WO2024102985A1 - Perturbations de complexe d'épissage et leurs utilisations - Google Patents

Perturbations de complexe d'épissage et leurs utilisations Download PDF

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WO2024102985A1
WO2024102985A1 PCT/US2023/079351 US2023079351W WO2024102985A1 WO 2024102985 A1 WO2024102985 A1 WO 2024102985A1 US 2023079351 W US2023079351 W US 2023079351W WO 2024102985 A1 WO2024102985 A1 WO 2024102985A1
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cell
cancer
subject
inhibiting
induces
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PCT/US2023/079351
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Thomas F. WESTBROOK
Kristen L. KARLIN
Calla OLSON
Elizabeth A. BOWLING
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Baylor College Of Medicine
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y306/00—Hydrolases acting on acid anhydrides (3.6)
    • C12Y306/04—Hydrolases acting on acid anhydrides (3.6) acting on acid anhydrides; involved in cellular and subcellular movement (3.6.4)
    • C12Y306/04013—RNA helicase (3.6.4.13)

Definitions

  • 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.
  • the present disclosure provides methods for treating a cancer in a subject.
  • said method comprises: selectively inhibiting an enzymatic component of a spliceosome in a cancer cell.
  • 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.
  • 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.
  • the present disclosure provides methods for treating a cancer in a subject.
  • said method comprises: inhibiting an enzymatic component of a spliceosome that is a RNA helicase in a cancer cell in said subject.
  • said inhibiting induces expression of one or more mis-spliced RNA in said cancer cell.
  • 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.
  • the present disclosure provides methods for treating a cancer in a subject.
  • said method comprises: inhibiting an enzymatic component of a spliceosome in a cancer cell of said subject.
  • said inhibiting induces an immune response to said cancer cell in said subject, thereby treating said subject.
  • the present disclosure provides methods for treating a cancer in a subject.
  • said method comprises: inducing degradation of an enzymatic component of a spliceosome in a cancer cell of said subject.
  • said degradation induces an immune response to said cancer cell in said subject, thereby treating said subject.
  • 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.
  • 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.
  • said cell is a cancer cell.
  • 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.
  • 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.
  • said enzymatic component is a RNA helicase.
  • said inhibiting comprises inducing degradation of the enzymatic component.
  • a method for treating a cancer in a subject 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.
  • SF3B1 splicing factor 3B subunit 1
  • a method for inducing an immune response in a subject 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.
  • SF3B1 splicing factor 3B subunit 1
  • said inhibiting comprises inducing degradation of SF3B1.
  • said inhibiting induces one or more mis-spliced RNA in said cancer cell.
  • said method further comprises inhibiting an enzymatic components of the spliceosome.
  • the method further comprises inhibiting a RNA helicase.
  • 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.
  • said one or more agents bind, degrade, and/or inhibit post-translational modification of said enzymatic component.
  • said one or more agents are a small molecule, protein, peptide, nucleic acid, carbohydrate, or a combination thereof.
  • said one or more agents are a siRNA, an antisense morphlino, an antisense oligonucleotide, a small molecule, or a combination thereof.
  • 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)).
  • PRRs Pattern Recognition Receptors
  • RLRs RIG-I-like receptors
  • NLRs Nod-like receptors
  • C-type lectin receptors C-type lectin receptors
  • the inhibiting induces a Jak-STAT signaling pathway.
  • the inhibiting results in activation of one or more interferon-stimulated genes (ISGs).
  • said inhibiting induces an increased expression or activity of a mitochondrial antiviral signaling protein.
  • said inhibiting induces an IFN signaling pathway in said cancer cell.
  • 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.
  • said inhibiting and said formation and/or an increase in level of said R loop results in activation of a cGAS-STING pathway.
  • 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.
  • said inhibiting comprises modifying a gene encoding the enzymatic component, wherein said modifying introduces a mutation in the enzymatic component.
  • said mutation is an amino acid substitution, deletion and/or insertion in said enzymatic component.
  • said modifying results in a decrease in the activity and/or level of said enzymatic component of the spliceosome.
  • 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.
  • said inhibiting comprises inhibiting the enzymatic activity of said enzymatic component of said spliceosome.
  • this disclosure provides methods for treating a cancer in a subject.
  • said method comprises: inducing degradation of an enzymatic component polypeptide of a spliceosome in a cancer cell in said subject.
  • said degradation induces expression of one or more mis-spliced RNA in said cancer cell.
  • 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.
  • 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.
  • 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)).
  • PRRs Pattern Recognition Receptors
  • the degradation induces a Jak-STAT signaling pathway.
  • the degradation results in activation of one or more interferon-stimulated genes (ISGs).
  • said degradation induces an increased expression or activity of a mitochondrial antiviral signaling protein.
  • said degradation induces an IFN signaling pathway in said cancer cell.
  • 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.
  • 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.
  • 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.
  • said degradation comprises inhibiting the enzymatic activity of said enzymatic component of said spliceosome.
  • said one or more mis-spliced RNA comprises one or more retained introns.
  • 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.
  • said enzymatic component e.g., a RNA helicase
  • said enzymatic component 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,
  • said enzymatic component is selected from the group consisting of DHX8, DHX15, DHX38, DHX8, DHX16, DDX46, DDX23, DDX41, DDX47, AQR, and DDX21.
  • said enzymatic component is DHX15, DHX38, DHX46, or DHX23.
  • said enzymatic component is DHX46, or DHX23.
  • said enzymatic component is DHX15, or DHX38.
  • said enzymatic component is DHX46.
  • said enzymatic component is DHX15, or DHX38.
  • 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.
  • said one or more mis-spliced RNA forms a dsRNA in said cancer cell.
  • said method comprises inducing expression of one or more mis-spliced RNA that forms a dsRNA in said cancer cell.
  • said dsRNA is located in cytoplasm of said cancer cell.
  • said dsRNA induces interferon signaling pathway.
  • said dsRNA induces an immune response.
  • the dsRNA induces an innate immune response.
  • said immune response is an anti-viral immune response.
  • the dsRNA induces interferon secretion.
  • the dsRNA induces interferon activation.
  • the dsRNA are detected by MDA5, RIG1, PKR, TLR3, or a combination thereof.
  • the dsRNA induces expression and/or activation of MDA5, RIG1, PKR, TLR3, or a combination thereof.
  • said immune response is an antiviral immune response.
  • said method comprises inducing expression of one or more mis-spliced RNA that encodes one or more neoantigens.
  • said one or more mis-spliced RNA encode said one or more neoantigens.
  • said one or more neoantigen comprises a neoepitope that binds to a HLA protein of said subject.
  • the method further comprises binding of a neoepitope of said one or more neoantigen to a HLA protein.
  • said binding to said HLA protein induced a T cell response (e.g., a cytotoxic T cell response or a helper T cell response).
  • said immune response comprises induction of a T cell response (e.g., a cytotoxic T cell response or a helper T cell response).
  • said immune response is a T-cell immune response.
  • said immune response is a memory immune response.
  • said immune response comprises an increase in level of one or more cytokine and/or chemokines in said subject.
  • 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.
  • said cancer is a solid tumor.
  • the cancer is a hematological tumor.
  • 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).
  • the one or more additional cancer therapeutic agent comprises a chemotherapeutic agent, radiation, or immunotherapy.
  • the method further comprises administering one or more anti immunosuppressive/immunostimulatory agents.
  • the one or more anti- immunosuppressive/immunostimulatory agent provides a CTLA4, a PD-1, a PD-L1 blockade, or a combination thereof.
  • 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.
  • 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.
  • 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,
  • 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,
  • said one or more additional cancer therapeutic agent is a CTLA4 modulator.
  • said one or more additional cancer therapeutic agent is a CD40L antibody, an OX-40 antibody, a CD28 antibody, or a combination thereof.
  • 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.
  • the breast cancer expresses an estrogen receptor with a mutation.
  • the method further comprises administering at least one additional therapeutic agent or modality.
  • said cancer cell comprises increased expression or activation of an oncogene (e.g., MYC) relative to that in a corresponding normal cell.
  • an oncogene e.g., MYC
  • said cancer is a myc dependent cancer.
  • said subject is a mammal, such as a human.
  • said cancer is a human cancer.
  • said immune response is long-term immune response.
  • the present disclosure provides a method of enhancing sensitivity of a cancer to one or more spliceosome targeted therapies (STT).
  • 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.
  • 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).
  • said subject is identified as responsive if said RNA expression profde matches said known mis-spliced RNA expression profde.
  • said subject is identified as non-responsive if said RNA expression profde is different than said known mis-spliced RNA expression profde.
  • 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.
  • 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.
  • said degradation induces expression of one or more mis-spliced RNA in said cancer cell.
  • 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.
  • 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.
  • said inhibiting induces expression of one or more mis-spliced RNA in said cancer cell.
  • said expression of said one or more mis-spliced RNA results in an immune response to said cancer cell, thereby treating said subject.
  • 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.
  • the dsRNA induces JAK/STAT signaling pathway.
  • 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).
  • the dsRNA induces expression of one or more NF-KB transcription factors.
  • the dsRNA induces Jak/Stat signaling pathway.
  • the dsRNA induces protein-kinase R-signaling pathway.
  • the dsRNA induces 2', 5 '-oligoadenylate synthetase (OAS)-RNase L pathway.
  • the one or more mis-spliced RNA induces JAK/STAT signaling pathway.
  • 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).
  • the one or more mis-spliced RNA induces expression of one or more NF-KB transcription factors.
  • 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.
  • OFS 2', 5 '-oligoadenylate synthetase
  • the immune response comprises activation JAK/STAT signaling pathway.
  • 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).
  • the immune response comprises expression of one or more NF-KB transcription factors.
  • the immune response comprises activation of Jak/Stat signaling pathway.
  • the immune response comprises activation of protein-kinase R-signaling pathway.
  • the immune response comprises activation of 2', 5 '-oligoadenylate synthetase (OAS)-RNase L pathway.
  • FIG. 1 shows exemplary enzymatic components of spliceosome and their role in splicing cycle.
  • 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.
  • 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.
  • splicing perturbations e.g., by inhibition or degradation of exemplary enzymatic components e.g., helicases of a spliceosome.
  • FIG. 4 shows fingerprints of inhibition (e.g., selective inhibition) of helicases.
  • 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).
  • 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
  • an immune response e.g., an antiviral immune
  • Perturbations of different exemplary enzymatic components results in a different immune response.
  • 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
  • an immune response e.g., an antiviral immune
  • Perturbations of different exemplary enzymatic components results in a different immune response.
  • FIG. 8 shows perturbation (e.g., inhibition or degradation) of spliceosome RBP-X in cancer cells induced anti-tumor activity.
  • FIG. 9 shows perturbation (e.g., inhibition or degradation) of RBP-X results in durable (i.e., a long term) immune response.
  • RBP-X is an enzymatic component of the spliceosome.
  • FIG. 10A and 10B shows perturbation (e.g., inhibition or degradation) of RBP-X-degradation results in immune memory response to a cancer.
  • 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.
  • 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.
  • 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.
  • 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).
  • FIGs. 12J and 12K show degradation of spliceosome targets results in increased RNA missplicing.
  • 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.
  • FIG. 14A shows degradation of specific spliceosome targets results in differential RNA mis- splicing.
  • 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.
  • 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.
  • 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
  • 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. 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.
  • 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.
  • 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.
  • 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.
  • FIG. 18K and FIG. 18L shows cryptic splice junction usage is increased in both SUM159 and LM2 cells upon DHX15 degradation.
  • 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.
  • FIGs. 19A-19J demonstrate effects of degradation of DHX15.
  • FIG. 19A shows DHX15 degradation induces cryptic splicing in SNAPCI.
  • 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.
  • FIG. 19C shows cryptic splicing of RBM17 is exacerbated by increased DHX15 degradation.
  • 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.
  • gray boxes represent read coverage
  • blue lines represent skipped coverage due to splicing
  • novel read coverage due to cryptic splicing is represented by maroon boxes.
  • 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.
  • 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.
  • FIG. 19G shows DHX15 degradation induces cryptic splicing in SNAPCI.
  • 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.
  • FIG. 19J shows DHX15 degradation induces cryptic splicing
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • FIGs. 21A-21C show effects of degradation of DHX15.
  • FIG. 21A shows intron length.
  • FIG. 2 IB shows cryptic splice sites are distributed throughout intronic space, with modestly increased frequency near the canonical splice junction.
  • Lines represent the kernel distribution estimation plot for SUM159 (blue) and LM2 (green).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. 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.
  • FIGs. 22H, and 221 show U2AF2 binding motif at sites that neither overlap cryptic or
  • 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.
  • 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.
  • 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.
  • FIG. 23A-23K shows Cancer hotspot mutation compromises DHX15 quality control function and results in increased cryptic splice site usage.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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. 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 25C shows increased levels of DHX15 degradation results in dose-dependent increase in DHX15 signature CSJ score.
  • FIG. 25D shows degradation of DHX15 uniquely increases DHX15 signature CSJ score.
  • 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.
  • 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.
  • 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.
  • FIG 251 shows DHX15 signature.
  • FIGs. 26A-26D show deletion of DHX15 allele correlates with increased DHX15 signature CSJ score.
  • 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.
  • FIG. 26B shows loss of DHX15 correlates with increased CSJ score in TCGA BRCA cohort.
  • FIG. 26C shows loss of SUGP1 correlates with increased CSJ score in TCGA BRCA cohort.
  • 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.
  • 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.
  • 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.
  • FIG. 28B is a negative control that shows no effect on cell number in the parental PyMT-M.
  • FIG. 29 shows spliceosome SF3B1 degradation leads to dose-dependent tumor regression.
  • Tumor cells 500,000 cells in 25uL PBS
  • Tumors 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.
  • 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.
  • 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.
  • mice 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • mice 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.
  • 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.
  • 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).
  • 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. 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),
  • 39B shows degradation of targets within the same cluster induces misprocessing of a unique set of introns.
  • RNA helicases e.g., DDX46, and DDX23
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • FIG. 44A shows expression of IL6.
  • FIG. 44B shows expression of IL1B.
  • FIG. 44C shows expression of CD80.
  • FIG. 44D shows expression of OAS1.
  • FIGs. 45A-45E show degradation of DDX46 leads to dose-dependent cell death (dependent on enzymatic activity)
  • FIG. 45A shows dose dependent degradation of DDX46 after dTagl3 treatment in SUM159 cells.
  • 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.
  • 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.
  • FIG. 45D shows DDX46 degradation causes intron retention (DYNCH1). DDX46 degradation results in intron retention.
  • 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.
  • FIGs. 46A-46E show degradation of DDX23 leads to dose-dependent cell death that is dependent on enzymatic activity.
  • FIG. 46A shows dose dependent degradation of DDX23 after dTagl3 treatment in SUM 159 cells.
  • FIG. 46B shows DDX23 degradation impairs SUM159 breast cancer cell growth. Cells were fixed and nuclei were counted 72 hours post-treatment with dTagl3.
  • FIG. 46C shows DDX23 degradation results in apoptotic cell death. Caspase 3/7 activity was measured at the indicated time after dTagl3 treatment.
  • 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.
  • FIG. 46E shows DDX23 enzymatic activity (ATPase) is required to support SUMI 59 breast cancer cell growth.
  • DDX23 enzymatic activity ATPase
  • 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.
  • the term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.
  • 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.
  • 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.
  • 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.
  • 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.
  • feline species e.g., domestic cat
  • 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.
  • 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.
  • 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).
  • 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.
  • improving can encompass a decrease, lessening, or diminishing of an undesirable effect or symptom.
  • the subject is suffering from, is at risk of suffering from a cancer or showing one or more symptoms of a cancer.
  • 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%,
  • “decrease”, “reduce”, “reduction”, “lower” or “lowering,” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount.
  • “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.
  • a 100% decrease e.g., absent level or non-detectable level as compared to a reference level
  • 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.
  • 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.
  • 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.
  • polypeptide 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.
  • a protein can be a portion of the protein, for example, a domain, a subdomain, or a motif of the protein.
  • 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.
  • Methods for detection and/or measurement of polypeptides in biological material 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.
  • RNA in biological material includes, 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.
  • 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.
  • snRNPs small nuclear ribonucleoproteins
  • 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).
  • rRNA ribosomal RNA
  • tRNA transfer RNA
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the RNA helicase is a SF2 RNA helicase.
  • the RNA helicase is a SF1 RNA helicase.
  • the RNA helicase is a SF2 RNA helicase.
  • 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.
  • the RNA helicase is an ATP dependent RNA helicase.
  • the RNA helicase is a DEAD-box helicase.
  • the RNA helicase is a DEAH-box helicase.
  • said enzymatic component e.g., a RNA helicase
  • said enzymatic component 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, e
  • the RNA helicase is selected from the group consisting of DHX8, DHX15, DHX38, DHX8, DHX16, DDX46, DDX23, DDX41, DDX47, AQR, and DDX21.
  • the RNA helicase is DHX15, or DHX38.
  • the RNA helicase is a Ski-21ike helicase.
  • the RNA helicase is Prp5, Sub2, Prp28, Prpl9, Brr2, Prpl6, Prp22, or Prp43.
  • the enzymatic component is a human enzymatic component.
  • 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:
  • 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.
  • 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.
  • the inhibiting of an enzymatic component in a 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.
  • a corresponding control cell is a cell that lacks or is not subject to perturbation (e.g., inhibition or degradation of the enzymatic component).
  • a corresponding control cell is a corresponding healthy cell, e.g., a corresponding non-cancerous cell.
  • 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.
  • 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
  • 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).
  • the inhibiting is transient.
  • the inhibiting is permanent.
  • the inhibiting is constitutive.
  • the inhibiting is inducible.
  • 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.
  • 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.
  • a level e.g., expression level
  • 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.
  • inhibiting of an enzymatic component comprises inhibiting an activity of the enzymatic component relative to that in a corresponding control cell.
  • 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.
  • the inhibiting comprises administering an effective amount of an agent capable of inhibiting the enzymatic component.
  • the inducing degradation comprises administering an effective amount of an agent capable of inducing degradation of the enzymatic component.
  • the agent binds, degrades, or inhibits post-translational modification of said enzymatic component.
  • the agent is a siRNA, an antisense morphlino, an antisense oligonucleotide, a small molecule, or a combination thereof.
  • the agent directly inhibits or degrades the enzymatic component.
  • 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.
  • nucleic acid sequence e.g., DNA or RNA
  • 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.
  • 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.
  • 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.
  • an agent e.g., a small molecule or peptides can interfere with an activity of the enzymatic component.
  • the activity is a catalytic activity.
  • the activity comprises binding and/or interacting with a downstream cofactor, subunit component, another spliceosomal factor, or RNA.
  • an agent prevents the activation or substrate binding of the enzymatic component.
  • an agent e.g., an agent that can inhibit or degrade the enzymatic component includes an antibody, an antibody fragment, or an aptamers.
  • the antibody or an antibody fragment binds an epitope of the enzymatic component.
  • 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).
  • an agent is capable of inhibiting a nucleic acid sequence (e.g., DNA or RNA; e.g., mRNA) that encodes the enzymatic component.
  • a nucleic acid sequence e.g., DNA or RNA; e.g., mRNA
  • 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.
  • the agent comprises an RNA silencing agent e.g., shRNA, siRNA, and miRNAs.
  • the agent is an antisense oligonucleotide e.g., that hybridizes to the RNA that encodes the enzymatic component.
  • the inhibiting comprises modifying a gene that encodes the enzymatic component.
  • the modifying introduces a mutation in the enzymatic component.
  • the mutation comprises an amino acid substitution, deletion and/or insertion.
  • the mutation is a loss of function mutation.
  • the modifying results in a decrease in the activity e.g., enzymatic, or catalytic activity of the enzymatic component.
  • the enzymatic component is DHX15, and the mutation is R222G amino acid substitution in said DHX15 relative to a corresponding wild type DHX15.
  • the enzymatic component is DDX46, and the mutation is D529A and/or D531 A in said DDX46 relative to a corresponding wild type DDX46.
  • the enzymatic component is DDX23, and the mutation is D549A and/or D552A in said DDX23 relative to a corresponding wild type DDX23.
  • 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.
  • 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
  • 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 for detecting sequence alteration 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.
  • 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.
  • the agent can be administered to an organism per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.
  • methods of the disclosure comprise degradation of the enzymatic component polypeptide.
  • the degradation comprises proteasomal degradation.
  • inducing degradation comprises ubiquitination of the enzymatic component.
  • the degradation results in a decrease in the level of the enzymatic component protein relative to that in a corresponding control cell.
  • inducing degradation comprises administering an effective amount of an agent capable of inducing degradation of the enzymatic component protein.
  • the inducing degradation comprises inducing transient degradation.
  • the inducing degradation comprises inducing permanent degradation.
  • the inducing degradation comprises inducing constitutive degradation.
  • the inducing degradation comprises inducing inducible degradation.
  • degradation is induced by a small molecule degrader (such as proteolysis targeting chimera (PROTAC) or molecular glue).
  • proteolysis targeting chimera such as proteolysis targeting chimera (PROTAC) or molecular glue).
  • 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.
  • PMID 36249565
  • PMCID PMC9535385; the contents of each are incorporated herein by reference in its entirety.
  • the methods of the disclosure comprise selectively inhibiting an enzymatic component of a spliceosome in a cancer cell.
  • 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.
  • selectively inhibiting refers to absence of inhibition of the non-selected enzymatic components of a spliceosome.
  • 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.
  • 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).
  • a small molecule degrader such as proteolysis targeting chimera (PROTAC) or molecular glue
  • 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.
  • the selectively inhibited enzymatic component is selected from the group consisting of DDX23, DDX46, DHX15, SF3B1, or a combination thereof.
  • the selectively inhibited enzymatic component is selected from the group consisting of DDX23, DDX46, SF3B1, or a combination thereof.
  • the selectively inhibited enzymatic component is selected from the group consisting of DDX23, DDX46, or a combination thereof.
  • the selectively inhibited enzymatic component is selected from the group consisting of DDX23.
  • the selectively inhibited enzymatic component is selected from the group consisting of DDX46.
  • 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).
  • the methods of the disclosure expresses the one or more mis-spliced RNA relative to the spliced RNA expressed in a corresponding control cell.
  • the expression of a mis-spliced RNA results in an immune response to the cell.
  • the expression of a mis-spliced RNA results in induction of an antiviral immune response.
  • the mis-spliced RNA forms a dsRNA.
  • the mis-spliced RNA encodes one or more neoantigens.
  • one or more mis-spliced RNA are expressed due to intron retention upon inhibition or degradation of the enzymatic component of the spliceosome.
  • the mis- spliced RNA comprises one or more retained introns of a pre-mRNA transcript.
  • 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.
  • 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.
  • “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.
  • 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.
  • 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.
  • the one or more mis-spliced RNA induces JAK/STAT signaling pathway.
  • 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)
  • the one or more mis-spliced RNA induces expression of one or more NF-KB transcription factors.
  • the one or more mis-spliced RNA induces Jak/Stat signaling pathway.
  • the one or more mis-spliced RNA induces protein-kinase R-signaling pathway.
  • the one or more mis-spliced RNA induces 2', 5 '-oligoadenylate synthetase (OAS)-RNase L pathway.
  • the one or more mis-spliced RNA comprises one or more cryptic junction.
  • 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.
  • the mis-spliced RNA transcripts can be detected and measured using deep sequencing, such as ILLUMINA® RNASeq, ILLUMINA® next generation sequencing (NGS), ION TORRENTTM RNA next generation sequencing, 454TM pyrosequencing, or Sequencing by Oligo Ligation Detection (SOLIDTM).
  • 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.
  • a statistical analysis or other analysis is performed on data from the assay utilized to measure an mis-spliced RNA transcript.
  • 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.
  • 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.
  • the stability of one or more RNA transcripts can be determined by Northern blots, RNase protection, or slot blots.
  • the inhibiting of an enzymatic component of a spliceosome in a cell results in an immune response to the cell.
  • the inhibiting of an enzymatic component of a spliceosome in a 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).
  • the degradation of an enzymatic component protein of a spliceosome in a cell results in an immune response to the cell.
  • the degradation of an enzymatic component of a spliceosome in a 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).
  • 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.
  • 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.
  • CTL cytotoxic T lymphocyte
  • 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.
  • an immune response is a T cell response, such as a CD4+ response or a CD8+ response.
  • 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 de
  • the immune response is a memory immune response.
  • 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.
  • 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.
  • the methods of the disclosure induces memory B cells, and/or memory T cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the immune response (e.g., an innate immune response) is an antiviral immune response.
  • the one or more mis-spliced RNA forms a double stranded RNA (dsRNA).
  • the methods of the disclosure comprise inducing one or more misspliced RNA that forms a dsRNA.
  • the dsRNA is located in the cytoplasm of the cancer cell. In some embodiments, the dsRNA induces the immune response.
  • Viral double stranded RNA 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)).
  • 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.
  • 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.
  • the dsRNA induces JAK/STAT signaling pathway.
  • 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).
  • the dsRNA induces expression of one or more NF-KB transcription factors.
  • the dsRNA induces Jak/Stat signaling pathway.
  • the dsRNA induces protein-kinase R-signaling pathway.
  • the dsRNA induces 2', 5 '-oligoadenylate synthetase (OAS)-RNase L pathway.
  • the immune response (e.g., an antiviral immune response) comprises production and/or an increase in level of an interferon and/or complement proteins.
  • the inhibiting and/or degradation induces production and/or an increase in level of an interferon and/or complement proteins.
  • the immune response (e.g., an antiviral immune response) comprises activation of IFN signaling pathway.
  • the inhibiting and/or degradation induces an IFN signaling pathway.
  • the interferon is a type I Interferon (e.g., IFN-a, IFN-P, IFN-a, IFN-K or IFN-co).
  • the interferon is an IFN-a, and/or IFN- .
  • the IFN signaling pathway is an IFN-a signaling pathway, and/or IFN-P signaling pathway.
  • 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.
  • Interferons 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).
  • IFN Interferons
  • 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.
  • 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.
  • the immune response e.g., an antiviral immune response
  • 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.
  • the immune response e.g., an antiviral immune response
  • the inhibiting and/or degradation induces expression (activation) of one or more nuclear factor KB (NF-KB)-responsive genes (e.g., TNF, IL1B).
  • the immune response (e.g., an antiviral immune response) comprises an increased expression and/or activation of a mitochondrial antiviral signaling protein.
  • the inhibiting and/or degradation induces an increased expression and/or activation of a mitochondrial antiviral signaling protein.
  • the immune response (e.g., an antiviral immune response) comprises an activation of a cGas-STING pathway.
  • the inhibiting and/or degradation induces activation cGas-STING pathway.
  • the immune response comprises activation JAK/STAT signaling pathway.
  • 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).
  • the immune response comprises expression of one or more NF-KB transcription factors.
  • the immune response comprises activation of Jak/Stat signaling pathway.
  • the immune response comprises activation of protein-kinase R-signaling pathway.
  • the immune response comprises activation of 2', 5 '-oligoadenylate synthetase (OAS)-RNase L pathway.
  • Methods to determine presence and level of a protein 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.
  • 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.
  • the one or more mis-spliced RNA encodes one or more neoantigen.
  • 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).
  • 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.
  • neoepitope 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.”
  • 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.
  • the immune response comprises an immune response to one or more neoantigen expressed by the cell.
  • the immune response to one or more neoantigen is a cellular immune response.
  • 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.
  • 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.
  • 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.
  • the one or more neoantigen comprises a neoepitope that binds to a HLA protein.
  • the methods of the disclosure further comprises binding of a neoepitope of said one or more neoantigen to a HLA protein.
  • 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 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.
  • the RNA expression profile refers to the expression of total RNA or a select candidate RNA sequences.
  • STT spliceosome targeted therapy
  • 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.
  • 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.
  • provided herein are methods of inducing an immune response in a subject.
  • a method of treating a cancer in a subject comprising inhibiting an enzymatic component of a spliceosome in a cancer cell.
  • the methods of the disclosure comprise inducing degradation of an enzymatic component of a spliceosome in a cancer cell.
  • the methods of the disclosure comprise inhibiting a DDX46, DDX23, DHX15, or a combination thereof in a subject.
  • the inhibiting of DDX46, DDX23, DHX15, or a combination thereof is in a cancer cell in a subject.
  • the methods of the disclosure comprise inducing degradation of a DDX46, DDX23, DHX15, or a combination thereof in a subject.
  • the inducing degradation of DDX46, DDX23, DHX15, or a combination thereof is in a cancer cell in a subject.
  • the methods of the disclosure comprise selectively inhibiting an enzymatic component of a spliceosome in a cell.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • a cell e.g., a cancer cell
  • 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.
  • 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.
  • an immune cell e.g., a T cell
  • an immunomodulatory molecule e.g., inflammatory cytokines, chemokines
  • 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.
  • 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.
  • the expression of MHC-class II, B220 and CD3 proteins by B-cell indicates that the B-cell has been activated.
  • the expression of IL-12, iNOS, Arg-1, or IL-1 proteins by macrophage indicates the macrophage has been activated.
  • 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.
  • 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.
  • 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.
  • Immune cell number and function may be monitored by assays that detect immune cells by an activity such as cytokine production, proliferation, or cytotoxicity.
  • 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.
  • 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.
  • expression kinetics for CD25, flow cytometric detection of intracellular perforin and granzyme B can be performed for memory T cells.
  • CD107/LAMP-1 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.
  • 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).
  • stimulator cells irradiated autologous or HLA matched antigen-presenting cells.
  • [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 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.
  • 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).
  • cytokine production by an ELISA
  • ELISPOT assay enumerating individual cytokine producing immune cells
  • 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.
  • 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.
  • samples e.g., serum or other body fluids
  • ELISPOT ELISA or ELISPOT
  • 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.
  • 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.
  • 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%.
  • 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.
  • 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.
  • 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.
  • 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.
  • the neoplastic tissue is a breast cancer tissue.
  • the neoplastic tissue is a breast tissue with atypical hyperplasia.
  • 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
  • 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 ulcer
  • 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, immuno
  • 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.
  • 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.
  • lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma
  • 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
  • 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).
  • 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.
  • 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.
  • treatment also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).
  • 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.
  • 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.
  • PFS duration of progression free survival
  • RR response rates
  • the therapy may delay the onset of cancer, reduce the severity of one or more symptoms of cancer, or both.
  • time to disease progression is defined as the time from treatment until disease progression or death.
  • the methods disclosed herein alone, or in combination with one or more additional therapeutic agents 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.
  • 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.
  • 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.
  • the methods described herein alleviate a symptom of a cancer.
  • "alleviating a symptom of a cancer” is ameliorating or reducing any condition or symptom associated with the cancer.
  • 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.
  • 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.
  • 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.
  • 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.
  • the methods of the present disclosure further comprises administering one or more additional therapeutic agents (e.g., additional cancer therapeutic agents).
  • additional therapeutic agents e.g., additional cancer therapeutic agents.
  • 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, CD27
  • said one or more additional therapeutic agents is a PD-1 modulator.
  • 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),
  • the one or more additional therapeutic agent is a PD-L1 modulator.
  • 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
  • the one or more additional therapeutic agent is a CTLA4 modulator.
  • the one or more additional therapeutic agent is a CD40L antibody, a OX- 40 antibody, or a CD28 antibody
  • 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.
  • 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 (
  • 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.
  • 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).
  • HDAC histone deacetylase
  • TKIs tyrosine kinase inhibitor
  • PI3K inhibitor
  • 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,
  • the second therapeutic agent can be antibodies currently used for the treatment of cancer.
  • 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.
  • 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.
  • 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-
  • 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.
  • 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.
  • 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.
  • 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.
  • 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;
  • 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.
  • Wilman "Prodrugs in Cancer Chemotherapy” Biochemical Society Transactions, 14, pp. 375-382, 615th Meeting Harbor (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.
  • cytotoxic drugs that can be derivatized into a prodrug form for use herein include, but are not limited to, those chemotherapeutic agents described above.
  • 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 .
  • 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.
  • 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).
  • sgRNA ThermoFisher LentiArray sgRNA library.
  • 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
  • target e.g., an enzymatic component of a spliceosome e.g., a helicase
  • 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.
  • 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
  • target e.g., an enzymatic component of a spliceosome e.g., a helicase
  • mice 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.
  • 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.
  • RNA splicing changes induced by degradation of core spliceosome components U2AF2, DDX46, SF3B1, PRPF8, AQR, DHX16, DHX38, and DHX15 were studied (FIG. 11).
  • 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.
  • 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).
  • 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.
  • DHX15 alone constitutes Cluster 3.
  • Example 5 - DHX15 suppresses 5’ and 3’ cryptic splicing and effects of degradation of DHX15.
  • DHX 15 uniquely leads to increases in all classifications of cryptic splicing (FIG. 18G).
  • 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.
  • 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.
  • 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).
  • Example 6 - DHX15 prevents cryptic splicing at weaker splice sites and effects of degradation of DHX15
  • Branch point prediction BPP
  • BPP Branch point prediction
  • Example 7 - DHX15 prevents cryptic splicing at sites with U2AF deposition but lacking SF3 complex
  • 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).
  • 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.
  • This model suggested that a larger proportion of SF3 complex peaks overlap with canonical splice sites than U2AF1/2.
  • 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.
  • 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).
  • RNAseq RNAseq
  • 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.
  • RNA sequencing 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).
  • 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
  • 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).
  • 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.
  • 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.

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Abstract

La présente invention concerne des perturbations de complexe d'épissage. L'invention concerne des procédés d'induction d'une réponse immunitaire à une cellule cancéreuse. L'invention concerne également des procédés pour traiter un sujet en ayant besoin, par exemple, un sujet souffrant d'un cancer.
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