untreated control, such reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique. Ideally, the cancer is completely cleared as detected by any standard method known in the art, in which case the cancer is considered to have been treated. A patient who is being treated for a cancer is one who a medical practitioner has diagnosed as having such a condition. Diagnosis can be by any suitable means. Diagnosis and monitoring can involve, for example, detecting the level of cancer cells in a biological sample (for example, a tissue or lymph node biopsy, blood test, or urine test), detecting the level of a surrogate marker of the cancer in a biological sample, detecting symptoms associated with the specific cancer, or detecting immune cells involved in the immune response typical of such a cancer.

[0253] The treatment and/or prevention of cancer includes, but is not limited to, alleviating symptoms associated with cancer, the inhibition of the progression of cancer, the promotion of the regression of cancer, the promotion of the immune response, inhibition of tumor growth, inhibition of tumor size, inhibition of metastasis, inhibition of cancer cell growth, inhibition of cancer cell proliferation, or cause cancer cell death.

[0254] In some embodiments, the methods of the present disclosure further comprises administering one or more additional therapeutic agents (e.g., additional cancer therapeutic agents).

[0255] In some embodiments, said one or more additional therapeutic agent is capable of binding to and/or inhibiting programmed cell death 1 (PDCD1, PD1, PD-1), CD274 (CD274, PDL1, PD-L1), PD-L2, cytotoxic T-lymphocyte associated protein 4 (CTLA4, CD152), CD276 (B7H3); V-set domain containing T cell activation inhibitor 1 (VTCN1, B7H4), CD272 (B and T lymphocyte associated (BTLA)), killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 1 (KIR, CD158E1), lymphocyte activating 3 (LAG3, CD223), hepatitis A virus cellular receptor 2 (HAVCR2, TIMD3, TIM3), V-set immunoregulatory receptor (VSIR, B7H5, VISTA), T cell immunoreceptor with Ig and ITIM domains (TIGIT), programmed cell death 1 ligand 2 (PDCD1LG2, PD-L2, CD273), immunoglobulin superfamily member 11 (IGSF11, VSIG3), TNFRSF14 (HVEM, CD270), TNFSF14 (HVEML), PVR related immunoglobulin domain containing (PVRIG, CD112R), galectin 9 (LGALS9), killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 1 (KIR2DL1); killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 2 (KIR2DL2); killer cell immunoglobulin like receptor, two Ig domains and long cytoplasmic tail 3 (KIR2DL3); and killer cell immunoglobulin like receptor, three Ig domains and long cytoplasmic tail 1 (KIR3DL1), killer cell lectin like receptor Cl (KLRC1, NKG2A, CD159A), killer cell lectin like receptor DI (KLRD1, CD94), killer cell lectin like receptor G1 (KLRG1, CLEC15A, MAP A, 2F1), sialic acid binding Ig like lectin 7 (SIGLEC7), SIGLEC, sialic acid binding Ig like lectin 9 (SIGLEC9), CEACAM (e.g., CEA CAM-1, CEACAM-3 and/or CEACAM-5), VISTA, LAIR1, CD160, 2B4, CD80, CD86, B7-H1, B7-H3 (CD276), B7-H4 (VTCN1), CD134 (OX40L), KIR, A2AR, A2BR, MHC class I, MHC class II, GAL9, adenosine, TGFR (e g., TGFR beta) , CD94/NKG2A, IDO, TDO, CD39, CD73, GARP, CD47, SIRP alpha, SIRP beta, PVRIG, CSF1R, orNOX.

[0256] In some embodiments, said one or more additional therapeutic agents is a PD-1 modulator. In some embodiments, a PD-1 modulator is Pembrolizumab (humanized antibody), Pidilizumab (CT-011, monoclonal antibody, binds DLL1 and PD-1), Spartalizumab (PDR001, monoclonal antibody), Nivolumab (BMS-936558, MDX-1106, human IgG4 monoclonal antibody), MEDI0680 (AMP-514, monoclonal antibody), Cemiplimab (REGN2810, monoclonal antibody), Dostarlimab (TSR-042, monoclonal antibody), Sasanlimab (PF- 06801591, monoclonal antibody), Tislelizumab (BGB-A317, monoclonal antibody), BGB-108 (antibody), Tislelizumab (BGB-A317, antibody), Camrelizumab (INCSHR1210, SHR-1210), AMP-224, Zimberelimab (AB 122, GLS-010, WBP-3055, monoclonal antibody), AK-103 (HX- 008, monoclonal antibody), AK-105 (anti-PD-1 antibody), CS1003 (monoclonal antibody), HLX10 (monoclonal antibody), Retifanlimab (MGA-012, anti-PD-1 monoclonal antibody), BI- 754091 (antibody), Balstilimab (AGEN2034, PD-1 antibody), toripalimab (JS-001, antibody), cetrelimab (JNJ-63723283, anti-PD-1 antibody), genolimzumab (CBT-501, anti-PD-1 antibody), LZM009 (anti-PD-1 monoclonal antibody), Prolgolimab (BCD- 100, anti-PD-1 monoclonal antibody), Sym021 (antibody), ABBV-181 (antibody), BAT- 1306 (antibody), JTX-4014, sintilimab (IBI-308), Tebotelimab (MGD013, PD-l/LAG-3 bispecific), MGD-019 (PD- 1/CTLA4 bispecific antibody), KN-046 (PD-1/CTLA4 bispecific antibody), MEDI-5752 (CTLA4/PD-1 bispecific antibody), RO7121661 (PD-l/TIM-3 bispecific antibody), XmAb20717 (PD-1/CTLA4 bispecific antibody), or AK-104 (CTLA4/PD-1 bispecific antibody)

[0257] In some embodiments, the one or more additional therapeutic agent is a PD-L1 modulator. In some embodiments, a PD-L1 modulator is Atezolizumab (MPDL3280A, monoclonal antibody), Avelumab (MSB0010718C, monoclonal antibody), Durvalumab (MEDI-4736, human immunoglobulin G1 kappa (IgGlx) monoclonal antibody), Envafolimab (KN035, single-domain PD-L1 antibody), AUNP12, CA-170 (small molecule targeting PD-L1 and VISTA), BMS-986189 (macrocyclic peptide), BMS-936559 (Anti-PD-Ll antibody), Cosibelimab (CK-301, monoclonal antibody), LY3300054 (antibody), CX-072 (antibody), CBT- 502 (antibody), MSB-2311 (antibody), BGB-A333 (antibody), SHR-1316 (antibody), CS1001 (WBP3155, antibody), HLX-20 (antibody), KL-A167 (HBM 9167, antibody), STI-A1014 (antibody), STI-A1015 (IMC-001, antibody), BCD-135 (monoclonal antibody), FAZ-053 (antibody), CBT-502 (TQB2450, antibody), MDX1105-01 (antibody), FS- 118 (LAG-3/PD-L1, bispecific antibody), M7824 (anti-PD-Ll/TGF-P receptor II fusion protein), CDX-527 (CD27/PD-L1 bispecific antibody), LY3415244 (TIM3/PD-L1 bispecific antibody), INBRX-105 (4-1BB/PD-L1 bispecific antibody)

[0258] In some embodiments, the one or more additional therapeutic agent is a CTLA4 modulator. [0259] In some embodiments, the one or more additional therapeutic agent is a CD40L antibody, a OX- 40 antibody, or a CD28 antibody

[0260] Examples of second therapy useful for treating cancer can include, but not limited to radiotherapy, cryotherapy, antibody therapy, chemotherapy, photodynamic therapy, surgery, hormonal therapy, immunotherapy, cytokine therapy, or a combination therapy with conventional drugs. In some embodiments, an additional therapeutic agent, can be a cytotoxic drug, tumor vaccine, a peptide, a pepti- body, a small molecule, a cytotoxic agent, a cytostatic agent, immunological modifier, interferon, interleukin, immunostimulatory growth hormone, cytokine, vitamin, mineral, aromatase inhibitor, RNAi, Histone Deacetylase Inhibitor, proteasome inhibitor, a cancer chemotherapeutic agent, Tregs targeting agent, another antibody, Immunostimulatory antibody, a NS AID, a corticosteroid, a dietary supplement such as an antioxidant, cisplatin, ifosfamide, paclitaxel, taxanes, topoisomerase I inhibitors (e.g., CPT-11, topotecan, 9-AC, and GG-211), gemcitabine, vinorelbine, oxaliplatin, 5 -fluorouracil (5-FU), leucovorin, vinorelbine, temodal, and taxol. In some embodiments, an additional therapeutic agent is a chemotherapeutic agent selected from a group consisting of platinum based compounds, antibiotics with anti-cancer activity, Anthracyclines, Anthracenediones, alkylating agents, antimetabolites, Antimitotic agents, Taxanes, Taxoids, microtubule inhibitors, Vinca alkaloids, Folate antagonists, Topoisomerase inhibitors, Antiestrogens, Antiandrogens, Aromatase inhibitors, GnRh analogs, and inhibitors of 5 a- reductase, biphosphonates. A "cytotoxic agent" refers to an agent that has a cytotoxic and/or cytostatic effect on a cell. A "cytotoxic effect" refers to the depletion, elimination and/or the killing of a target cell(s). A "cytostatic effect" refers to the inhibition of cell proliferation.

[0261] In some embodiments, an additional agent can be a PD- 1 inhibitor, histone deacetylase (HDAC) inhibitor, proteasome inhibitor, mTOR pathway inhibitor, JAK2 inhibitor, tyrosine kinase inhibitor (TKIs), PI3K inhibitor, Protein kinase inhibitor, Inhibitor of serine/threonine kinases, inhibitor of intracellular signaling, inhibitors of Ras/Raf signaling, MEK inhibitor, AKT inhibitor, inhibitor of survival signaling proteins, cyclin dependent kinase inhibitor, therapeutic monoclonal antibodies, TRAIL pathway agonist, anti-angiogenic agent, metalloproteinase inhibitor, cathepsin inhibitor, inhibitor of urokinase plasminogen activator receptor function, immunoconjugate, antibody drug conjugate, antibody fragments, bispecific antibodies, bispecific T cell engagers (BiTEs). In some embodiments, the additional therapeutic agent is an antibody that is selected from cetuximab, panitumumab, nimotuzumab, trastuzumab, pertuzumab, rituximab, ofatumumab, veltuzumab, alemtuzumab, labetuzumab, adecatumumab, oregovomab, onartuzumab; apomab, mapatumumab, lexatumumab, conatumumab, tigatuzumab, catumaxomab, blinatumomab, ibritumomab triuxetan, tositumomab, brentuximab vedotin, gemtuzumab ozogamicin, clivatuzumab tetraxetan, pemtumomab, trastuzumab emtansine, bevacizumab, etaracizumab, volociximab, ramucirumab, aflibercept. In yet another embodiment, the second therapeutic agent can be antibodies currently used for the treatment of cancer. Examples of such antibodies include, but are not limited to, HERCEPTIN®, RETUXAN®, OvaRex, Panorex, BEC2, IMC-C225, Vitaxin, Campath I/H, Smart MI95, LymphoCide, Smart I DIO, and Oncolym. In some embodiments, the another antibody is an immunostimulatory antibody is selected from antagonistic antibodies targeting one or more of CTLA4, PD-1, PDL-1, LAG-3, TIM-3, BTLA, B7-H4, B7-H3, VISTA, and/or Agonistic antibodies targeting one or more of CD40, CD137, 0X40, GITR, CD27, CD28, ICOS or a combination thereof. In some embodiments, an additional therapeutic agent targeting immunosuppressive cells Tregs and/or MDSCs is selected from antimitotic drugs, cyclophosphamide, gemcitabine, mitoxantrone, fludarabine, thalidomide, thalidomide derivatives, COX-2 inhibitors, depleting or killing antibodies that directly target Tregs through recognition of Treg cell surface receptors, anti-CD25 daclizumab, basiliximab, ligand- directed toxins, denileukin diftitox (ONTAK®) — a fusion protein of human IL-2 and diphtheria toxin, or LMB-2 — a fusion between an scFv against CD25 and the pseudomonas exotoxin, antibodies targeting Treg cell surface receptors, TLR modulators, agents that interfere with the adenosinergic pathway, ectonucleotidase inhibitors, or inhibitors of the A2A adenosine receptor, TGF-P inhibitors, chemokine receptor inhibitors, retinoic acid, all-trans retinoic acid (ATRA), Vitamin D3, phosphodiesterase 5 inhibitors, sildenafil, ROS inhibitors and nitroaspirin. In some embodiments, an additional therapeutic agent is cytokine therapy selected from one or more of the following cytokines such as IL-2, IL-7, IL- 12, IL-15, IL-17, IL-18 and IL-21, IL23, IL-27, GM-CSF, IFNa (interferon alpha), IFNa-2b, IFNP, IFNy, and their different strategies for delivery. In some embodiments, the an additional therapeutic agent is a therapeutic cancer vaccine selected from a group consisting of exogenous cancer vaccines including proteins or peptides used to mount an immunogenic response to a tumor antigen, recombinant virus and bacteria vectors encoding tumor antigens, DNA-based vaccines encoding tumor antigens, proteins targeted to dendritic cell-based vaccines, whole tumor cell vaccines, gene modified tumor cells expressing GM-CSF, ICOS and/or Flt3-ligand, oncolytic virus vaccines. [0262] In some embodiments, an additional therapeutic agent includes EPO, G-CSF, ganciclovir; antibiotics, leuprolide; meperidine; zidovudine (AZT); interleukins 1 through 18, including mutants and analogues; interferons or cytokines, such as interferons a, ' and y hormones, such as luteinizing hormone releasing hormone (LHRH) and analogues and, gonadotropin releasing hormone (GnRH); growth factors, such as transforming growth factor, fibroblast growth factor (FGF), nerve growth factor (NGF), growth hormone releasing factor (GHRF), epidermal growth factor (EGF), fibroblast growth factor homologous factor (FGFHF), hepatocyte growth factor (HGF), and insulin growth factor (IGF); tumor necrosis factoralpha (TNF-a); invasion inhibiting factor-2 (IIF-2); bone morphogenetic proteins 1-7 (BMP 1-7); somatostatin; thymosin-a-1; y-globulin; superoxide dismutase (SOD); complement factors; antiangiogenesis factors; antigenic materials; and pro-drugs. Prodrug refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic or non-cytotoxic to tumor cells compared to the parent drug and is capable of being enzymatically activated or converted into an active or the more active parent form. See, e.g., Wilman, "Prodrugs in Cancer Chemotherapy" Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Belfast (1986) and Stella et al., "Prodrugs: A Chemical Approach to Targeted Drug Delivery," Directed Drug Delivery, Borchardt et al., (ed.), pp. 247-267, Humana Press (1985). Prodrugs include, but are not limited to, phosphate- containing prodrugs, thiophosphate-containing prodrugs, sulfate -containing prodrugs, peptide-containing prodrugs, D-amino acid-modified prodrugs, glycosylated prodrugs, lactam-containing prodrugs, optionally substituted phenoxyacetamide -containing prodrugs or optionally substituted phenylacetamide -containing prodrugs, 5- fluorocyto- sine and other 5 -fluorouridine prodrugs which can be converted into the more active cytotoxic free drug. Examples of cytotoxic drugs that can be derivatized into a prodrug form for use herein include, but are not limited to, those chemotherapeutic agents described above.

EXAMPLES

[0263] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

Example 1 - Degradable Cell Line Generation

[0264] SUM159 human breast cancer cells were cultured in F12 media supplemented with 5% FBS, lOmM HEPES, 5ug/mL insulin, and lug/mL hydrocortisone. MDA-MB-231-LM2 breast cancer and 293T cells were cultured in DMEM media supplemented with 10% FBS. MYC-ER HME1 cells were cultured in MEGM (Lonza). PyMT-M and E0771 mouse breast cancer cells were cultured in DMEM media supplemented with 10% FBS and 1% Penicillin Streptomycin. All cell lines were incubated at 37C and 5% CO ₂ . [0265] Lentiviruses were generated by transfection of 293Ts with appropriate sgRNA or cDNA construct with packaging plasmids using Minis Bio’s TransIT transfection reagent. Viral supernatants were harvested 48 hours after transfection.

[0266] Cas9 was amplified from pCW-Cas9 and cloned into pINDUCER20 to allow for dox-inducible Cas9 expression. This vector was transduced into SUM159 and MDA-MB-231-LM2 cells and infected cells were selected with neomycin. A clone from each parental cell line was selected to generate SUM159-Cas9 and MDA-MB-231-LM2-Cas9 cells with homogeneous Cas9 expression and inducibility. SUM159 and MDA-MB-231-LM2 cells stably expressing dox-inducible Cas9 were transduced with the FKBP12 ^F36V -tagged helicase lentivirus and then selected with puromycin. SUM159-Cas9 FKBP12 ^F36V - tagged helicase and MDA-MB-231-LM2-Cas9 FKBP12 ^F36V -tagged helicase cells were transduced with the appropriate helicase targeting sgRNA (ThermoFisher LentiArray sgRNA library). To knock out the endogenous helicase of interest, cells were cultured in 500ng/mL doxycycline for 6 days to induce Cas9 expression followed by 6 days of culture in complete media to allow Cas9 to be turned off. A clone from each parental cell line was selected to generate SUM159-Cas9 FKBP12 ^F36V -tagged helicase + endogenous helicase depleted and MDA-MB-231-LM2-Cas9 FKBP12 ^F36V -tagged helicase + endogenous helicase depleted cells with homogeneous FKBP12 ^F36V -tagged helicase expression. Endogenous depletion was confirmed via western blot analysis and sanger sequencing of the genomic locus. Exogenous expression of FKBP12 ^F36V -tagged helicase and dTagl3 or dTagVl -mediate degradation of FKBP12 ^F36V -tagged helicase was confirmed via western blot analysis.

Example 2 - Degradation of target (e.g., an enzymatic component of a spliceosome e.g., a helicase) to assess RNA misprocessing phenotypes

[0267] SUM159-Cas9 FKBP12 ^F36V -tagged helicase + endogenous helicase depleted and MDA-MB-231- LM2-Cas9 FKBP12 ^F36V -tagged helicase + endogenous helicase depleted cells were treated with targetspecific doses of dTagl3 or dTagVl for 6, 9, 12 & 24hrs to elicit helicase degradation. At each time point, an RNA lysate (Trizol, in biologic triplicate), a protein lysate (lx Bolt LDS Sample Buffer, to confirm degradation) and a cell count were taken.

[0268] Total RNA was isolated using the Direct-zol-96 Kit (Zymo). Synthesis of cDNA was done using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems). RT-qPCR was performed using the SYBR Select Master Mix (Applied Biosystems) on 2ng of input cDNA. For intron retention analysis, primer sets were designed to measure intron-containing transcripts and fully spliced transcripts. Intron retention was calculated as the ratio of intron-containing transcripts over fully spliced transcripts. Data were calculated as fold change relative to control data using the AACt method. All experiments were performed in biological triplicate.

[0269] Cell-count normalized total RNA was used as input for the TruSeq Stranded mRNA HT Prep Kit (Illumina). Libraries were made following Illumina’s recommend protocol, successful production confirmed by Aligent RNA ScreenTape analysis and quantified using NEBNext Library Quant Kit for Illumina. Libraries were sequenced on the Illumina NovaSeq 6000 as 150bp paired end reads.

Example 3 - RNA misprocessing analysis and signature generation

[0270] To quantify misprocessing (mis-splicing) the following algorithm was developed. Eirst, a custom annotation was generated using the ENSEMBL genome annotation (hg38 version 92). Each gene was divided into intronic and exonic regions by aggregating across isoforms. In case of genomic overlap, genes were merged into a ‘multi -gene’. The algorithm requires two inputs: 1) custom annotation file and 2) BAM alignment file. The algorithm then classifies each read-pair based on the mapping properties of each read mate. Each mate can be classified as purely exonic, purely intronic or both. Read-pairs are summed per mapping class and specified region in the custom annotation file. The algorithm allows the user to specify whether to perform the aggregation at the intron or gene level.

[0271] Given the total count of processed and misprocessed read-pairs a binomial test was applied to assess statistical significance for each gene/intron. To quantify the magnitude, the algorithm calculates the ‘delta ratio’ which represents difference between the average misprocessing rate in treated compared to untreated samples for each gene/intron.

[0272] To discover helicase degradation specific biomarkers the following analyses were developed. For each gene/intron and each helicase delta ratios were calculated. For each gene/intron the delta ratios were compared across helicases across multiple conditions using a linear model. Gene/intron with statistically significant effects were defined as helicase-specific.

[0273] In addition, the following approach was applied for intron analysis only. RNA sequencing read densities spanning each intron were calculated for all libraries. For each intron, read densities from all libraries were reduced to two dimensions using a convolutional autoencoder. A random forest model was applied to associate the embedding with the helicase label. If the embedding separated a specific helicase from all others with high accuracy, the intron was considered helicase-specific.

Example 4 - Degradation of target (e.g., an enzymatic component of a spliceosome e.g., a helicase) to assess immune signaling activation

[0274] SUM159-Cas9 FKBP12 ^F36V -tagged helicase + endogenous helicase depleted and MDA-MB-231- LM2-Cas9 FKBP12 ^F36V -tagged helicase + endogenous helicase depleted cells were treated with targetspecific doses of dTagl3 or dTagVl for 48 & 72hrs to elicit helicase degradation. At each time point, an RNA lysate (Trizol, in biologic triplicate), a protein lysate (lx Bolt LDS Sample Buffer, to confirm degradation) and a cell count were taken.

[0275] Total RNA was isolated using the Direct-zol-96 Kit (Zymo). Synthesis of cDNA was done using the High-Capacity RNA-to-cDNA Kit (Applied Biosystems). RT-qPCR was performed using the SYBR Select Master Mix (Applied Biosystems) on 2ng of input cDNA. Relative transcript abundance was normalized (GAPDH). Data were calculated as fold change relative to control data using the AACt method. All experiments were performed in biological triplicate.

Example 5. In vivo tumor studies

[0276] All animal protocols related to mouse experiments were approved by the Baylor College of Medicine Institutional Animal Care and Use Committee (protocol AN-6672). 4-5-week-old female C57BL/6J and BALB/c AnNHsd female mice were obtained from The Jackson Laboratory (000664) and Envigo (470 IF), respectively. Mice were housed in ventilated cages in a pathogen-free animal facility under a 14hr light/ lOhr dark cycle. Tumor chunks were transplanted into the cleared mammary fat pad of 4-5 week old female C57BL/6J or BALB/c AnNHsd female mice. PyMT-M and E0771 tumors were randomized onto vehicle or dTagl3 at 150-250mm ³ for long term response studies or 300-500mm ³ for short term studies. Animals were allocated into treatment groups so that the average tumor size in both groups was similar. Tumor growth was monitored twice weekly.

[0277] For studies to assess immune memory, tumor chunks were transplanted into the cleared mammary fat pad of previously tumor bearing C57BL/6J or BALB/c AnNHsd female mice or age-matched tumor naive controls. Tumor growth was monitored twice weekly.

Example 4 Perturbation of spliceosome components leads to distinct patterns of RNA mis-splicing [0278] RNA splicing changes induced by degradation of core spliceosome components U2AF2, DDX46, SF3B1, PRPF8, AQR, DHX16, DHX38, and DHX15 were studied (FIG. 11). To specifically perturb spliceosome proteins, target cDNA fused to FKBP12 ^F36V were expressed in SUM159 cells in which the endogenous loci of the corresponding spliceosome component was knocked out via CRISPR/Cas9 (FIG. 12A-12H), thus enabling selective and dose-dependent target perturbation using heterobifunctional degrader molecules. Changes in RNA splicing induced by maximum target degradation were measured using paired-end poly(A)+ RNAseq followed by classification of misprocessed fragments across annotated introns (FIG. 12I-12K). Fidelity of RNA splicing was quantified for every intron as the ratio of improperly spliced reads (those containing intronic sequences or aberrant splice junctions) to properly spliced reads (those containing only exonic sequences). Degradation of these core spliceosome components resulted in significantly increased misprocessing as compared to baseline (FIG. 13). The data shows that maximum degradation of all targets leads to misprocessing of a large fraction of introns, the number of misprocessed introns (FIG 14A-14B) varies between targets indicating that introns display differential sensitivity to inhibition of certain steps of splicing.

[0279] To investigate overarching differences in misprocessing between targets, dimension reduction analysis on RNA misprocessing events that occur upon target degradation was performed. Surprisingly, this analysis revealed distinct clusters (FIG. 15) which segregated targets by function in the spliceosome. Cluster 1 is made up of proteins associated with 3 ’splice site (ss) and branchpoint (BP) identification as part of the U2 snRNP: U2AF2, SF3B1, and DDX46. Cluster 2 consists of proteins that function to activate the spliceosome or mediate catalytic steps in the splicing cycle: PRPF8, AQR, DHX16, and DHX38. Surprisingly, DHX15 alone constitutes Cluster 3. To further understand the features underlying this functional clustering, a linear model was utilized to identify introns that constitute a misprocessing signature unique to each functional cluster (FIG 16 and FIG. 17). Importantly, while dimension reduction analysis reveals similar signatures associated with functional groups, deeper analysis highlights individual events that are selectively misprocessed upon single target degradation demonstrating that there are differences in intron response to spliceosome perturbation even within splicing step (FIG. 14B). Collectively, these data reveal that while degradation of spliceosome components spanning the splicing cascade results in rampant RNA misprocessing, a subset of these events are indeed unique to functional subgroups and/or individual components of the spliceosome.

Example 5 - DHX15 suppresses 5’ and 3’ cryptic splicing and effects of degradation of DHX15.

[0280] The differential misprocessing signatures upon inhibition of individual spliceosome components as disclosed above indicates that DHX15 plays a unique role in RNA splicing. Investigation of individual DHX 15 -specific misprocessing events revealed generation of cryptic splice junctions within misprocessed introns (Figure 18A-18B, FIG. 19A-19J). Cryptic splicing observed in these DHX 15 -specific introns is exacerbated as DHX 15 protein levels are reduced with increasing concentrations of dTagl3, indicating that DHX15 plays a dose-dependent role in cryptic splice site suppression (FIG. 18C, FIG. 18D FIGs. 19A-19J). Global quantification of cryptic splice junctions reveals pervasive cryptic splicing upon DHX 15 degradation, with cryptic splicing occurring in thousands of introns (FIG. 18E) . Notably, these cryptic splice junctions are observed in the context of full-length transcripts (FIGs. 19A-19J) and map to reads identified in circular RNA sequencing (FIGs. 19A-19J), indicating that cryptic junctions identified in short-read RNAseq datasets are not degradation products and are formed by canonical splicing reactions. To further investigate the nature of these cryptic splice junctions, junctions were classified based on the cryptic site used (FIG. 18F). Importantly, and unexpectedly, degradation of DHX 15 uniquely leads to increases in all classifications of cryptic splicing (FIG. 18G). In contrast to the diversity of DHX 15 -associated cryptic splicing, degradation of AQR leads to specific increase in cryptic 3’ss utilization (FIG. 18G). Degradation of other tested spliceosome components does not lead to an increase in any type of cryptic splicing. Instead, degradation of SF3B1, PRPF8, DHX16, and DHX38 leads to a significant decrease in cryptic splicing across classifications which is indicates degradation of these splicing factors leads to intron retention. Together, these data show that DHX 15 plays a unique role in global suppression of cryptic splice site usage, and its degradation results in loss of suppression leading to unique RNA mis-spilicing pattern.

[0281] To test if the role of DHX 15 in suppression of cryptic splicing, and effects of DHX 15 degradation are conserved, an MDA-MB-231-LM2 cell line was generated with exogenous expression of FKBP-DHX15 and knockout of endogenous DHX 15 and performed RNAseq (FIGs. 19A-19J). Degradation of DHX 15 similarly induces cryptic splicing of all junction classes in LM2 cells (FIG. 18H). Importantly, individual cryptic splice junctions measured in SUM 159 cells upon DHX15 degradation are also detected in LM2 cells (FIG. 181). To determine whether similar cryptic splice sites are globally utilized across cell lines, cryptic splice junction usage was calculated as the ratio of reads mapping to a specific cryptic splice site relative to those mapping to the canonical splice junction. Differential cryptic splice junction usage in SUM159 and LM2 cells -/+ DHX15 degradation was highly correlated, with a majority of junctions increased in usage in both cell lines following depletion of DHX 15 (FIG. 18J, 18K). Consistent with this conserved role of DHX15 in suppressing cryptic splicing, and conserved effects upon degradation of DHX15, analysis of published DHX15 knockdown datasets similarly reveals increased cryptic splicing (FIGs 19A-19J). Together, these data support a conserved role of DHX15 in suppressing usage of specific cryptic splice sites, and conserved effect of the loss of DHX15 on RNA splicing.

Example 6 - DHX15 prevents cryptic splicing at weaker splice sites and effects of degradation of DHX15

[0282] As these data show role of wild type DHX15 in suppression of cryptic splicing, the characteristics of cryptic splice sites used was determined. The results showed that cryptic splice sites show generally similar sequence properties to canonical splice sites. Both 5’ and 3’ cryptic splice sites resemble canonical splice sites by k-mer motif analysis, with the requisite GT and AG needed for splicing present at the 5 ’ and 3’ splice sites, respectively (FIG. 20A, 20B). Similarly, by using Branch point prediction (BPP) to identify the putative branch point utilized with cryptic 3’ splice sites, it was observed that branch points with similar sequence motifs and predicted strength for putative branch points for canonical and cryptic splice sites (FIGs. 20C, 20D) at similar distance from the 3’ splice site (FIGs. 21A-21C) To perform more quantitative analysis of the strength of cryptic 5 ’ and 3 ’ splice sites, MaxEntScan, a kmer-based approach that predicts splice site strength based on adjacent and non-adjacent dependencies was utilized. Analysis with this model similarly indicated that both cryptic 5 ’ and 3 ’ splice sites utilized upon DHX 15 degradation are well-scoring splice sites, albeit slightly weaker than canonical splice sites (FIGs. 20E-20F).

[0283] The difference in magnitude between the subtle shift in splice site k-mer strength and dramatic difference in splicing seen upon DHX 15 degradation suggested that features beyond the local splice site sequence might drive this distinction. To test this, additional analysis was performed of predicted strength of cryptic 3 ’ splice sites using SpliceAI, a deep neural network that predicts splice sites using sequence information from greater genomic space (5kb for SpliceAI compared to <25bp for MaxEntScore). Analysis of a single intron of SNAPCI using SpliceAI analysis revealed that cryptic splice junctions occur at sites that are indeed predicted to be splice sites (FIG. 20G). While these cryptic sites have lower predicted scores than the canonical 3’ splice site in this SNAPCI intron, 100 randomly selected cryptic 5’ and 3’ splice sites have higher predicted splice scores compared to surrounding nucleotides (FIG. 20H), suggesting nonrandom splicing upon DHX 15 degradation. [0284] These results show that cryptic sites used in SNAPCI are weaker than the canonical splice site (FIG. 20F, FIG. 20G). The SpliceAI score of these cryptic splice sites on a more global scale was calculated. In contrast to MaxEntScan, SpliceAI analysis predicts that cryptic splice sites are remarkably weaker than canonical splice sites (FIG. 201, 20 J).

Example 7 - DHX15 prevents cryptic splicing at sites with U2AF deposition but lacking SF3 complex [0285] The recognition of cryptic splice sites utilized upon DHX15 degradation by the splicing machinery was investigated. ENCODE eCLIP data was utilized to assess binding frequency in the vicinity of both canonical and cryptic splice sites, specifically a window covering 75nt of exonic space and 250nt of intronic space (FIG. 22A). RBPs associated with the A or later spliceosomal complexes, including SF3B4 and PRPF8 (proteins reflective of branch point recognition and activated spliceosome, respectively) had specific enrichment for peaks at canonical splice sites but lacked binding peaks at cryptic splice junctions, consistent with the lack of usage seen at these sites in wild-type cells with active DHX15 (FIG. 22B). This analysis was extended to RBPs previously identified to bind near canonical splice junctions and observed a similar pattern for a number of additional spliceosome-associated RBPs, including SF3A3, AQR, and others (FIG. 22C).

[0286] Surprisingly, and similar to the previous short- versus long-range sequence analysis, a small subset of RBPs showed highly similar enrichment at both canonical and cryptic sites (FIG. 22C). Notably, this included the U2 Small Nuclear RNA Auxiliary Factors U2AF1 and U2AF2, which recognize the polypyrimidine tract and 3 splice site acceptor ‘AG’ sequence as part of the transition to the pre- spliceosomal E complex. Despite the large difference in splice site usage in wild-type cells between canonical and cryptic 3’ splice sites, eCLIP data for U2AF2 and U2AF1 showed comparable binding in wild-type cells (FIG. 22D). These results indicate that cryptic splice sites not only show similar 3’ splice site sequence motifs but are already recognized and bound by the U2AF factors in cells expressing DHX15. [0287] The data indicated that DHX15 suppresses cryptic splice site usage by acting as a quality control step that bridges the gap between wide-spread recognition of 3 ’ splice site-like sequences by U2AF 1/2, and subsequent selection of bona fide acceptor sites based on other, longer-range features (including binding of other RBPs, RNA structures, and other features that feed into exon definition). This model suggested that a larger proportion of SF3 complex peaks overlap with canonical splice sites than U2AF1/2. Indeed, 70% of SF3B4 compared to only 55% of U2AF2 peaks overlap with canonical splice sites (FIG. 22E). Second, this model and data shows that upon DHX 15 degradation, splice sites bound by U2AF2 peaks that do not overlap with canonical splice junctions would be activated to generate cryptic splice junctions. Indeed, splicing at U2AF2 peaks not associated with canonical splice sites is increased 8-fold with DHX 15 degradation (FIG. 22F). Investigation of RNA sequences bound by U2AF2 revealed that the frequency of pyrimidine-rich and 3’ splice sites containing 6-mers is similar between U2AF2 peaks overlapping both canonical and cryptic splice sites (FIGs, 22G-22I). Strikingly, comparison of U2AF2 peaks overlapping canonical splice sites and those not overlapping cryptic or canonical reveal that peaks overlapping neither have increased frequency of pyrimidine rich sequences and deceased frequency of 3’ splice site (FIG. 22H, 221). Consistent with this finding, sequences bound by U2AF2 that are neither cryptic or canonical splice sites have markedly lower MaxENT 3’ splice site score (FIG. 22J).

[0288] To directly assess the role of WT DHX15 in regulating SF3 complex binding to suppress cryptic splicing, and effects of DHX15 degradation, eCLIP was performed of SF3B4 in SUM159 FKBP-DHX15 cells with DMSO and dTAG13 treatment. Consistent with SF3B4 eCLIP in HepG2 and K562 cells, SF3B4 is not bound in the vicinity of cryptic splice sites in DMSO treated cells (FIG. 22K). Strikingly, SF3B4 binding is evident at cryptic 3’ splice sites following DHX15 degradation (FIG. 22L). This result indicates that A complex formation is promoted in the absence of DHX15. The data shows that WT DHX15 acts to suppress splicing at cryptic 3’ splice sites through regulation of SF3 binding to sequences recognized by U2AF1/2, and loss of effect is observed upon DHX15 degradation.

Example 8 - Cancer hotspot mutation R222G of DHX15 results in similar RNA mis-splicing pattern as degradation of DHX15 and compromises DHX15-mediated splicing quality control and results in increased cryptic splice site usage.

[0289] Recent genome sequencing of acute myelogenous leukemia (AML) revealed a hotspot mutation (R222G) in DHX15 (FIG. 23 A). It was determined whether this mutation compromises the function of DHX15 and have effects (e.g., a mis-spliced RNA signature) similar to degradation ofDHX15. Consistent with evolutionary trace analysis suggesting evolutionary importance of amino acids in the RNA binding domain, evolutionary action analysis predicts that the R222G mutation has significant impact on DHX15 function, with a score of 92.60. Indeed, structural modeling of RNA binding by DHX15 showed that the R222G mutation significantly impacts interaction with RNA (FIG. 23B). To evaluate the effect of R222G mutation on DHX 15 -mediated splicing quality control, LM2 FKBP-DHX15 cells were transduced with either GFP, DHX15 ^WT , or DHX15 ^R222G cDNA and frequency of RNA misprocessing events were measured with RNAseq (FIG. 23C). Expression of DHX15 ^WT suppressed misprocessing upon degradation of FKBP- DHX15, whereas expression of DHX15 ^R222G led to a similar increase in RNA misprocessing as GFP control, indicating that this cancer hotspot mutation impairs DHX15 function (FIG. 23D). Importantly, DHX15 ^R222G expression does not suppress cryptic splicing upon FKBP-DHX15 degradation (FIG. 23E, 23F). These results support a critical role for the RecAl RNA binding domain, and specifically the R222 residue, of DHX15 in suppressing cryptic splice site usage in LM2 TNBC cells.

[0290] While these result show that the R222G mutation impairs the quality control function of DHX 15 in cancer, this function was investigated in the context of AML, where the R222G mutation was first identified. Thus, a murine model was developed of endogenous R222G mutation that allows for inducible recombination and introduction of the R222G mutation (FIG. 23G). Fetal liver cells were isolated from DHX15 ^+/+ Rosa26CreER, DHX15 ^R222G/+ Rosa26CreER, and DHX15 ^R222G/R222G Rosa26CreER embryos. These cells were transduced to express AML-ET09a and transformed cells were sorted by flow cytometry (FIG. 24). Transformed cells were treated with ethanol or tamoxifen for 24 or 48hrs to induce allele recombination before harvesting for RNA sequencing (FIG. 23G, FIG. 24). Principle component analysis of RNA misprocessing analysis revealed that both homozygous and heterozygous DHX15 ^R222G mutation induced a similar misprocessing signature, distinct from DHX15 ^WT (FIG. 231, FIG. 24). Importantly, DHX15 ^R222G AML-ET09a cells also have increased cryptic splice site usage compared to DHX15 ^WT AML- ETO9a cells (FIGs. 23J, 23K). Together, these data show that the AML-associated DHX15 ^R222G hotspot mutation compromises DHX15-mediated control of cryptic splicing, and have effects similar to degradation of DHX15.

Example 9 - DHX15 copy number loss leads to cryptic splice site usage across cancers similar to degradation of DHX15

[0291] These results demonstrate tha6t DHX15 R222G hotspot mutation compromises quality control of splice site selection, and may lead to aberrant cryptic splicing similar to degradation of DHX15 in cancer (e.g., AML). A signature of aberrant cryptic splicing was developed that is induced upon DHX15 inactivation, and then this signature was leveraged to (1) investigate whether similar patterns of mis-splicing occur in cancer, and (2) identify underlying features of cancer cells that correlate with such aberrant cryptic splicing. Because multiple mechanisms may contribute to aberrant cryptic splicing across heterogeneous tumor types (that may or may not involve DHX15), a signature based on cryptic splicing events was assembled that are (a) robustly induced by DHX15 degradation across two independent TNBC cell lines (FIGs. 25A-25C) and (b) induced selectively by DHX15 perturbation but not by perturbation of other spliceosome components (FIG. 25D). Importantly, this cryptic splicing signature is suppressed by expression of WT DHX15 cDNA but not R222G (FIG. 25E), indicating its is similar to and represents a proxy of DHX15 loss of function.

[0292] To assess the extent of cryptic splicing in cancer, this signature was quantified in RNAseq datasets across cancer cell lines. Surprisingly, the DHX15-derived cryptic splicing signature was elevated in cancer cell lines compared to normal cells (FIG. 25F). Likewise, TNBC cell lines (SUM159 and LM2) exhibited higher DHX 15 -associated cryptic splicing than non-transformed mammary epithelial cells (FIG. 25G). Notably, cancer cell lines with higher levels of such aberrant cryptic splicing also exhibited greater sensitivity to DHX 15 perturbation (shRNA) in both pan-cancer and breast cancer analyses (Figure 25G- 25H).

[0293] As DHX 15 mutations are infrequent in cancer, aberrant cryptic splicing was profded in the context of human tumors to determine if there are genomic features that associate with aberrant cryptic splicing. To this end, the association between the DHX 15 mis-splicing signature and DNA copy number across all genomic loci was tested in 1,070 BRCA patient samples profiled by TCGA (FIG. 25H). Intriguingly, several loci that are commonly deleted in cancer are associated with increased cryptic splicing signature score, suggesting there may be several mechanisms driving dysregulation of splicing quality control during cancer evolution. Notably, one of these loci is 4p 15 that harbors the DHX 15 gene (FIG. 25H) . More directed analysis of DHX 15 gene copy number revealed that DHX 15 loss (hemizygous) is correlated with upregulation of the DHX 15 mis-splicing signature (FIG. 251), implicating DHX 15 copy number loss as one of several mechanisms driving splicing dysregulation in some cancer cell types. 4pl5 harbors several tumor suppressors and hemizygous 4pl5 loss is a frequent event in several common cancer types including breast. DHX 15 -mediated splicing quality control may be frequently compromised in a large proportion of BRCA patients as a collateral side-effect of 4pl5 deletion.

[0294] Further analysis of loci correlated with cryptic splicing signature score revealed that loss of SUGP1, a recently identified GPATCH activator of DHX 15, correlates with increased cryptic splicing signature score (FIG. 25J). Increased cryptic splice site usage upon loss of either DHX15 or the SF3B1 -interacting SUGP1 supports our proposed model that DHX 15 regulates 3’ splice site selection through quality control of motifs recognized by U2AF1/2 before stable SF3 complex integration (FIG. 22). Interestingly, some tumors with copy number loss of DHX 15 have gain of SUGP1, suggesting a potential compensatory mechanism in which increased SUGP1 expression attempts to rescue loss of DHX15 and restore DHX15- mediated quality control . Consistent with this hypothesis, tumors with SUGP 1 gain in the context of DHX 15 loss have significantly lower cryptic splicing signature score than those without (FIG. 25K). Collectively, the data indicates that copy-number loss of DHX15, a collateral side-effect of 4pl5 deletion, leads to aberrant RNA splicing in tumor cells similar to the degradation of DHX 15.