Selection-based high-throughput screening platform to identify protein degraders

Abstract

The application provides methods for identifying a degrader of a target protein comprising contacting cells with a test compound wherein the cells express an exogenous fusion protein comprising the target protein and an inducible cell suicide protein and wherein the cell suicide protein induces the cells to undergo apoptosis in the presence of a suicide inducer, as well as polynucleotides encoding such fusion protein, recombinant vectors comprising such polynucleotide, and cells comprising such polynucleotide or recombinant vector.

Description

Attorney Docket No: 243735.000473SELECTION-BASED HIGH-THROUGHPUT SCREENING PLATFORM TO IDENTIFY PROTEIN DEGRADERSCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application Nos. 63 / 771,376, filed March 13, 2025, and 63 / 971,281, filed January 29, 2026, the disclosures of which are herein incorporated by reference in their entireties.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML file copy, created on March 3. 2026, is named 243735_000473_SL.xml and is 68,463 bytes in size.FIELD OF THE INVENTION

[0003] The application relates to methods for identifying a degrader of a target protein comprising contacting cells with a test compound wherein the cells express an exogenous fusion protein comprising the target protein and an inducible cell suicide protein and wherein the cell suicide protein induces the cells to undergo apoptosis in the presence of a suicide inducer, as well as polynucleotides encoding said fusion protein, recombinant vectors comprising said polynucleotide, and cells comprising said polynucleotide or said recombinant vector.BACKGROUND

[0004] Over 80% of disease-related pathogenic proteins cannot be targeted by current conventional drug discovery methods. Identification or discovery of small molecule protein degraders from vast chemical space remains a challenging task. While a few phenotypic screening approaches, including both “up” and “down” assays, have been introduced to address this bottleneck issue, the screening approaches often suffer from issues like poor signal-to-noise ratio, compatibility with a particular set of E3 ligases (e.g., Cullin-RING E3 ubiquitin ligases (CRLs)), and end-assay readout requiring long timeframes (e.g., days). Therefore, it becomes1326495150V1Attorney Docket No; 243735.000473difficult to distinguish a degrader’s effectiveness, potentially missing valuable compounds. Hence, there remains a great need for improved screening approaches, in particular large-scale screening platforms and target-centric screening platforms, to accelerate the discovery of protein degraders from large chemical libraries (Hanzl, et al., J. Am. Chem. Soc., 2023, 745(2); Koduri, et al., Sci. Adv., 2021, 7(6)).SUMMARY OF THE INVENTION

[0005] In some aspects, provided herein is a method for identifying a degrader of a target protein, comprising:(a) contacting a population of cells with a test compound in a test cell culture, wherein the population of cells expresses an exogenous fusion protein, wherein the fusion protein comprises the target protein and an inducible cell suicide protein, and wherein the cell suicide protein induces the cells to undergo apoptosis in the presence of a suicide inducer;(b) incubating the test cell culture under conditions and for a time sufficient for degradation of the target protein to occur;(c) adding a suicide inducer to the test cell culture, and incubating the test cell culture under conditions and for a time sufficient for the cells to undergo apoptosis;(d) determining the level of cell viability using a chemical cell viability assay;(e) comparing the level of cell viability determined in step (d) to a control level of cell viability, wherein the control level of cell viability is a predetermined control cell viability level or the level of cell viability determined under the same conditions in a control cell culture; and (f) (i) determining the test compound is a degrader of the target protein when the level of cell viability determined in step (d) is higher than the control level of cell viability, or (ii) determining the test compound is not a degrader of the target protein when the level of cell viability determined in step (d) is not higher than the control level of cell viability.

[0006] In some embodiments, the target protein is positioned at the N-terminus relative to the inducible cell suicide protein.

[0007] In some embodiments, the target protein is positioned at the C-terminus relative to the inducible cell suicide protein.

[0008] In some embodiments, the target protein is fused to the inducible cell suicide protein via a linker.2326495150V1Attorney Docket No: 243735.000473

[0009] In some embodiments, the linker comprises about 5-20 amino acids.

[0010] In some embodiments, the linker comprises about 5-15 amino acids.

[0011] In some embodiments, the linker comprises about 10-15 amino acids.

[0012] In some embodiments, the linker comprises the amino acid sequence of GGSGGGSGGG (SEQ ID NO: 12), GGSGG (SEQ ID NO: 47), SGGGSG (SEQ ID NO: 48), (SGGGS)n (SEQ ID NO: 20), wherein n is 1, 2, or 3. (GGGGS)n (SEQ ID NO: 21), wherein n is 1, 2, or 3, GGGGGG (SEQ ID NO: 16), GGGGGGGG (SEQ ID NO: 17), GSAGSAAGSGEF (SEQ ID NO: 18), or EGKSSGSGSESKST (SEQ ID NO: 19).

[0013] In some embodiments, the inducible cell suicide protein comprises a ligand binding domain fused to a cell suicide domain.

[0014] In some embodiments, the ligand binding domain comprises an FKBP12 polypeptide, an FRB 1 polypeptide, or a variant, fragment, or combination thereof.

[0015] In some embodiments, the ligand binding domain comprises a variant of the FKBP12 polypeptide comprising an F36V substitution.

[0016] In some embodiments, the variant of the FKBP12 polypeptide comprising an F36V substitution comprises the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 3), or an amino acid sequence having at least about 90% sequence identity thereof.

[0017] In some embodiments, the ligand binding domain comprises two tandem copies of the variant of the FKBP12 polypeptide comprising an F36V substitution.

[0018] In some embodiments, the ligand binding domain comprises a variant of the FKBP12 polypeptide comprising G89P and I90K substitutions.

[0019] In some embodiments, the ligand binding domain comprises an FRB1 polypeptide, or a variant or fragment thereof.

[0020] In some embodiments, the ligand binding domain comprises a variant of the FRB1 polypeptide comprising a T2098L substitution.

[0021] In some embodiments, the ligand binding domain comprises a variant of the FRB1 polypeptide comprising a T2098L substitution and a variant of the FKBP12 polypeptide comprising G89P and I90K substitutions.3326495150V1Attorney Docket No: 243735.000473

[0022] In some embodiments, the variant of the FKBP12 polypeptide comprising G89P and I90K substitutions comprises the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPPKIPPHATLVFDVELLKLE (SEQ ID NO: 49), or an amino acid sequence having at least about 90% sequence identity thereof.

[0023] In some embodiments, the variant of the FRB1 polypeptide comprising a T2098L substitution comprises the amino acid sequence of EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEA QEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK (SEQ ID NO: 50), or an amino acid sequence having at least about 90% sequence identity thereof.

[0024] In some embodiments, the cell suicide domain comprises a caspase-9 polypeptide, a caspase-8 polypeptide, a caspase- 1 polypeptide, a caspase-3 polypeptide, a FADD polypeptide, a Fas polypeptide, or a mBax polypeptide, or a variant, fragment, or combination thereof.

[0025] In some embodiments, the cell suicide domain comprises a variant of the caspase-9 polypeptide comprising Q221R substitution.

[0026] In some embodiments, the variant of the caspase-9 polypeptide comprising Q221R substitution comprises the amino acid sequence of GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRF SSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVY GTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNP EPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQ WAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 2), or an amino acid sequence having at least about 90% sequence identity thereof.

[0027] In some embodiments, the cell suicide domain comprises a variant of the caspase-8 polypeptide comprising amino acids 216-479 of a wild-type caspase-8 protein.

[0028] In some embodiments, the variant of the caspase-8 polypeptide comprising amino acids 216-479 of a wild-type caspase-8 protein comprises the amino acid sequence of SESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKLHSIRDRNGTHLDAGALTTTFE ELHFEIKPHDDCTVEQIYEILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPIYELT SQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSEEQPYLEMDLSSPQTRYIPDEA DFLLGMATVNNCVSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNYEVSNKDDK4326495150vlAttorney Docket No: 243735.000473KNMGKQMPQPTFTLRKKLVFPSD (SEQ ID NO: 51), or an amino acid sequence having at least about 90% sequence identity thereof.

[0029] In some embodiments, the caspase- 1 polypeptide comprises the amino acid sequence of MADKVLKEKRKLFIRSMGEGTINGLLDELLQTRVLNKEEMEKVKRENATVMDKTRALI DSVIPKGAQACQICITYICEEDSYLAGTLGLSADQTSGNYLNMQDSQGVLSSFPAPQAVQ DNPAMPTSSGSEGNVKLCSLEEAQRIWKQKSAEIYPIMDKSSRTRLALIICNEEFDSIPRR TGAEVDITGMTMLLQNLGYSVDVKKNLTASDMTTELEAFAHRPEHKTSDSTFLVFMSH GIREGICGKKHSEQVPDILQLNAIFNMLNTKNCPSLKDKPKVIIIQACRGDSPGVVWFKD SVGVSGNLSLPTTEEFEDDAIKKAHIEKDFIAFCSSTPDNVSWRHPTMGSVFIGRLIEHMQ EYACSCDVEEIFRKVRFSFEQPDGRAQMPTTERVTLTRCFYLFPGH (SEQ ID NO: 52), or an amino acid sequence having at least about 90% sequence identity thereof.

[0030] In some embodiments, the caspase-3 polypeptide comprises the amino acid sequence of MENTENSVDSKSIKNLEPKIIHGSESMDSGISLDNSYKMDYPEMGLCIIINNKNFHKSTG MTSRSGTDVDAANLRETFRNLKYEVRNKNDLTREEIVELMRDVSKEDHSKRSSFVCVL LSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLTGKPKLFIIQACRGTELDCGIETDSGVD DDMACHKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCAMLKQYADKLEFMHILT RVNRKVATEFESFSFDATFHAKKQIPCIVSMLTKELYFYH (SEQ ID NO: 53), or an amino acid sequence having at least about 90% sequence identity thereof.

[0031] In some embodiments, the cell suicide domain comprises a variant of the FADD polypeptide comprising amino acids 1-125 of a wild-type FADD protein.

[0032] In some embodiments, the variant of the FADD polypeptide comprising amino acids 1-125 of a wild-type FADD protein comprises the amino acid sequence of MDPFLVLLHSVSSSLSSSELTELKFLCLGRVGKRKLERVQSGLDLFSMLLEQNDLEPGHT ELLRELLASLRRHDLLRRVDDFEAGAAAGAAPGEEDLCAAFNVICDNVGKDWRRLARQ LKVSDTK (SEQ ID NO: 54), or an amino acid sequence having at least about 90% sequence identity thereof.

[0033] In some embodiments, the cell suicide domain comprises a variant of the Fas polypeptide comprising amino acids 175-304 of a wild-type Fas protein.

[0034] In some embodiments, the variant of the Fas polypeptide comprising amino acids 175-304 of a wild-type Fas protein comprises the amino acid sequence of IHSGQCPAAIVGGINVLLKPNTSVQFLRLGMLSPEGTCKAFDTAGNGYCRSEGVVAVLL5326495150vlAttorney Docket No: 243735.000473TKKSLARRVYATILNAGTNTDGFKEQGVTFPSGDIQEQLIRSLYQSAGVAPESFEYIEAH GTGTKVGDPQE (SEQ ID NO: 55), or an amino acid sequence having at least about 90% sequence identity thereof.

[0035] In some embodiments, the mBax polypeptide comprises the amino acid sequence of MDGSGEQLGSGGPTSSEQIMKTGAFLLQGFIQDRAGRMAGETPELTLEQPPQDASTKKL SECLRRIGDELDSNMELQRMIADVDTDSPREVFFRVAADMFADGNFNWGRVVALFYFA SKLVLKALCTKVPELIRTIMGWTLDFLRERLLVWIQDQGGWEGLLSYFGTPTWQTVTIF VAGVLTASLTIWKKMG (SEQ ID NO: 56). or an amino acid sequence having at least about 90% sequence identity thereof.

[0036] In some embodiments, the ligand binding domain is fused to the cell suicide domain via a linker.

[0037] In some embodiments, the linker comprises the amino acid sequence of GGSGGGSGGG (SEQ ID NO: 12), GGSGG (SEQ ID NO: 47), SGGGSG (SEQ ID NO: 48), (SGGGS)n (SEQ ID NO: 20), wherein n is 1, 2. or 3. (GGGGS)n (SEQ ID NO: 21). wherein n is 1, 2, or 3, GGGGGG (SEQ ID NO: 16), GGGGGGGG (SEQ ID NO: 17), GSAGSAAGSGEF (SEQ ID NO: 18), or EGKSSGSGSESKST (SEQ ID NO: 19). In one embodiment, the linker comprises the amino acid sequence of SGGGS (SEQ ID NO: 9).

[0038] In some embodiments, the inducible cell suicide protein further comprises an HA tag at the C-terminus.

[0039] In some embodiments, the HA tag comprises the amino acid sequence of YPYDVPDYALD (SEQ ID NO: 6).

[0040] In some embodiments, the inducible cell suicide protein comprise the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGF GDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSS LHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGT DGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEP DATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWA HSEDLQSLLLRV AN AVSVKGIYKQMPGCFNFLRKKLFFKTSVD YPYDVPDYALD (SEQ ID NO: 7), or an amino acid sequence having at least about 90% sequence identity thereof.6326495150V1Attorney Docket No: 243735.000473

[0041] In some embodiments, the suicide inducer is AP20187, AP1903, FK1012, AP1510, or AP22783.

[0042] In some embodiments, the chemical cell viability assay is a luminescence-based cell viability assay.

[0043] In some embodiments, the luminescence-based cell viability assay measures the number of viable cells by quantifying adenosine triphosphate (ATP).

[0044] In some embodiments, the luminescence-based cell viability assay comprises a one-step reaction.

[0045] In some embodiments, the one-step reaction comprises addition of a recombinant luciferase reaction mix.

[0046] In some embodiments, the recombinant luciferase reaction mix comprises a recombinant luciferase, luciferin substrate, one or more components to lyse cell membranes, and one or more components to inhibit endogenous ATPases.

[0047] In some embodiments, the one or more components to lyse cell membranes comprises TCA (trichloroacetic acid), DMSA (dimercaptosuccinic acid), or CTAB (cetyltrimethylammonium bromide) .

[0048] In some embodiments, the one or more components to inhibit endogenous ATPases comprises NaF.

[0049] In some embodiments, the chemical cell viability assay is an absorbance cell viability assay.

[0050] In some embodiments, the absorbance cell viability assay is a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay or a MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay.

[0051] In some embodiments, the chemical cell viability assay is a fluorescence cell viability assay.

[0052] In some embodiments, the fluorescence cell viability assay is a resazurin assay.

[0053] In some embodiments, the control cell culture comprises a population of cells expressing the inducible cell suicide protein which is not fused to the target protein or which is fused to a protein that is not the target protein, and the control cell culture is subjected to the same test compound treatment.7326495150V1Attorney Docket No: 243735.000473

[0054] In some embodiments, the control cell culture comprises a population of cells expressing the same exogenous fusion protein as the test cell culture, but the control cell culture is not subjected to the test compound treatment.

[0055] In some embodiments, in step (b), the test compound is incubated with the population of cells for about 3-4 hours.

[0056] In some embodiments, in step (c), the test cell culture is incubated with the suicide inducer for about 12-16 hours.

[0057] In some embodiments, the total time for conducting the method is about 15-20 hours.

[0058] In some embodiments, the degrader is a small molecule, a nucleic acid molecule, a peptide, or a protein.

[0059] In some embodiments, the degrader is an exogenous degrader or an endogenous degrader.

[0060] In some embodiments, the exogenous degrader is a monovalent degrader, bifunctional degrader, or a destabilizer.

[0061] In some embodiments, the monovalent degrader is a molecular glue.

[0062] In some embodiments, the bifunctional degrader is a proteolysis targeting chimera (PROTAC).

[0063] In some embodiments, the degrader comprises a library of compounds.

[0064] In some embodiments, the target protein is a proto-oncoprotein.

[0065] In some embodiments, the target protein is selected from Ikaros ZF23, Kras, Skpl, Cyclin DI, Skp2, cMyc, Ringol, FAM96B, PDCD1, SEC61A1, HMGCS1, ANAPC10, IRF4, CCND2, N-myc, SOX10. SEH1L, TBCB. KLF4. CEBPA. RPP25L, POU2AF1. Timl7-A, SMARCB1, S0CS3, CDC26, FOXA1, LRR1, FAU, GATA3, SYF2, SOX2, and mutants or fragments thereof.

[0066] In some embodiments, the target protein comprises the amino acid sequence of Ikaros ZF23 (SEQ ID NO: 8), Kras (SEQ ID NO: 57), Skpl (SEQ ID NO: 58), Cyclin DI (SEQ ID NO: 59), Skp2 (SEQ ID NO: 60), cMyc (SEQ ID NO: 61), Ringol (SEQ ID NO: 62), FAM96B (SEQ ID NO: 22), PDCD1 (SEQ ID NO: 23), SEC61A1 (SEQ ID NO: 24), HMGCS1 (SEQ ID NO: 25), ANAPC10 (SEQ ID NO: 26), IRF4 (SEQ ID NO: 27), CCND2 (SEQ ID NO: 28), N-myc (SEQ ID NO: 29), SOX10 (SEQ ID NO: 30), SEH1L (SEQ ID NO: 31), TBCB (SEQ ID NO: 32), KLF4 (SEQ ID NO: 33), CEBPA (SEQ ID NO: 34), RPP25L (SEQ ID NO: 35), POU2AF18326495150vlAttorney Docket No: 243735.000473(SEQ TD NO: 36), Timl7-A (SEQ ID NO: 37), SMARCB1 (SEQ ID NO: 38), SOCS3 (SEQ ID NO: 39), CDC26 (SEQ ID NO: 40), FOXA1 (SEQ ID NO: 41), LRR1 (SEQ ID NO: 42), FAU (SEQ ID NO: 43), GATA3 (SEQ ID NO: 44). SYF2 (SEQ ID NO: 45). or SOX2 (SEQ ID NO: 46).

[0067] In some embodiments, the target protein is a Kras (G12C) mutant.

[0068] In some embodiments, the exogenous fusion protein is expressed from a transgene integrated into the chromosome of the cells.

[0069] In some embodiments, the population of cells comprises mammalian cells.

[0070] In some embodiments, the population of cells comprises adherent cells and / or suspension cells.

[0071] In some embodiments, the adherent cells comprise HEK293, CHO, MCF7. or HeLa cells.

[0072] In some embodiments, the suspension cells comprise Jurkat, Expi293F, K562 cells, or HEK293 suspension-adapted cells.

[0073] In some embodiments, the method is conducted in a high-throughput format.

[0074] In some embodiments, the test compound represents a library of compounds.

[0075] In a further aspect, provided herein is a polynucleotide encoding a fusion protein comprising a target protein and an inducible cell suicide protein, wherein when the fusion protein is expressed in a cell, the cell suicide protein induces the cell to undergo apoptosis in the presence of a suicide inducer.

[0076] In some embodiments, the fusion protein is expressed from a transgene integrated into the chromosome of the cell.

[0077] In some embodiments, the target protein is positioned at the N-terminus relative to the inducible cell suicide protein.

[0078] In some embodiments, the target protein is positioned at the C-terminus relative to the inducible cell suicide protein.

[0079] In some embodiments, the target protein is fused to the inducible cell suicide protein via a linker.

[0080] In some embodiments, the linker comprises about 5-20 amino acids.

[0081] In some embodiments, the linker comprises about 5-15 amino acids.

[0082] In some embodiments, the linker comprises about 10-15 amino acids.9326495150V1Attorney Docket No: 243735.000473

[0083] In some embodiments, the linker comprises the amino acid sequence of GGSGGGSGGG (SEQ ID NO: 12), GGSGG (SEQ ID NO: 47), SGGGSG (SEQ ID NO: 48), (SGGGS)n (SEQ ID NO: 20), wherein n is 1, 2. or 3. (GGGGS)n (SEQ ID NO: 21). wherein n is 1, 2, or 3, GGGGGG (SEQ ID NO: 16), GGGGGGGG (SEQ ID NO: 17), GSAGSAAGSGEF (SEQ ID NO: 18), or EGKSSGSGSESKST (SEQ ID NO: 19).

[0084] In some embodiments, the inducible cell suicide protein comprises a ligand binding domain fused to a cell suicide domain.

[0085] In some embodiments, the ligand binding domain comprises an FKBP12 polypeptide, an FRB 1 polypeptide, or a variant, fragment, or combination thereof.

[0086] In some embodiments, the ligand binding domain comprises a variant of the FKBP12 polypeptide comprising an F36V substitution.

[0087] In some embodiments, the variant of the FKBP12 polypeptide comprising an F36V substitution comprises the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 3), or an amino acid sequence having at least about 90% sequence identity thereof.

[0088] In some embodiments, the ligand binding domain comprises two tandem copies of the variant of the FKBP12 polypeptide comprising an F36V substitution.

[0089] In some embodiments, the ligand binding domain comprises a variant of the FKBP12 polypeptide comprising G89P and I90K substitutions.

[0090] In some embodiments, the ligand binding domain comprises an FRB1 polypeptide, or a variant or fragment thereof.

[0091] In some embodiments, the ligand binding domain comprises a variant of the FRB1 polypeptide comprising a T2098L substitution.

[0092] In some embodiments, the ligand binding domain comprises a variant of the FRB1 polypeptide comprising a T2098L substitution and a variant of the FKBP12 polypeptide comprising G89P and KOK substitutions.

[0093] In some embodiments, the variant of the FKBP12 polypeptide comprising G89P and I90K substitutions comprises the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRG10326495150vlAttorney Docket No: 243735.000473WEEGVAQMSVGQRAKLTISPDYAYGATGHPPKIPPHATLVFDVELLKLE (SEQ ID NO: 49), or an amino acid sequence having at least about 90% sequence identity thereof.

[0094] In some embodiments, the variant of the FRB 1 polypeptide comprising a T2098L substitution comprises the amino acid sequence of EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEA QEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK (SEQ ID NO: 50), or an amino acid sequence having at least about 90% sequence identity thereof.

[0095] In some embodiments, the cell suicide domain comprises a caspase-9 polypeptide, a caspase-8 polypeptide, a caspase- 1 polypeptide, a caspase-3 polypeptide, a FADD polypeptide, a Fas polypeptide, or a mBax polypeptide, or a variant, fragment, or combination thereof.

[0096] In some embodiments, the cell suicide domain comprises a variant of the caspase-9 polypeptide comprising Q221R substitution.

[0097] In some embodiments, the variant of the caspase-9 polypeptide comprising Q221R substitution comprises the amino acid sequence of GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRF SSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVY GTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNP EPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQ WAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 2), or an amino acid sequence having at least about 90% sequence identity thereof.

[0098] In some embodiments, the cell suicide domain comprises a variant of the caspase-8 polypeptide comprising amino acids 216-479 of a wild-type caspase-8 protein.

[0099] In some embodiments, the variant of the caspase-8 polypeptide comprising amino acids 216-479 of a wild-type caspase-8 protein comprises the amino acid sequence of SESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKLHSIRDRNGTHLDAGALTTTFE ELHFEIKPHDDCTVEQIYEILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPIYELT SQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSEEQPYLEMDLSSPQTRYIPDEA DFLLGMATVNNCVSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNYEVSNKDDK KNMGKQMPQPTFTLRKKLVFPSD (SEQ ID NO: 51), or an amino acid sequence having at least about 90% sequence identity thereof.11326495150V1Attorney Docket No: 243735.000473

[0100] In some embodiments, the caspase- 1 polypeptide comprises the amino acid sequence of MADKVLKEKRKLFIRSMGEGTINGLLDELLQTRVLNKEEMEKVKRENATVMDKTRALI DSVIPKGAQACQICITYICEEDSYLAGTLGLSADQTSGNYLNMQDSQGVLSSFPAPQAVQ DNPAMPTSSGSEGNVKLCSLEEAQRIWKQKSAEIYPIMDKSSRTRLALIICNEEFDSIPRR TGAEVDITGMTMLLQNLGYSVDVKKNLTASDMTTELEAFAHRPEHKTSDSTFLVFMSH GIREGICGKKHSEQVPDILQLNAIFNMLNTKNCPSLKDKPKVIIIQACRGDSPGVVWFKD SVGVSGNLSLPTTEEFEDDAIKKAHIEKDFIAFCSSTPDNVSWRHPTMGSVFIGRLIEHMQ EYACSCDVEEIFRKVRFSFEQPDGRAQMPTTERVTLTRCFYLFPGH (SEQ ID NO: 52), or an amino acid sequence having at least about 90% sequence identity thereof.

[0101] In some embodiments, the caspase-3 polypeptide comprises the amino acid sequence of MENTENSVDSKSIKNLEPKIIHGSESMDSGISLDNSYKMDYPEMGLCIIINNKNFHKSTG MTSRSGTDVDAANLRETFRNLKYEVRNKNDLTREEIVELMRDVSKEDHSKRSSFVCVL LSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLTGKPKLFIIQACRGTELDCGIETDSGVD DDMACHKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCAMLKQYADKLEFMHILT RVNRKVATEFESFSFDATFHAKKQIPCIVSMLTKELYFYH (SEQ ID NO: 53), or an amino acid sequence having at least about 90% sequence identity thereof.

[0102] In some embodiments, the cell suicide domain comprises a variant of the FADD polypeptide comprising amino acids 1-125 of a wild- type FADD protein.

[0103] In some embodiments, the variant of the FADD polypeptide comprising amino acids 1-125 of a wild-type FADD protein comprises the amino acid sequence of MDPFLVLLHSVSSSLSSSELTELKFLCLGRVGKRKLERVQSGLDLFSMLLEQNDLEPGHT ELLRELLASLRRHDLLRRVDDFEAGAAAGAAPGEEDLCAAFNVICDNVGKDWRRLARQ LKVSDTK (SEQ ID NO: 54), or an amino acid sequence having at least about 90% sequence identity thereof.

[0104] In some embodiments, the cell suicide domain comprises a variant of the Fas polypeptide comprising amino acids 175-304 of a wild-type Fas protein.

[0105] In some embodiments, the variant of the Fas polypeptide comprising amino acids 175-304 of a wild-type Fas protein comprises the amino acid sequence of IHSGQCPAAIVGGINVLLKPNTSVQFLRLGMLSPEGTCKAFDTAGNGYCRSEGVVAVLL TKKSLARRVYATILNAGTNTDGFKEQGVTFPSGDIQEQLIRSLYQSAGVAPESFEYIEAH12326495150vlAttorney Docket No: 243735.000473GTGTKVGDPQE (SEQ ID NO: 55), or an amino acid sequence having at least about 90% sequence identity thereof.

[0106] In some embodiments, the mBax polypeptide comprises the amino acid sequence of MDGSGEQLGSGGPTSSEQIMKTGAFLLQGFIQDRAGRMAGETPELTLEQPPQDASTKKL SECLRRIGDELDSNMELQRMIADVDTDSPREVFFRVAADMFADGNFNWGRVVALFYFA S KLVLKALCTKVPELIRTIMGWTLDFLRERLLVWIQDQGGWEGLLS YFGTPTWQTVTIF VAGVLTASETIWKKMG (SEQ ID NO: 56), or an amino acid sequence having at least about 90% sequence identity thereof.

[0107] In some embodiments, the ligand binding domain fused to a cell suicide domain via a linker.

[0108] In some embodiments, the linker comprises the amino acid sequence of GGSGGGSGGG (SEQ ID NO: 12), GGSGG (SEQ ID NO: 47), SGGGSG (SEQ ID NO: 48), (SGGGS)n (SEQ ID NO: 20), wherein n is 1, 2, or 3, (GGGGS)n (SEQ ID NO: 21), wherein n is 1, 2, or 3. GGGGGG (SEQ ID NO: 16), GGGGGGGG (SEQ ID NO: 17), GSAGSAAGSGEF (SEQ ID NO: 18), or EGKSSGSGSESKST (SEQ ID NO: 19). In one embodiment, the linker comprises the amino acid sequence of SGGGS (SEQ ID NO: 9).

[0109] In some embodiments, the inducible cell suicide protein further comprises an HA tag at the C-terminus.

[0110] In some embodiments, the HA tag comprises the amino acid sequence of YPYDVPDYALD (SEQ ID NO: 6).

[0111] In some embodiments, the inducible cell suicide protein comprise the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGF GDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSS LHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGT DGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEP DATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWA HSEDLQSLLLRV AN AVSVKGIYKQMPGCFNFLRKKLFFKTSVD YPYDVPDYALD (SEQ ID NO: 7), or an amino acid sequence having at least about 90% sequence identity thereof.13326495150V1Attorney Docket No: 243735.000473

[0112] In an additional aspect, provided herein is a recombinant vector comprising the polynucleotide as described herein.

[0113] In some embodiments, the vector is a viral vector.

[0114] In some embodiments, the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, a baculoviral vector, or a vaccinia virus vector.

[0115] In some embodiments, the vector is a non-viral vector.

[0116] In some embodiments, the non-viral vector is a minicircle plasmid, a Sleeping Beauty transposon, a PiggyBac transposon, or a single or double stranded DNA molecule that is used as a template for homology directed repair (HDR)-based gene editing.

[0117] In a further aspect, provided herein is a cell comprising the polynucleotide as described herein or the recombinant vector as described herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figures 1A-1C show schematics of methods to target disease-related proteins. Figure 1A shows a schematic of druggable proteins of interest (POI) that may be targeted with traditional drugs and undruggable POIs that cannot be targeted by traditional drugs. Figure IB shows a schematic of targeted protein degradation (TPD) technology employing monovalent (e.g., molecular glues (MGs)) or bifunctional (e.g., Proteolysis Targeting Chimeras (PROTACs)) small molecule degraders that hijack the cell’s degradation machinery to enable immediate loss of the protein of interest (POI). Figure 1C shows a schematic of plant hormone and synthetic molecule examples that act as molecular glues.

[0119] Figures 2A-2B show the concept behind the inducible caspase-9 (iCasp9) suicidal gene system. Figure 2A shows the composition of the iCasp9 gene system. The iCasp9 system consists of a fusion construct designed for precise regulation of apoptotic signaling. The construct includes the catalytic domain of human caspase-9 (residues 135-416), referred to as ACaspase9, which lacks its physiological dimerization CARD domain (caspase activation and recruitment domain). ACaspase9 is fused to a dimerizer domain, a variant of the FKBP12 polypeptide comprising an F36V substitution (also known as the DmrB or dTAG domain), through a flexible SGGGS (SEQ ID NO: 9) linker. The absence of the CARD domain in ACaspase9 prevents its dimerization under physiological conditions, thereby inhibiting14326495150V1Attorney Docket No: 243735.000473downstream apoptotic signaling. The iCasp9 system allows for controlled activation of caspase-9 upon the addition of a specific dimerizing ligand, enabling targeted induction of apoptosis.Figure 2B shows the iCasp9 gene system as a death switch or suicide switch. The system functions as a highly effective death or suicide switch, designed to induce controlled apoptosis. The system can be retrovirally transduced into cells to enable stable expression. The inclusion of the FKBP12 F36V domain allows the system to be activated by synthetic chemical inducers of dimerization (CIDs), such as AP20187 (CAS No. 195514-80-8) or AP1903 (Rimiducid). Figure discloses “SGGGS” as SEQ ID NO: 9.

[0120] Figures 3A-3D show the rationale for the iCADD platform. Figure 3A shows the composition of the iCADD system. The system comprises a mutant form caspase-9 (iCasp9M) suicide gene fused to a target protein of interest (POI) via a flexible SGGGS (SEQ ID NO: 9) linker. This fusion construct can be retrovirally transduced into cells for stable expression. The inclusion of the FKBP12 F36V domain enables dimerization upon the addition of synthetic CIDs. such as AP20187 or AP1903 (Rimiducid). This dimerization activates the apoptotic cascade, leading to the cleavage of effector caspase-3 and caspase-7 and ultimately resulting in cell death. Figure 3B shows the working principle of the iCADD system. It is envisioned that if a chemical compound (e.g., molecular glue, PROTAC, or a destabilizer) induces degradation of the target POI, the fused iCasp9M protein would be co-degraded. This co-degradation would impair or abolish the apoptotic signaling typically initiated by iCasp9M upon API 903 or AP20187 treatment, thereby resulting in a survival phenotype. A compound capable of reversing cell death in this manner would be identified as a candidate degrader of the target POI. Figure 3C shows the possibility that a chemical compound might interfere with the transcription or translation of the POI-iCasp9M fusion construct, preventing its synthesis. This interference would similarly diminish AP1903- or AP20187-induced apoptosis and result in a survival phenotype. Such a compound could indicate disruption of POI-iCasp9M expression rather than degradation of the POI itself. Figure 3D shows a schematic comparing the signals in an “up assay” (marked with a check) versus a “down assay” (marked with an “X”).

[0121] Figures 4A-4D show the iCADD construct design and initial testing. Figure 4A shows amplification of the FKBP12 F36V-ACaspase9M (iCasp9M)-HA fusion protein and cloning of the fusion protein along with various target POIs, including Ikaros ZF23, Kras, Skpl, Cyclin DI, Skp2, and cMyc, into the retroviral vector MSCV-puro. The POIs were positioned at the N-15326495150V1Attorney Docket No: 243735.000473terminus of iCasp9M, separated by an SGGGS (SEQ ID NO: 9) linker. These fusion constructs were transfected into HEK293T cells. Then, the virus-containing supernatant was collected and used to transduce HeLa cells. The transduced cells were then selected using puromycin. Stable cell lines expressing the desired POI-iCasp9M fusion proteins were confirmed via western blotting. Additionally, HeLa stable cell lines expressing either iCasp9M alone or an empty vector (EV) were generated as controls. Figure 4B shows initial testing of HeLa stable cell lines expressing either target POLiCasp9M-HA or iCasp9M / empty vector (EV) as controls that was performed in a 6-well plate format. For each stable cell line, 200,000 cells per well were seeded and treated with either DMSO or 10 nM AP1903 over time intervals ranging from 0 to 24 hours. Images were captured using the EVOS cell imaging system. Figure 4C shows initial testing of HeLa stable cell lines expressing either target POI-iCasp9M-HA or iCasp9M / empty vector (EV) as controls that was conducted in a 96-well plate format. Cells were treated with either DMSO or 10 nM API 903 at various time intervals ranging from 0 to 24 hours. After treatment, CellTiter-Glo 2.0 reagent was added to each well. Figure 4D shows increasing the linker length between the target POI and the iCasp9M-HA fusion constructs improved the killing efficiency of AP1903. The linker length was extended between the target POI and iCasp9M-HA genes by two (L2)- or three (L3)-fold, particularly for large molecular weight proteins such as Skp2 and cMyc. The longer linker markedly enhanced the killing efficiency of API 903 in the 96-well plate format, following the same experimental protocol described in Figure 4C.

[0122] Figures 5A-5C show the characterization of iCADD by using knockdown and cellular degradation experiments. Figure 5A shows genetic knockdown experiments in HeLa-KrasWT-iCasp9M-HA and HeLa-iCasp9M-HA (control) cells. Each cell line was seeded at 35.000 cells per well in a 96-well microplate. Cells were transfected with either specific (si-Kras), nonspecific (si-Skpl), or control (si-NT) siRNAs and incubated for 48 hours. Media was then replaced with fresh media containing either DMSO or AP1903, followed by a 24-hour incubation. Subsequently, CellTiter-Glo 2.0 reagent was added to each well to create a luminescent signal that was measured. Pretreatment with si-Kras in HeLa-KrasWT-iCasp9M-HA cells rescued AP1903-induced cell death, while pretreatment with si-Skpl had no effect. In HeLa-iCasp9M-HA control cells, neither si-Kras nor si-Skpl pretreatment rescued AP1903-induced cell death. siRNA knockdown efficiency was confirmed through western blot analysis of cellular lysates. Figure 5B shows a rescue experiment conducted using Hela-Ikaros-ZF23-16326495150V1Attorney Docket No: 243735.000473iCasp9M-HA cells, with Hela-iCasp9M-HA cells serving as a control. For this experiment, 35,000 cells were seeded per well in a 96-well microplate. Cells were treated with increasing concentrations of Ikaros molecular-glue degraders (Iberdomide (Iberdo.). Pomalidomide (Poma.), and Lenalidomide (Lena.)) or the translational inhibitor cycloheximide (CHX) for 6 hours. The media was then replaced with fresh media containing either DMSO or AP1903, and cells were incubated for an additional 12 hours. CellTiter-Glo 2.0 was then added to each well to create a luminescent signal that was then measured. Pretreatment of Hela-Ikaros-ZF23-iCasp9M-HA cells with Ikaros molecular glues rescued AP1903-induced cell death in a dose-dependent manner, resulting in a survival phenotype. The iCADD assay effectively distinguished the potency differences among the degraders (e.g., Iberdomide as the most potent Ikaros degrader exhibited better rescue effects at 0.1 pM, 1 pM, and 5 pM compared to Pomalidomide and Lenalidomide, which are less potent degraders). Similarly, CHX also rescued AP1903-induced cell death. No rescue effect was observed in Hela-iCasp9M-HA cells, which lack the target POI (Ikaros ZF23), upon pretreatment with Ikaros degraders, even at concentrations up to 10 pM. Parallel western blot analyses confirmed that Ikaros degraders (Iberdomide and Pomalidomide) induced dose-dependent degradation of Ikaros-ZF23-iCasp9M-HA within 6 hours in Hela cells.Figure 5C shows the same rescue experiment as described in Figure 5B using the PROTAC degrader LC-2. This experiment employed Hela-Kras-G72C-iCasp9M-H A and Hela-KrasWT-iCasp9M-HA cell lines as positive and negative controls, respectively. LC-2 is a specific degrader of the Kras (G72C) mutant. Pretreatment with LC-2 dose-dependently rescued AP1903-induced cell death in Hela-Kras-GT2C-iCasp9M-HA cells. In contrast, no rescue effect was observed in Hela-KrasWT-iCasp9M-HA cells, which served as the negative control. Consistently, western blot analysis confirmed that LC-2 induced Kras degradation only in Hela-Kras-G72C-iCasp9M-HA cells, with no detectable effect in Hela-KrasWT-iCasp9M-HA cells.

[0123] Figures 6A-6C show small-scale high-throughput test screens to validate the iCADD platform. Figure 6A shows a small-scale test screen conducted in a 384- well plate format. For this study, an FDA-approved compound library was used, comprising -3,800 compounds including known Ikaros degraders. The iCADD system was adapted to K562 suspension cells, and a stably expressing K562-Ikaros-ZF23-iCasp9M-HA cell line was successfully established. Cells were seeded at a density of 1,000 cells per well. Subsequently, media containing each compound was added to achieve a final concentration of 1 pM or 10 pM. After a 3-hour17326495150V1Attorney Docket No: 243735.000473incubation, API 903 was added to reach a final concentration of 20 nM. Following an overnight incubation, CellTiter-Glo 2.0 was added per well to induce cell lysis and luminescence was measured at 1-hour and 3-hour intervals. The data were analyzed as the percentage inhibition of AP1903-induced activity. Figure 6B shows the data collected from the test screen conducted in Figure 6A. Assay measurement 1 hour after cell lysis using Promega CellTiter-Glo 2.0 revealed significant findings. At a 1 pM concentration, the screen identified CC-92480 / Mezigdomide as the second top hit, causing >60% inhibition of AP1903 activity (left panel). Additionally, Iberdomide and Pomalidomide were identified, each causing 41% inhibition at the same concentration. At 10 pM, the screen again highlighted Iberdomide, CC-92480 / Mezigdomide, and Pomalidomide among the top hits, all causing >60% inhibition of AP1903 activity (right panel). Furthermore, Avadomide was identified, causing 57% inhibition. The screen also identified procaspase inhibitors (e.g., Emricasan and Q-VD-Oph), FKBP12 binders (e.g., Rapamycin), and compounds that disrupt transcription or translation. Figure 6C shows the numbers of identified specific hits in additional small-scale screens comprising cMyc, Cyclin DI, and Kras as the target POIs using a threshold of at least 60% inhibition of cell death.DETAILED DESCRIPTION

[0124] The human proteome comprises about 20,000 proteins of which about 500 are druggable proteins out of over 3,500 disease-related proteins. The druggable proteins may be targeted with traditional drugs, however, disease-related proteins that are considered undruggable cannot be targeted by traditional drags (Figure 1A).

[0125] Investing in new technologies and adopting innovative approaches in the drag discovery arsenal could lead to new methods to overcome the issue of challenging drag targets. For example, targeted protein degradation (TPD) technology has emerged as a breakthrough strategy to target undruggable / challenging disease targets allowing for drug development against the undruggable / challenging targets (Figure IB). TPD primarily employs monovalent (e.g., molecular glues (MGs)) or bifunctional (e.g., Proteolysis Targeting Chimeras (PROTACs)) small molecule degraders that utilize the cell’s own degradation machinery to enable immediate selective degradation of the protein of interest (POI) (e.g., a disease-driving protein) rather than merely inhibiting its function.18326495150V1Attorney Docket No; 243735.000473

[0126] It has been discovered that critical plant hormones (e.g., auxin / indole-3-acetic acid (Tan et al. Nature, 446, 640-5, 2007) and jasmonate isoleucine (Sheard et al. Nature, 468, 400-5, 2010)) and synthetic anticancer molecules (e.g.. immunomodulatory imide drugs (IMiDs) such as lenalidomide (Petzold et al. Nature, 532, 127-30, 2016) and sulfonamide drugs such as indisulam (Du et al. Structure, 27, 1625-33. e3, 2019; Bussiere et al., Nat Chem Biol, 16, 15-23, 2020; Faust et al., Nat Chem Biol, 16, 7-14, 2020)) act as molecular glues (Figure 1C). IMiDs act via the molecular glue mechanism to degrade zinc finger transcription factors. This highlights the importance of molecular glues in nature and in the clinical field. Given the lack of systematic ways to identify small molecule protein degraders, companies often rely on use of IMiD derivatives.

[0127] TPD faces certain limitations despite its rapid rise as a drug discovery strategy. One major limitation is its reliance on only a small set of amenable E3 ligases, such as Cereblon and von Hippel-Lindau (VHL). Additionally, the discovery of degraders has been largely serendipitous (e.g., as with MGs) or requires complex chemistry protocols (e.g., as with PROTACs).

[0128] The present application provides, among many other things, a method for identifying a degrader of a target protein. The iCasp9 (inducible caspase-9) is a cellular suicide gene system which comprises the catalytic domain of human caspase-9, ACaspase9 (caspase-9 without its physiological dimerization CARD domain), fused to a dimerizer FKBP12 F36V domain (also called the DmrB domain or dTAG) via a flexible SGGGS (SEQ ID NO: 9) linker (Figure 2A).Due to the presence of the FKBP12 F36V domain, the system can be dimerized by synthetic chemical inducers of dimerization, such as AP20187 or API 903 (Rimiducid). Thus, the iCasp9 system operates on the principle of chemically induced proximity (CIP), where the conditional dimerization and activation of iCasp9 using a small molecule (e.g., AP1903 or AP20187) triggers the apoptosis pathway, leading to efficient induction of cell death (Figure 2B).

[0129] The iCasp9 system has shown particularly promising results in alleviating the events of toxicity associated with adoptive T-cell therapies and has been used to mitigate chimeric antigen receptor (CAR)-T cell toxicity (Clackson, et al., Proc. Natl. Acad. Sci. U.S.A., 1998, 95(18); Di Stasi, et al., N. Engl. J. Med., 2011, 365(18); Gargett, et al., Front. Pharmacol., 2014, 5: 235; Straathof, et al., Blood, 2005, 705(11), each of which is incorporated by reference in its entirety for all purposes).19326495150V1Attorney Docket No: 243735.000473

[0130] iCADD (iCasp9M-Assisted Degrader Discovery), as an exemplary system, is a selection-based high-throughput screening platform to identify protein degraders (e.g., molecular glue degraders or PROTACs). For example, the “up”, positive- selection, cell-based assay screening platform iCADD can leverage the suicidal activity of a mutant form caspase-9 (iCasp9M) (Figure 3A). In the exemplary target-centric iCADD system, the iCasp9M suicide gene can be expressed in fusion with a target protein of interest (POI). If a chemical compound induces the degradation of the target POI, the fused iCasp9M protein is co-degraded as well. Consequently, this impairs or diminishes the iCasp9M-derived apoptotic signaling upon treatment with a suicide inducer (e.g., AP20187 or AP1903), resulting in a survival phenotype (Figure 3B). Such a compound causing reversal of cell death can then be considered a candidate degrader of the target POI. Nonetheless, it is possible that a chemical compound may interfere with transcription or translation of the POI-iCasp9M fusion construct, thereby preventing the synthesis of the fusion protein. This would also impair or diminish the small molecule-induced cell death and result in a survival phenotype (Figure 3C). Given this scenario, the identities of the specific degrader(s) could then be revealed by a counter assay against cells expressing only iCasp9M without the POI. In this case, a specific degrader of POI will not be expected to cause reversal of small molecule-induced cell death.

[0131] The validated iCADD system described herein may transform the elusive question of whether a target POI is degraded by certain compound(s) in a large chemical library into an easy-to-interpret death / survival phenotype in just a few hours (e.g., 8 hrs). The iCADD platform may implement a robust and straightforward plate end-assay readout methodology, involving a one-step addition of cell lysis reagent (e.g.. a recombinant luciferase reaction mix (e.g.. CellTiter-Glo 2.0 from Promega)) followed by luminescence measurement. These advantages can enable a high-throughput screen with a tremendously shorter timeframe for end-assay readout compared to the currently existing methods. Further, compared to other degrader screening platforms, the iCADD platform may have additional advantages. The iCADD platform may produce a rapid death outcome that can readily identify chemical degraders for the target POI while markedly enhancing the signal-to-noise ratio. The iCADD platform is considered an “up assay”, which can result in better signal to noise ratios and fewer false positives versus a “down assay” (Figure 3D). Additionally, the iCADD platform may be used as a genetic-screen method for target deconvolution of identified hit molecule(s) from the “up” assay screen. Moreover, the iCADD20326495150V1Attorney Docket No: 243735.000473platform can harness the cell’s endogenous degradation machinery to identify degraders, which may broaden compatibility and potential utility for a wide range of E3 ligases.Definitions

[0132] The term “about” or “approximately” means within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%. and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.

[0133] The terms “a,” “an,” and “the” do not denote a limitation of quantity, but rather denote the presence of “at least one” of the referenced item.

[0134] The terms “polypeptide” and “protein” used interchangeably herein encompass native or artificial proteins, protein fragments, and polypeptide analogs of a protein sequence. A polypeptide or protein may be monomeric or polymeric. The terms encompass all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins, including without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, PEGylation, biotinylation, etc.). Small polypeptides of less than 100 amino acids, preferably less than 50 amino acids, may be referred to as “peptides”.

[0135] The term “fragment” in regard to peptides or polypeptides refers to a peptide or a polypeptide that has an amino-terminal and / or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the full-length naturally-occurring sequence. Also, fragments according to the invention may be made by truncation, e.g., by removal of one or more amino acids from the N and / or C-terminal ends of a polypeptide. Up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up tolO, up to 20, up to 30, up to 40 or more amino acids may be removed from the N and / or C terminal in this way. Fragments may also be generated by one or more internal deletions. In some embodiments, fragments are at least 5, 6, 8 or 10 amino acids long. In other embodiments, the fragments are at least 14, at least 20, at least 50. or at least 70, 80, 90, 100, 150, 200. or 400 amino acids long.21326495150V1Attorney Docket No: 243735.000473

[0136] The term “variant” as used herein refers to a peptide, polypeptide, or polynucleotide, or a derivative, analog thereof, comprising one or more mutations and / or chemical modifications as compared to a reference peptide, polypeptide or polynucleotide. Mutations and / or chemical modifications can include, for example, insertions, substitutions, deletions, transversions, and / or inversions at one or more locations in the amino acid or nucleotide sequence.

[0137] In certain embodiments, amino acid substitutions of a peptide or a protein or portion thereof are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, or (4) confer or modify other physicochemical or functional properties. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the normally-occurring sequence.

[0138] A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution.Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson, Methods Mol. Biol. 243:307-31 (1994). Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and 7) sulfur-containing side chains: cysteine and methionine.Conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalaninetyrosine, lysine-arginine, alanine- valine, glutamate-aspartate, and asparagine-glutamine.

[0139] A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence. Examples of art-recognized polypeptide secondary and tertiary structures are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et22326495150V1Attorney Docket No: 243735.000473al., Nature 354:105 (1991), which are each incorporated herein by reference in their entirety for all purposes.

[0140] As used herein, the twenty naturally occurring amino acids and their abbreviations follow conventional usage. See Immunology — A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.

[0141] The term “polynucleotide” as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxyribonucleotides or a modified form of either type of nucleotide. The term includes single and double stranded forms.

[0142] The term “percent sequence identity” means a ratio, expressed as a percent of the number of identical residues over the total number of residues compared.

[0143] Sequence identity for nucleic acid sequences may be analyzed over a stretch of at least about nine nucleotides, usually at least about 18 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36, 48 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA, which includes, e.g.. the programs FASTA2 and FASTA3, provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000): Pearson, Methods Enzymol. 266:227-258 (1996); Pearson, J. Mol. Biol. 276:71-84 (1998); herein incorporated by reference). Unless otherwise specified, default parameters for a particular program or algorithm are used. For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference.

[0144] A reference to a nucleotide sequence encompasses its complement unless otherwise specified. Thus, a reference to a nucleic acid having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence.23326495150V1Attorney Docket No: 243735.000473

[0145] Sequence identity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches sequences using measures of similarity assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Bestfif ’ which can be used with default parameters, as specified with the programs, to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g.. GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, see GCG Version 6.1. (University of Wisconsin Wis.) FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000)). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially blastp or tblastn, using default parameters, as supplied with the programs. See, e.g., Altschul et al., J. Mol. Biol.215:403-410 (1990): Altschul et al., Nucleic Acids Res. 25:3389-402 (1997), each of which is incorporated by reference in its entirety for all purposes.

[0146] The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences.

[0147] The term “substantial similarity” or “substantial sequence similarity,” when referring to a nucleic acid or fragment thereof, means that when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 80%, preferably at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.

[0148] As applied to polypeptides, the term “substantial identity” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, as supplied with the programs, share at least 70%, 75%, 80% or 85% sequence identity,24326495150V1Attorney Docket No: 243735.000473preferably at least 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or 99% sequence identity. In certain embodiments, residue positions that are not identical differ by conservative amino acid substitutions.

[0149] The term “vector”, as used herein, means a vehicle capable of transporting a nucleic acid into a host cell. In some embodiments, the vector is a plasmid, i.e., a circular double stranded DNA loop into which additional DNA segments may be ligated. In some embodiments, the vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome, hi some embodiments, the vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). In other embodiments, the vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).

[0150] The term “recombinant host cell” (or simply “host cell”), as used herein, means a cell into which an exogenous nucleic acid and / or recombinant vector has been introduced. It should be understood that “recombinant host cell” and “host cell” mean not only the particular subject cell but also the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term “host cell” as used herein.

[0151] The practice of the present invention employs, unless otherwise indicated, conventional techniques of statistical analysis, molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such tools and techniques are described in detail in e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein25326495150V1Attorney Docket No; 243735.000473Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ. Additional techniques are explained, e.g.. in U.S. Patent No. 7,912,698 and U.S. Patent Appl. Pub. Nos. 2011 / 0202322 and 2011 / 0307437, each of which is herein incorporated by reference in its entirety for all intended purposes.Methods

[0152] In some aspects, provided herein is a method for identifying a degrader of a target protein, comprising:(a) contacting a population of cells with a test compound in a test cell culture, wherein the population of cells expresses an exogenous fusion protein, wherein the fusion protein comprises the target protein and an inducible cell suicide protein, and wherein the cell suicide protein induces the cells to undergo apoptosis in the presence of a suicide inducer;(b) incubating the test cell culture under conditions and for a time sufficient for degradation of the target protein to occur;(c) adding a suicide inducer to the test cell culture, and incubating the test cell culture under conditions and for a time sufficient for the cells to undergo apoptosis;(d) determining the level of cell viability using a chemical cell viability assay;(e) comparing the level of cell viability determined in step (d) to a control level of cell viability, wherein the control level of cell viability is a predetermined control cell viability level or the level of cell viability determined under the same conditions in a control cell culture; and (f) (i) determining the test compound is a degrader of the target protein when the level of cell viability determined in step (d) is higher than the control level of cell viability, or (ii) determining the test compound is not a degrader of the target protein when the level of cell viability determined in step (d) is not higher than the control level of cell viability.

[0153] In some embodiments, the exogenous fusion protein is expressed from a transgene integrated into the chromosome of the cells. In some embodiments, the population of cells expresses an exogenous fusion protein via integration of a transgene encoding said exogenous fusion protein into a chromosome of the population of cells. In some embodiments, the population of cells expresses an exogenous fusion protein via an episomal vector expressing a transgene encoding said exogenous fusion protein. In some embodiments, the population of cells expresses an exogenous fusion protein that remains stable within the cells.26326495150V1Attorney Docket No: 243735.000473

[0154] In some embodiments, the target protein is positioned at the N-terminus relative to the inducible cell suicide protein.

[0155] In some embodiments, the target protein is positioned at the C-terminus relative to the inducible cell suicide protein.

[0156] In some embodiments, the target protein is fused to the inducible cell suicide protein via a linker.

[0157] Fusion proteins described herein may comprise a linker that connects the target protein to the inducible cell suicide protein. In some embodiments, a linker comprises at least one covalent bond. In some embodiments, a linker may comprise a single bond, e.g., a disulfide bond or disulfide bridge, that connects the target protein to the inducible cell suicide protein. However, in some embodiments, a linker may connect the target protein to the inducible cell suicide protein through multiple covalent bonds. In some embodiments, a linker is generally stable in vitro and in vivo, and may be stable in certain cellular environments. Additionally, generally a linker does not negatively impact the functional properties of either the target protein or the inducible cell suicide protein. In some embodiments, non-cleavable linkers may be used.Generally, a non-cleavable linker cannot be readily degraded in a cellular or physiological environment.

[0158] In some embodiments, the linker comprises about 5-20 amino acids. In some embodiments, the linker comprises 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids.

[0159] In some embodiments, the linker comprises about 5-18 amino acids, about 5-15 amino acids, about 5-12 amino acids, about 5-10 amino acids, about 5-8 amino acids, about 10-20 amino acids, about 10-15 amino acids, about 10-12 amino acids, or about 15-20 amino acids. In some embodiments, the linker comprises about 5-15 amino acids. In some embodiments, the linker comprises about 10-15 amino acids.

[0160] In some embodiments, the linker length between the target protein and the inducible cell suicide protein may vary. In some embodiments, a linker length of 15 amino acids is used for target proteins with a molecular weight of greater than 30 kDa. In some embodiments, a27326495150V1Attorney Docket No: 243735.000473linker length of 15 amino acids is used for target proteins with a molecular weight of greater than 35 kDa.

[0161] The linker length may be variable and may be customized according to the nature of the target protein. In some embodiments, the linker length may be correlated to the size of the target protein (e.g., the use of longer linkers between the target protein and the inducible cell suicide protein correlates with the use of target proteins of larger molecular weights). Without wishing to be bound by theory, the use of longer linkers with the use of target proteins of larger molecular weights leads to less inhibition of degradation by the target protein (e.g., leads to greater killing efficiency) due to less steric hinderance. In some embodiments, the linker length may be correlated to the structure (e.g., structural characteristics) of the target protein (e.g., flexible / disordered proteins vs. globular / compact proteins vs. fibrous / strand-like proteins).

[0162] In some embodiments, the linker comprises the amino acid sequence of GGSGGGSGGG (SEQ ID NO: 12), GGSGG (SEQ ID NO: 47), SGGGSG (SEQ ID NO: 48), (SGGGS)n (SEQ ID NO: 20), wherein n is 1, 2. or 3. (GGGGS)n (SEQ ID NO: 21). wherein n is 1, 2, or 3, GGGGGG (SEQ ID NO: 16), GGGGGGGG (SEQ ID NO: 17), GSAGSAAGSGEF (SEQ ID NO: 18), or EGKSSGSGSESKST (SEQ ID NO: 19), or a variant or combination thereof.

[0163] In some embodiments, the linker between the target protein and the inducible cell suicide protein is GGSGGGSGGG (SEQ ID NO: 12). In some embodiments, the linker between the target protein and the inducible cell suicide protein is GGSGG (SEQ ID NO: 47). In some embodiments, the linker between the target protein and the inducible cell suicide protein is SGGGSG (SEQ ID NO: 48). In some embodiments, the linker between the target protein and the inducible cell suicide protein is SGGGS (SEQ ID NO: 9). In some embodiments, the linker between the target protein and the inducible cell suicide protein is SGGGSSGGGS (SEQ ID NO: 10). In some embodiments, the linker between the target protein and the inducible cell suicide protein is SGGGS SGGGS SGGGS (SEQ ID NO: 11). In some embodiments, the linker between the target protein and the inducible cell suicide protein is GGGGS (SEQ ID NO: 13). In some embodiments, the linker between the target protein and the inducible cell suicide protein is GGGGSGGGGS (SEQ ID NO: 14). In some embodiments, the linker between the target protein and the inducible cell suicide protein is GGGGSGGGGSGGGGS (SEQ ID NO: 15). In some embodiments, the linker between the target protein and the inducible cell suicide protein is28326495150V1Attorney Docket No: 243735.000473GGGGGG (SEQ ID NO: 16). In some embodiments, the linker between the target protein and the inducible cell suicide protein is GGGGGGGG (SEQ ID NO: 17). In some embodiments, the linker between the target protein and the inducible cell suicide protein is GSAGSAAGSGEF (SEQ ID NO: 18). In some embodiments, the linker between the target protein and the inducible cell suicide protein is EGKSSGSGSESKST (SEQ ID NO: 19).

[0164] In some embodiments, the linker between the target protein and the inducible cell suicide protein is a flexible linker. Without wishing to be bound by theory, a flexible linker lacks rigid structure and comprises small and / or polar (e.g., glycine and serine) amino acid residues. A flexible linker may be essential when movement between fusion protein domains is necessary (e.g., the flexible linker provides mobility for connected functional protein domains while maintaining an adequate distance between the protein domains). As used herein, a certain level of flexibility may be required to prevent structural hinderance (e.g., particularly between the ligand binding domain (e.g., FKBP12 F36V) when fused to a large molecular weight target protein given the proximity between the two entities). In some embodiments, the linker between the target protein and the inducible cell suicide protein is not a rigid linker. Without wishing to be bound by theory, a rigid linker may comprise an a-helical structure and may be stiffer (e.g., compared to a flexible linker).

[0165] In some embodiments, the inducible cell suicide protein comprises a ligand binding domain fused to a cell suicide domain.

[0166] In some embodiments, the ligand binding domain comprises an FKBP12 polypeptide, an FRB 1 polypeptide, or a variant, fragment, or combination thereof.

[0167] In some embodiments, the ligand binding domain comprises a variant of the FKBP12 polypeptide comprising an F36V substitution.

[0168] In some embodiments, the variant of the FKBP12 polypeptide comprising an F36V substitution comprises the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 3), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the variant of the FKBP12 polypeptide comprising an F36V substitution comprises the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG29326495150vlAttorney Docket No: 243735.000473WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 3), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0169] In some embodiments, the ligand binding domain comprises two tandem copies of the variant of the FKBP12 polypeptide comprising an F36V substitution (Fan, et al., Hum. Gene. Ther., 1999, 10(14); Mallet, et al., Nat. Biotechnol., 2002, 20(12), each of which is incorporated by reference in its entirety for all purposes).

[0170] In some embodiments, the ligand binding domain comprises a variant of the FKBP12 polypeptide comprising G89P and I90K substitutions (Shahi, et al., PLoS One, 2012, 7(1);Amara, et al., Proc. Natl. Acad. Sei. U.S.A., 1997, 94(20), each of which is incorporated by reference in its entirety for all purposes).

[0171] In some embodiments, wherein the ligand binding domain comprises a variant of the FKBP12 polypeptide comprising G89P and I90K substitutions, the suicide inducer is FK1012 and / or AP1510.

[0172] In some embodiments, the ligand binding domain comprises an FRB 1 polypeptide, or a variant or fragment thereof. Without wishing to be bound by theory, the FRB 1 polypeptide (FKBP12-rapamycin associated protein (FRAP) rapamycin binding domain) comprises residues 2025-2113 of mTOR.

[0173] In some embodiments, the ligand binding domain comprises a variant of the FRB 1 polypeptide comprising a T2098L substitution.

[0174] In some embodiments, the ligand binding domain comprises a variant of the FRB 1 polypeptide comprising a T2098L substitution and a variant of the FKBP12 polypeptide comprising G89P and I90K substitutions.

[0175] In some embodiments, the variant of the FKBP12 polypeptide comprising G89P and I90K substitutions comprises the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPPKIPPHATLVFDVELLKLE (SEQ ID NO: 49), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the variant of the FKBP12 polypeptide comprising G89P and I90K substitutions30326495150V1Attorney Docket No: 243735.000473comprises the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPPKIPPHATLVFDVELLKLE (SEQ ID NO: 49), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%. or at least about 99% sequence identity thereof.

[0176] In some embodiments, the variant of the FRB1 polypeptide comprising a T2098L substitution comprises the amino acid sequence of EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEA QEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK (SEQ ID NO: 50), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the variant of the FRB1 polypeptide comprising a T2098L substitution comprises the amino acid sequence of EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEA QEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK (SEQ ID NO: 50), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%. or at least about 99% sequence identity thereof.

[0177] In some embodiments, wherein the ligand binding domain comprises a variant of the FRB1 polypeptide comprising a T2098L substitution and a variant of the FKBP12 polypeptide comprising G89P and I90K substitutions, the suicide inducer is AP22783 (Li, et al., Gene. Ther., 2002, 9(4); Shahi, et al., PLoS One, 2012, 7(1); Pollock, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97(24); Chen, et al.. Proc. Natl. Acad. Sci. U.S.A., 1995, 92(11); Falcon, et al., Front. Immunol., 2022, 13. each of which is incorporated by reference in its entirety for all purposes).

[0178] In some embodiments, the cell suicide domain comprises a caspase-9 polypeptide, a caspase-8 polypeptide, a caspase- 1 polypeptide, a caspase-3 polypeptide, a FADD polypeptide, a Fas polypeptide, or a mBax polypeptide, or a variant, fragment or combination thereof (Fan, et al., Hum. Gene. Ther., 1999, 10(14), which is incorporated by reference in its entirety for all purposes).31326495150V1Attorney Docket No: 243735.000473

[0179] In some embodiments, the cell suicide domain comprises a variant of the caspase-9 polypeptide comprising Q221R substitution.

[0180] In some embodiments, the variant of the caspase-9 polypeptide comprising Q221R substitution comprises the amino acid sequence of GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRF SSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVY GTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNP EPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQ WAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 2), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the variant of the caspase-9 polypeptide comprising Q221R substitution comprises the amino acid sequence of GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRF SSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVY GTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNP EPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQ WAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 2), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0181] In some embodiments, the caspase-9 polypeptide is a truncated form, comprising only the catalytic domain of human caspase-9 (e.g., amino acid residues 135-416), referred to as ACaspase9. Without wishing to be bound by theory, ACaspase9 lacks the physiological dimerization CARD domain (caspase activation and recruitment domain) of human caspase-9, which prevents dimerization under physiological conditions. In some embodiments, the sequence of the caspase-9 polypeptide comprises SEQ ID NO: 4 (iCasp9). In some embodiments, the sequence of the caspase-9 Q221R polypeptide comprises SEQ ID NO: 5 (iCasp9M). iCasp9 may be referred to as wild-type caspase-9 polypeptide and iCasp9M may be referred to as a mutant form caspase-9 polypeptide. The iCasp9M sequence comprises a Q221R mutation in the ACaspase9 domain, notated as a mutant form ACaspase9M domain, compared to the wild-type32326495150V1Attorney Docket No: 243735.000473ACaspase9 domain of iCasp9. Without wishing to be bound by theory, the Q221R mutation in the iCasp9M variant may cause a conformational change modifying the affinity of caspase-9 to Apaf-1. potentially reducing its basal activity. Moreover, the Q221R mutation does not affect the functional activity of caspase-9 (Xu, et al., PLoS ONE, 2012, 7(5); Avrutsky, et al., Front.Pharmacol., 2021, 12, each of which is incorporated by reference in its entirety for all purposes).

[0182] In some embodiments, the cell suicide domain comprises a variant of the caspase-8 polypeptide comprising amino acids 216-479 of a wild-type caspase-8 protein.

[0183] In some embodiments, the variant of the caspase-8 polypeptide comprising amino acids 216-479 of a wild-type caspase-8 protein comprises the amino acid sequence of SESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKLHSIRDRNGTHLDAGALTTTFE ELHFEIKPHDDCTVEQIYEILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPIYELT SQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSEEQPYLEMDLSSPQTRYIPDEA DFLLGMATVNNCVSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNYEVSNKDDK KNMGKQMPQPTFTLRKKLVFPSD (SEQ ID NO: 51), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the variant of the caspase-8 polypeptide comprising amino acids 216-479 of a wild-type caspase-8 protein comprises the amino acid sequence of SESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKLHSIRDRNGTHLDAGALTTTFE ELHFEIKPHDDCTVEQIYEILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPIYELT SQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSEEQPYLEMDLSSPQTRYIPDEA DFLLGMATVNNCVSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNYEVSNKDDK KNMGKQMPQPTFTLRKKLVFPSD (SEQ ID NO: 51), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, caspase-8216-479 comprises the catalytically active domain of the human caspase-8 protein (Carlotti, et al.. Cancer Gene Ther., 2005, 12(7); Fan, et al., Hum. Gene. Ther., 1999, 10(14), each of which is incorporated by reference in its entirety for all purposes).

[0184] In some embodiments, the caspase- 1 polypeptide comprises the amino acid sequence of MADKVLKEKRKLFIRSMGEGTINGLLDELLQTRVLNKEEMEKVKRENATVMDKTRALI DSVIPKGAQACQICITYICEEDSYLAGTLGLSADQTSGNYLNMQDSQGVLSSFPAPQAVQ33326495150vlAttorney Docket No: 243735.000473DNPAMPTSSGSEGNVKLCSLEEAQRIWKQKSAEIYPIMDKSSRTRLALIICNEEFDSIPRR TGAEVDITGMTMLLQNLGYSVDVKKNLTASDMTTELEAFAHRPEHKTSDSTFLVFMSH GIREGICGKKHSEQVPDILQLNAIFNMLNTKNCPSLKDKPKVIIIQACRGDSPGVVWFKD SVGVSGNLSLPTTEEFEDDAIKKAHIEKDFIAFCSSTPDNVSWRHPTMGSVFIGRLIEHMQ EYACSCDVEEIFRKVRFSFEQPDGRAQMPTTERVTLTRCFYLFPGH (SEQ ID NO: 52), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the caspase- 1 polypeptide comprises the amino acid sequence of MADKVLKEKRKLFIRSMGEGTINGLLDELLQTRVLNKEEMEKVKRENATVMDKTRALI DSVIPKGAQACQICITYICEEDSYLAGTLGLSADQTSGNYLNMQDSQGVLSSFPAPQAVQ DNPAMPTSSGSEGNVKLCSLEEAQRIWKQKSAEIYPIMDKSSRTRLALIICNEEFDSIPRR TGAEVDITGMTMLLQNLGYSVDVKKNLTASDMTTELEAFAHRPEHKTSDSTFLVFMSH GIREGICGKKHSEQVPDILQLNAIFNMLNTKNCPSLKDKPKVIIIQACRGDSPGVVWFKD SVGVSGNLSLPTTEEFEDDAIKKAHIEKDFIAFCSSTPDNVSWRHPTMGSVFIGRLIEHMQ EYACSCDVEEIFRKVRFSFEQPDGRAQMPTTERVTLTRCFYLFPGH (SEQ ID NO: 52). or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0185] In some embodiments, the caspase-3 polypeptide comprises the amino acid sequence of MENTENSVDSKSIKNLEPKIIHGSESMDSGISLDNSYKMDYPEMGLCIIINNKNFHKSTG MTSRSGTDVDAANLRETFRNLKYEVRNKNDLTREEIVELMRDVSKEDHSKRSSFVCVL LSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLTGKPKLFIIQACRGTELDCGIETDSGVD DDMACHKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCAMLKQYADKLEFMHILT RVNRKVATEFESFSFDATFHAKKQIPCIVSMLTKELYFYH (SEQ ID NO: 53), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the caspase-3 polypeptide comprises the amino acid sequence of MENTENSVDSKSIKNLEPKIIHGSESMDSGISLDNSYKMDYPEMGLCIIINNKNFHKSTG MTSRSGTDVDAANLRETFRNLKYEVRNKNDLTREEIVELMRDVSKEDHSKRSSFVCVL LSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLTGKPKLFIIQACRGTELDCGIETDSGVD DDMACHKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCAMLKQYADKLEFMHILT RVNRKVATEFESFSFDATFHAKKQIPCIVSMLTKELYFYH (SEQ ID NO: 53), or an amino34326495150vlAttorney Docket No: 243735.000473acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%. at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0186] In some embodiments, the cell suicide domain comprises a variant of the FADD polypeptide comprising amino acids 1-125 of a wild- type FADD protein.

[0187] In some embodiments, the variant of the FADD polypeptide comprising amino acids 1-125 of a wild- type FADD protein comprises the amino acid sequence of MDPFLVLLHSVSSSLSSSELTELKFLCLGRVGKRKLERVQSGLDLFSMLLEQNDLEPGHT ELLRELLASLRRHDLLRRVDDFEAGAAAGAAPGEEDLCAAFNVICDNVGKDWRRLARQ LKVSDTK (SEQ ID NO: 54), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the variant of the FADD polypeptide comprising amino acids 1-125 of a wild- type FADD protein comprises the amino acid sequence of MDPFLVLLHSVSSSLSSSELTELKFLCLGRVGKRKLERVQSGLDLFSMLLEQNDLEPGHT ELLRELLASLRRHDLLRRVDDFEAGAAAGAAPGEEDLCAAFNVICDNVGKDWRRLARQ LKVSDTK (SEQ ID NO: 54), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the FADD 125 polypeptide comprises the amino terminal of the FADD protein (Fan, et al., Hum. Gene. Ther., 1999, 10(14), which is incorporated by reference in its entirety for all purposes).

[0188] In some embodiments, the cell suicide domain comprises a variant of the Fas polypeptide comprising amino acids 175-304 of a wild-type Fas protein.

[0189] In some embodiments, the variant of the Fas polypeptide comprising amino acids 175-304 of a wild-type Fas protein comprises the amino acid sequence of IHSGQCPAAIVGGINVLLKPNTSVQFLRLGMLSPEGTCKAFDTAGNGYCRSEGVVAVLL TKKSLARRVYATILNAGTNTDGFKEQGVTFPSGDIQEQLIRSLYQSAGVAPESFEYIEAH GTGTKVGDPQE (SEQ ID NO: 55), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the variant of the Fas polypeptide comprising amino acids 175-304 of a wild-type Fas protein comprises the amino acid sequence of IHSGQCPAAIVGGINVLLKPNTSVQFLRLGMLSPEGTCKAFDTAGNGYCRSEGVVAVLL35326495150vlAttorney Docket No: 243735.000473TKKSLARRVYATILNAGTNTDGFKEQGVTFPSGDIQEQLIRSLYQSAGVAPESFEYIEAH GTGTKVGDPQE (SEQ ID NO: 55), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%. at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, Fas 175-304 comprises the cell-killing domain of the Fas protein (Amara. et al., Proc. Natl. Acad. Sci. U.S.A..1997, 94(20); Fan, et al., Hum. Gene. Ther., 1999, 10(14); Amara, et al., Hum Gene Ther., 1999, 10(16), each of which is incorporated by reference in its entirety for all purposes).

[0190] In some embodiments, the mBax polypeptide comprises the amino acid sequence of MDGSGEQLGSGGPTSSEQIMKTGAFLLQGFIQDRAGRMAGETPELTLEQPPQDASTKKL SECLRRIGDELDSNMELQRMIADVDTDSPREVFFRVAADMFADGNFNWGRVVALFYFA S KLVLKALCTKVPELIRTIMGWTLDFLRERLLVWIQDQGGWEGLLS YFGTPTWQTVTIF VAGVLTASLTIWKKMG (SEQ ID NO: 56), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the mBax polypeptide comprises the amino acid sequence of MDGSGEQLGSGGPTSSEQIMKTGAFLLQGFIQDRAGRMAGETPELTLEQPPQDASTKKL SECLRRIGDELDSNMELQRMIADVDTDSPREVFFRVAADMFADGNFNWGRVVALFYFA SKLVLKALCTKVPELIRTIMGWTLDFLRERLLVWIQDQGGWEGLLSYFGTPTWQTVTIF VAGVLTASLTIWKKMG (SEQ ID NO: 56), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof. In some embodiments, the mBax polypeptide is a murine Bax protein (Xiao, et al., J Biomol Screen., 2006, 11(3), which is incorporated by reference in its entirety for all purposes).

[0191] In some embodiments, the ligand binding domain is fused to the cell suicide domain via a linker. In some embodiments, the linker comprises about 5-20 amino acids. In some embodiments, the linker comprises 5 amino acids, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, or 20 amino acids.36326495150V1Attorney Docket No: 243735.000473

[0192] In some embodiments, the linker comprises about 5-18 amino acids, about 5-15 amino acids, about 5-12 amino acids, about 5-10 amino acids, about 5-8 amino acids, about 10-20 amino acids, about 10-15 amino acids, about 10-12 amino acids, or about 15-20 amino acids. In some embodiments, the linker comprises about 5-15 amino acids. In some embodiments, the linker comprises about 10-15 amino acids.

[0193] In some embodiments, the linker comprises the amino acid sequence of GGSGGGSGGG (SEQ ID NO: 12), GGSGG (SEQ ID NO: 47), SGGGSG (SEQ ID NO: 48), (SGGGS)n (SEQ ID NO: 20), wherein n is 1, 2. or 3, (GGGGS)n (SEQ ID NO: 21), wherein n is 1, 2, or 3, GGGGGG (SEQ ID NO: 16), GGGGGGGG (SEQ ID NO: 17), GSAGSAAGSGEF (SEQ ID NO: 18), or EGKSSGSGSESKST (SEQ ID NO: 19), or a variant or combination thereof.

[0194] In some embodiments, the linker between the ligand binding domain and the cell suicide domain is GGSGGGSGGG (SEQ ID NO: 12). In some embodiments, the linker between the ligand binding domain and the cell suicide domain is GGSGG (SEQ ID NO: 47). In some embodiments, the linker between the ligand binding domain and the cell suicide domain is SGGGSG (SEQ ID NO: 48). In some embodiments, the linker between the ligand binding domain and the cell suicide domain is SGGGS (SEQ ID NO: 9). In some embodiments, the linker between the ligand binding domain and the cell suicide domain is SGGGSSGGGS (SEQ ID NO: 10). In some embodiments, the linker between the ligand binding domain and the cell suicide domain is SGGGS SGGGS SGGGS (SEQ ID NO: 11). In some embodiments, the linker between the ligand binding domain and the cell suicide domain is GGGGS (SEQ ID NO: 13). In some embodiments, the linker between the ligand binding domain and the cell suicide domain is GGGGSGGGGS (SEQ ID NO: 14). In some embodiments, the linker between the ligand binding domain and the cell suicide domain is GGGGSGGGGSGGGGS (SEQ ID NO: 15). In some embodiments, the linker between the ligand binding domain and the cell suicide domain is GGGGGG (SEQ ID NO: 16). In some embodiments, the linker between the ligand binding domain and the cell suicide domain is GGGGGGGG (SEQ ID NO: 17). In some embodiments, the linker between the ligand binding domain and the cell suicide domain is GSAGSAAGSGEF (SEQ ID NO: 18). In some embodiments, the linker between the ligand binding domain and the cell suicide domain is EGKSSGSGSESKST (SEQ ID NO: 19).37326495150vlAttorney Docket No: 243735.000473

[0195] In some embodiments, the inducible cell suicide protein further comprises an HA tag at the C-terminus.

[0196] In some embodiments, the HA tag comprises the amino acid sequence of YPYDVPDYALD (SEQ ID NO: 6).

[0197] In some embodiments, the inducible cell suicide protein is linked to a protein tag. In some embodiments, the protein tag is a hemagglutinin (HA) tag. In some embodiments, the protein tag is attached to the C-terminus of the inducible cell suicide protein. Without wishing to be bound by theory, the protein tag (e.g., HA tag) may be used for detection and / or purification of the inducible cell suicide protein. In some embodiments, the detection method comprises Western blotting, immunoprecipitation, immunofluorescence, and / or ELISA.

[0198] In some embodiments, fusion proteins of the disclosure may comprise one or more affinity tags, e.g., to allow for affinity purification or coupling to another molecule. Examples of affinity tags include, but are not limited to, an Avi-tag, a biotin, a calmodulin tag, a hexahistidine tag (SEQ ID NO: 63), a hemagglutinin (HA) tag, a Myc tag, a GST tag. a green fluorescent protein (GFP), YFP, RFP, CFP, a MBP tag, a chitin binding protein tag, a FLAG tag, a V5 tag, a streptavidin binding tag, mCherry, tdTomato, SUMO tag, and Ubiquitin tag.

[0199] In some embodiments, the protein tag is used to facilitate purification of the tagged inducible cell suicide protein. In preferred embodiments, the protein tag is a HA tag, which corresponds to an epitope derived from the Influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767, which is incorporated by reference in its entirety for all purposes). Alternatively, the protein tag may be a “flag” tag or a hexa-histidine peptide (SEQ ID NO: 63) (i.e., His-tag), such as the tag provided in a pQE vector (QIAGEN, Inc.), among others, many of which are commercially available. As described in Gentz et al., 1989, Proc. Natl. Acad. Sci. USA 86:821-824, which is incorporated by reference in its entirety for all purposes, for instance, hexahistidine (SEQ ID NO: 63) provides for convenient purification of the fusion protein.

[0200] In some embodiments, the inducible cell suicide protein comprise the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGF GDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSS LHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGT38326495150vlAttorney Docket No: 243735.000473DGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEP DATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWA HSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSVDYPYDVPDYALD (SEQ ID NO: 7), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the inducible cell suicide protein comprise the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGF GDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSS LHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGT DGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEP DATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWA HSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSVDYPYDVPDYALD (SEQ ID NO: 7), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%. at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0201] In some embodiments, the suicide inducer is AP20187 (( 1R, l'R)-(((((2-((dimethylamino)methyl)propane-l,3-diyl)bis(azanediyl))bis(2-oxoethane-2,l-diyl))bis(oxy))bis(3,l-phenylene))bis(3-(3,4-dimethoxyphenyl)propane-l,l-diyl) (2S,2'S)-bis(l-((S ) -2- (3 ,4,5 -trimethoxyphenyl)butanoyl)piperidine-2-carboxylate)), AP 1903 (( 1 R, 1 'R)-((((ethane-l,2-diylbis(azanediyl))bis(2-oxoethane-2,l-diyl))bis(oxy))bis(3,l-phenylene))bis(3-(3,4-dimethoxyphenyl)propane-l,l-diyl) (2S,2'S)-bis(l-((S)-2-(3,4,5-trimethoxyphenyl)butanoyl)piperidine-2-carboxylate)), FK1012 (a synthetic dimer comprising two molecules of FK506 (e.g., tacrolimus) linked by their vinyl groups, (3S.3'S.4R.4'R,5S.5'S.8S.8'S.9Z,9'Z.12R,12'R,14R.14'R.15S,15'S.16S,16'S,18R.18'R.19R,19'R,2 6aR,26a'R)-8,8'-((Z)-but-2-ene-l,4-diyl)bis(5,19-dihydroxy-3-((E)-l-((lR,3R,4R)-4-hydroxy-3-methoxycyclohexyl)prop- 1 -en-2-yl)- 14, 16-dimethoxy-4, 10,12, 18-tetramethyl-5,6,8,ll,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-3H-15,19-epoxypyrido[2,l-c][l]oxa[4]azacyclotricosine-l,7,20,21(4H,23H)-tetraone)), AP1510 (CAS No. 178446-42-9, 3-(3,4-dimethoxyphenyl)-l-(3-(((2-((2-(3-((R)-3-(3,4-dimethoxyphenyl)-l-(((S)-l-(3,3-dimethyl-2-oxopentanoyl)piperidine-2-carbonyl)oxy)propyl)phenoxy)-2-39326495150V1Attorney Docket No; 243735.000473oxoethyl)amino)ethyl)glycyl)oxy)phenyl)propyl l-(3,3-dimethyl-2-oxopentanoyl)piperidine-2-carboxylate) (Shahi, et al., PLoS One, 2012, 7(1); Amara, et al., Proc. Natl. Acad. Sci. U.S.A., 1997, 94(20), each of which is incorporated by reference in its entirety for all purposes), or AP22783 (Rapalog-B, Li, et al., Gene. Ther., 2002, 9(4); Shahi, et al., PLoS One, 2012, 7(1); Pollock, et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97(24); Chen, et al., Proc. Natl. Acad. Sci. U.S.A., 1995. 92(11); Falcon, et al., Front. Immunol., 2022, 13, each of which is incorporated by reference in its entirety for all purposes). In some embodiments, AP22783 is considered a rapalog. In some embodiments, a rapalog is a highly specific heterodimerizing ligand based on the immunosuppressant drag, rapamycin. In some embodiments, a rapalog has an O-substitution at the C-40 position of rapamycin.

[0202] In some embodiments, the chemical cell viability assay is a luminescence-based cell viability assay.

[0203] In some embodiments, the luminescence-based cell viability assay measures the number of viable cells by quantifying adenosine triphosphate (ATP).

[0204] In some embodiments, the luminescence-based cell viability assay comprises a one-step reaction.

[0205] In some embodiments, the one-step reaction comprises addition of a recombinant luciferase reaction mix.

[0206] In some embodiments, the recombinant luciferase reaction mix comprises a CellTiter-Glo™ 2.0 reagent.

[0207] In some embodiments, the recombinant luciferase reaction mix comprises recombinant luciferase, luciferin substrate, one or more components to lyse cell membranes, and one or more components to inhibit endogenous ATPases.

[0208] In some embodiments, the recombinant luciferase reaction mix comprises Mg2+ e.g., as a cofactor, 1-2 mM EDTA (ethylenediaminetetraacetic acid) e.g., as an enzyme chelator, 0.05-2% DTAB (dodecyltrimethylammonium bromide) and sulfobetaine 3-10 e.g., as detergents, 1-20 mM NaF e.g., as an ATPase inhibitor, and / or TCA (trichloroacetic acid), DMSA (dimercaptosuccinic acid), or CTAB (cetyltrimethylammonium bromide) e.g., as cell-lysing agents.40326495150V1Attorney Docket No: 243735.000473

[0209] In some embodiments, the one or more components to lyse cell membranes comprises TCA (trichloroacetic acid), DMSA (dimercaptosuccinic acid), or CTAB (cetyltrimethylammonium bromide).

[0210] In some embodiments, the one or more components to inhibit endogenous ATPases comprises NaF.

[0211] In a preferred embodiment, the chemical cell viability assay is a luminescent cell viability assay. The luminescent cell viability assay may be optimized to minimize both time and protocol complexity, critical factors to distinguish between a degrader effect and a cytotoxic (i.e., killing) effect of a test compound (e.g., prolonged incubation times increase the risk of losing the classification of a true degrader due to cell death). In a preferred embodiment, the luminescent cell viability assay measures the number of viable cells by quantifying adenosine triphosphate (ATP) via the use of a recombinant luciferase reaction mix (e.g., CellTiter-Glo ™ 2.0 reagent). Quantifying adenosine triphosphate (ATP) via the use of a recombinant luciferase reaction mix (e.g., CellTiter-Glo ™ 2.0 reagent) can offer high sensitivity, few steps, rapid performance, and minimal interference.

[0212] In some embodiments, the chemical cell viability assay is an absorbance cell viability assay.

[0213] In some embodiments, the absorbance cell viability assay is a MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay or a MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. In some embodiments, the MTT assay comprises addition of the MTT reagent, incubation of about 1 to 4 hours, addition of about 100 pL solubilization solution to dissolve the formazan crystals, and recording the absorbance at 570 nm. In some embodiments, the MTS assay comprises addition of about 20 pL MTS reagent, incubation of about 1 to 4 hours, and recording the absorbance at 490 nm.

[0214] In some embodiments, the chemical cell viability assay is a fluorescence cell viability assay.

[0215] In some embodiments, the fluorescence cell viability assay is a resazurin assay. In some embodiments, the resazurin assay comprises addition of about 20 pL resazurin reagent, incubation of about 1 to 4 hours, and recording the fluorescence using a 560 nm excitation 1590 nm emission filter set.41326495150V1Attorney Docket No: 243735.000473

[0216] An absorbance cell viability assay or a fluorescence cell viability assay may require incubation of the substrate (e.g., tetrazolium (e.g., MTT or MTS) or resazurin) with viable cells at 37 °C for a prolonged period of time to generate a signal. The incubation of the substrate with cells may increase the possibility of artifacts resulting from chemical interactions (e.g., among the assay chemistry, the compounds being tested, and the biochemistry of the cell). Further, incubation introduces an extra plate-handling step that is not required in a luminescent cell viability assay that quantifies adenosine triphosphate (ATP) via the use of a recombinant luciferase reaction mix (e.g., CellTiter-Glo™ 2.0 reagent).

[0217] In some embodiments, the assay comprises a plate end-assay readout. In some embodiments, the readout is enabled by addition of a cell lysis reagent. In some embodiments, the cell lysis reagent comprises a recombinant luciferase reaction mix (e.g.. CellTiter-Glo™ 2.0 from Promega). In some embodiments, the readout comprises measurement of luminescence. In some embodiments, the reagents comprise luciferin and luciferase to create luminescence. In some embodiments, the readout is qualitative (e.g., light vs. no light).

[0218] In some embodiments, the control cell culture comprises a population of cells expressing the inducible cell suicide protein which is not fused to the target protein or which is fused to a protein that is not the target protein, and the control cell culture is subjected to the same test compound treatment.

[0219] In some embodiments, the control cell culture comprises a population of cells expressing the same exogenous fusion protein as the test cell culture, but the control cell culture is not subjected to the test compound treatment.

[0220] In some embodiments, in step (b), the test compound is incubated with the population of cells for about 3-4 hours.

[0221] In some embodiments, in step (b), the time for test compound (e.g., compound library) treatment (i.e., incubation) is about 3-4 hours. In some embodiments, the time for test compound (e.g., compound library) treatment (i.e., incubation) is about 2 to about 8 hours. In some embodiments, the time for test compound (e.g., compound library) treatment (i.e., incubation) is about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, about 5 hours, about 5.5 hours, about 6 hours, about 6.5 hours, about 7 hours, about 7.5 hours, or about 8 hours.42326495150vlAttorney Docket No: 243735.000473

[0222] In some embodiments, the test compound is incubated with the population of cells before addition of the suicide inducer and the test compound remains in the test cell culture until the determining the level of cell viability.

[0223] In some embodiments, in step (c), the test cell culture is incubated with the suicide inducer for about 12-16 hours.

[0224] In some embodiments, in step (c), the time for the suicide inducer (e.g.. API 903) treatment (i.e., incubation) is about 12-16 hours. In some embodiments, the time for the suicide inducer (e.g., AP1903) treatment (i.e., incubation) is about 6 to about 16 hours. In some embodiments, the time for the suicide inducer (e.g., AP1903) treatment (i.e., incubation) is about 6 hours, about 6.5 hours, about 7 hours, about 7.5 hours, about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours, about 10 hours, about 10.5 hours, about 11 hours, about 11.5 hours, about 12 hours, about 12.5 hours, about 13 hours, about 13.5 hours, about 14 hours, about 14.5 hours, about 15 hours, about 15.5 hours, or about 16 hours.

[0225] In some embodiments, the total time for conducting the method is about 15-20 hours.

[0226] In some embodiments, the total assay time is about 15 to about 16 hours. In some embodiments, the total assay time is about 16 hours. In some embodiments, the total assay time is about 15 hours. In some embodiments, the total assay time is about 8 to about 20 hours. In some embodiments, the total assay time is about 8 hours, about 8.5 hours, about 9 hours, about 9.5 hours, about 10 hours, about 10.5 hours, about 11 hours, about 11.5 hours, about 12 hours, about 12.5 hours, about 13 hours, about 13.5 hours, about 14 hours, about 14.5 hours, about 15 hours, about 15.5 hours, about 16 hours, about 16.5 hours, about 17 hours, about 17.5 hours, about 18 hours, about 18.5 hours, about 19 hours, about 19.5 hours, or about 20 hours. In some embodiments, the total assay time comprises the time for test compound (e.g., compound library) treatment (i.e., incubation) plus the time for the suicide inducer (e.g., AP1903) treatment (i.e., incubation).

[0227] In some embodiments, the degrader is a small molecule, a nucleic acid molecule, a peptide, or a protein.

[0228] In some embodiments, the small molecule comprises a molecular glue, a PROTAC, or a destabilizer. In some embodiments, the nucleic acid molecule, the peptide, or the protein is considered a down-regulator or a modulator of the target protein.43326495150V1Attorney Docket No: 243735.000473

[0229] In some embodiments, the degrader is an exogenous degrader or an endogenous degrader.

[0230] In some embodiments, the exogenous degrader is a monovalent degrader, bifunctional degrader, or a destabilizer.

[0231] In some embodiments, the monovalent degrader is a molecular glue.

[0232] In some embodiments, the bifunctional degrader is a proteolysis targeting chimera (PROTAC).

[0233] In some embodiments, the degrader comprises a library of compounds.

[0234] In some embodiments, the target protein is a proto-oncoprotein.

[0235] In some embodiments, the target protein is selected from Ikaros ZF23, Kras, Skpl, Cyclin DI, Skp2, cMyc. Ringol, FAM96B, PDCD1, SEC61A1. HMGCS1, ANAPC10. IRF4, CCND2, N-myc, SOX10, SEH1L, TBCB, KLF4, CEBPA, RPP25L, POU2AF1, Timl7-A, SMARCB1, S0CS3, CDC26, FOXA1, LRR1, FAU, GATA3, SYF2, SOX2, and mutants or fragments thereof.

[0236] In some embodiments, the target protein comprises the amino acid sequence of Ikaros ZF23 (SEQ ID NO: 8), Kras (SEQ ID NO: 57), Skpl (SEQ ID NO: 58), Cyclin DI (SEQ ID NO: 59), Skp2 (SEQ ID NO: 60), cMyc (SEQ ID NO: 61), Ringol (SEQ ID NO: 62), FAM96B (SEQ ID NO: 22), PDCD1 (SEQ ID NO: 23), SEC61A1 (SEQ ID NO: 24), HMGCS1 (SEQ ID NO: 25), ANAPC10 (SEQ ID NO: 26), IRF4 (SEQ ID NO: 27), CCND2 (SEQ ID NO: 28), N-myc (SEQ ID NO: 29), SOX10 (SEQ ID NO: 30), SEH1L (SEQ ID NO: 31), TBCB (SEQ ID NO: 32), KLF4 (SEQ ID NO: 33), CEBPA (SEQ ID NO: 34), RPP25L (SEQ ID NO: 35), POU2AF1 (SEQ ID NO: 36), Timl7-A (SEQ ID NO: 37), SMARCB1 (SEQ ID NO: 38), S0CS3 (SEQ ID NO: 39), CDC26 (SEQ ID NO: 40), FOXA1 (SEQ ID NO: 41), LRR1 (SEQ ID NO: 42), FAU (SEQ ID NO: 43), GATA3 (SEQ ID NO: 44), SYF2 (SEQ ID NO: 45), or SOX2 (SEQ ID NO: 46).

[0237] In some embodiments, the target protein is a Kras (G12C) mutant.

[0238] In some embodiments, the target protein is an oncoprotein. In some embodiments, the target protein comprises Ikaros ZF23, Kras, Skpl, Cyclin DI, Skp2, cMyc, Ringol, FAM96B, PDCD1, SEC61A1, HMGCS1, ANAPC10, IRF4, CCND2, N-myc, SOX10, SEH1L, TBCB, KLF4, CEBPA, RPP25L, POU2AF1, Timl7-A, SMARCB1, S0CS3. CDC26, FOXA1. LRR1. FAU, GATA3, SYF2, or SOX2. In some embodiments, the target protein is a protein that forms44326495150V1Attorney Docket No: 243735.000473dimers naturally in its wild-type state, but the dimerization domain of the target protein is mutated or removed to prevent dimerization.

[0239] In some embodiments, the target protein has a molecular weight between about 15 kilodaltons (kDa) and about 75 kilodaltons (kDa). In some embodiments, the target protein has a molecular weight of about 15 kDa, about 16 kDa, about 17 kDa, about 18 kDa, about 19 kDa, about 20 kDa, about 21 kDa, about 22 kDa, about 23 kDa, about 24 kDa, about 25 kDa. about 26 kDa, about 27 kDa, about 28 kDa, about 29 kDa, about 30 kDa, about 31 kDa, about 32 kDa, about 33 kDa, about 34 kDa, about 35 kDa, about 36 kDa, about 37 kDa, about 38 kDa, about 39 kDa, about 40 kDa, about 41 kDa, about 42 kDa, about 43 kDa, about 44 kDa, about 45 kDa, about 46 kDa, about 47 kDa, about 48 kDa, about 49 kDa, about 50 kDa, about 51 kDa, about 52 kDa, about 53 kDa, about 54 kDa, about 55 kDa, about 56 kDa, about 57 kDa, about 58 kDa, about 59 kDa, about 60 kDa, about 61 kDa, about 62 kDa, about 63 kDa, about 64 kDa, about 65 kDa, about 66 kDa, about 67 kDa, about 68 kDa, about 69 kDa, about 70 kDa, about 71 kDa, about 72 kDa, about 73 kDa, about 74 kDa, or about 75 kDa.

[0240] In some embodiments, the exogenous fusion protein is expressed from a transgene integrated into the chromosome of the cells.

[0241] In some embodiments, the population of cells comprises mammalian cells.

[0242] In some embodiments, the population of cells comprises adherent cells and / or suspension cells.

[0243] In some embodiments, the adherent cells comprise HEK293, CHO, MCF7, or HeLa cells. In some embodiments, the adherent cells comprise HeLa cells.

[0244] In some embodiments, the suspension cells comprise Jurkat. Expi293F, K562 cells, or HEK293 suspension-adapted cells. In some embodiments, the suspension cells comprise K562 cells.

[0245] In some embodiments, the method is conducted in a high-throughput format.

[0246] In some embodiments, the test compound represents a library of compounds.

[0247] In some embodiments, the method described herein is used in combination with a genetic screening for target deconvolution of the identified hit molecules. It is contemplated that with this system, a loss-of-function screening approach using a CRISPR single-guide RNA (sgRNA) library will enrich for cells where the target of the hit molecule (following compound treatment) has been knocked out. The control group would be cultured without hit molecule45326495150V1Attorney Docket No: 243735.000473treatment. The drug targets and other essential genes required for the activity of the hit molecule can then be identified by sequencing and comparing the data from the treated versus untreated groups of hit molecule.Polynucleotides

[0248] In a further aspect, provided herein is a polynucleotide encoding a fusion protein comprising a target protein and an inducible cell suicide protein, wherein when the fusion protein is expressed in a cell, the cell suicide protein induces the cell to undergo apoptosis in the presence of a suicide inducer.

[0249] In some embodiments, the fusion protein is expressed from a transgene integrated into the chromosome of the cell.

[0250] In some embodiments, the target protein is positioned at the N-terminus relative to the inducible cell suicide protein.

[0251] In some embodiments, the target protein is positioned at the C-terminus relative to the inducible cell suicide protein.

[0252] In some embodiments, the target protein is fused to the inducible cell suicide protein via a linker.

[0253] In some embodiments, the linker comprises about 5-20 amino acids.

[0254] In some embodiments, the linker comprises about 5-15 amino acids.

[0255] In some embodiments, the linker comprises about 10-15 amino acids.

[0256] In some embodiments, the linker comprises the amino acid sequence of GGSGGGSGGG (SEQ ID NO: 12), GGSGG (SEQ ID NO: 47), SGGGSG (SEQ ID NO: 48), (SGGGS)n (SEQ ID NO: 20), wherein n is 1, 2. or 3. (GGGGS)n (SEQ ID NO: 21). wherein n is 1, 2, or 3, GGGGGG (SEQ ID NO: 16), GGGGGGGG (SEQ ID NO: 17), GSAGSAAGSGEF (SEQ ID NO: 18), or EGKSSGSGSESKST (SEQ ID NO: 19).

[0257] In some embodiments, the inducible cell suicide protein comprises a ligand binding domain fused to a cell suicide domain.

[0258] In some embodiments, the ligand binding domain comprises an FKBP12 polypeptide, an FRB 1 polypeptide, or a variant, fragment, or combination thereof.

[0259] In some embodiments, the ligand binding domain comprises a variant of the FKBP12 polypeptide comprising an F36V substitution.46326495150V1Attorney Docket No: 243735.000473

[0260] In some embodiments, the variant of the FKBP12 polypeptide comprising an F36V substitution comprises the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 3), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the variant of the FKBP12 polypeptide comprising an F36V substitution comprises the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 3), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0261] In some embodiments, the ligand binding domain comprises two tandem copies of the variant of the FKBP12 polypeptide comprising an F36V substitution.

[0262] In some embodiments, the ligand binding domain comprises a variant of the FKBP12 polypeptide comprising G89P and I90K substitutions.

[0263] In some embodiments, the ligand binding domain comprises an FRB 1 polypeptide, or a variant or fragment thereof.

[0264] In some embodiments, the ligand binding domain comprises a variant of the FRB 1 polypeptide comprising a T2098L substitution.

[0265] In some embodiments, the ligand binding domain comprises a variant of the FRB 1 polypeptide comprising a T2098L substitution and a variant of the FKBP12 polypeptide comprising G89P and I90K substitutions.

[0266] In some embodiments, the variant of the FKBP12 polypeptide comprising G89P and I90K substitutions comprises the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPPKIPPHATLVFDVELLKLE (SEQ ID NO: 49), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the variant of the FKBP12 polypeptide comprising G89P and I90K substitutions comprises the amino acid sequence of47326495150V1Attorney Docket No: 243735.000473GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPPKIPPHATLVFDVELLKLE (SEQ ID NO: 49), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0267] In some embodiments, the variant of the FRB1 polypeptide comprising a T2098L substitution comprises the amino acid sequence of EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEA QEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK (SEQ ID NO: 50), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the variant of the FRB1 polypeptide comprising a T2098L substitution comprises the amino acid sequence of EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEA QEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK (SEQ ID NO: 50), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0268] In some embodiments, the cell suicide domain comprises a caspase-9 polypeptide, a caspase-8 polypeptide, a caspase- 1 polypeptide, a caspase-3 polypeptide, a FADD polypeptide, a Fas polypeptide, or a mBax polypeptide, or a variant, fragment, or combination thereof.

[0269] In some embodiments, the cell suicide domain comprises a variant of the caspase-9 polypeptide comprising Q221R substitution.

[0270] In some embodiments, the variant of the caspase-9 polypeptide comprising Q221R substitution comprises the amino acid sequence of GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRF SSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVY GTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNP EPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQ WAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 2), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments,48326495150V1Attorney Docket No: 243735.000473the variant of the caspase-9 polypeptide comprising Q221R substitution comprises the amino acid sequence of GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRF SSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVY GTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNP EPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQ WAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 2), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0271] In some embodiments, the cell suicide domain comprises a variant of the caspase-8 polypeptide comprising amino acids 216-479 of a wild-type caspase-8 protein.

[0272] In some embodiments, the variant of the caspase-8 polypeptide comprising amino acids 216-479 of a wild-type caspase-8 protein comprises the amino acid sequence of SESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKLHSIRDRNGTHLDAGALTTTFE ELHFEIKPHDDCTVEQIYEILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPIYELT SQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSEEQPYLEMDLSSPQTRYIPDEA DFLLGMATVNNCVSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNYEVSNKDDK KNMGKQMPQPTFTLRKKLVFPSD (SEQ ID NO: 51), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the variant of the caspase-8 polypeptide comprising amino acids 216-479 of a wild-type caspase-8 protein comprises the amino acid sequence of SESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKLHSIRDRNGTHLDAGALTTTFE ELHFEIKPHDDCTVEQIYEILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPIYELT SQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSEEQPYLEMDLSSPQTRYIPDEA DFLLGMATVNNCVSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNYEVSNKDDK KNMGKQMPQPTFTLRKKLVFPSD (SEQ ID NO: 51), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.49326495150V1Attorney Docket No: 243735.000473

[0273] In some embodiments, the caspase- 1 polypeptide comprises the amino acid sequence of MADKVLKEKRKLFIRSMGEGTINGLLDELLQTRVLNKEEMEKVKRENATVMDKTRALI DSVIPKGAQACQICITYICEEDSYLAGTLGLSADQTSGNYLNMQDSQGVLSSFPAPQAVQ DNPAMPTSSGSEGNVKLCSLEEAQRIWKQKSAEIYPIMDKSSRTRLALIICNEEFDSIPRR TGAEVDITGMTMLLQNLGYSVDVKKNLTASDMTTELEAFAHRPEHKTSDSTFLVFMSH GIREGICGKKHSEQVPDILQLNAIFNMLNTKNCPSLKDKPKVIIIQACRGDSPGVVWFKD SVGVSGNLSLPTTEEFEDDAIKKAHIEKDFIAFCSSTPDNVSWRHPTMGSVFIGRLIEHMQ EYACSCDVEEIFRKVRFSFEQPDGRAQMPTTERVTLTRCFYLFPGH (SEQ ID NO: 52), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the caspase- 1 polypeptide comprises the amino acid sequence of MADKVLKEKRKLFIRSMGEGTINGLLDELLQTRVLNKEEMEKVKRENATVMDKTRALI DSVIPKGAQACQICITYICEEDSYLAGTLGLSADQTSGNYLNMQDSQGVLSSFPAPQAVQ DNPAMPTSSGSEGNVKLCSLEEAQRIWKQKSAEIYPIMDKSSRTRLALIICNEEFDSIPRR TGAEVDITGMTMLLQNLGYSVDVKKNLTASDMTTELEAFAHRPEHKTSDSTFLVFMSH GIREGICGKKHSEQVPDILQLNAIFNMLNTKNCPSLKDKPKVIIIQACRGDSPGVVWFKD SVGVSGNLSLPTTEEFEDDAIKKAHIEKDFIAFCSSTPDNVSWRHPTMGSVFIGRLIEHMQ EYACSCDVEEIFRKVRFSFEQPDGRAQMPTTERVTLTRCFYLFPGH (SEQ ID NO: 52), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0274] In some embodiments, the caspase-3 polypeptide comprises the amino acid sequence of MENTENSVDSKSIKNLEPKIIHGSESMDSGISLDNSYKMDYPEMGLCIIINNKNFHKSTG MTSRSGTDVDAANLRETFRNLKYEVRNKNDLTREEIVELMRDVSKEDHSKRSSFVCVL LSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLTGKPKLFIIQACRGTELDCGIETDSGVD DDMACHKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCAMLKQYADKLEFMHILT RVNRKVATEFESFSFDATFHAKKQIPCIVSMLTKELYFYH (SEQ ID NO: 53), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the caspase-3 polypeptide comprises the amino acid sequence of MENTENSVDSKSIKNLEPKIIHGSESMDSGISLDNSYKMDYPEMGLCIIINNKNFHKSTG MTSRSGTDVDAANLRETFRNLKYEVRNKNDLTREEIVELMRDVSKEDHSKRSSFVCVL50326495150vlAttorney Docket No: 243735.000473LSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLTGKPKLFIIQACRGTELDCGIETDSGVD DDMACHKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCAMLKQYADKLEFMHILT RVNRKVATEFESFSFDATFHAKKQIPCIVSMLTKELYFYH (SEQ ID NO: 53), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%. at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0275] In some embodiments, the cell suicide domain comprises a variant of the FADD polypeptide comprising amino acids 1-125 of a wild-type FADD protein.

[0276] In some embodiments, the variant of the FADD polypeptide comprising amino acids 1-125 of a wild-type FADD protein comprises the amino acid sequence of MDPFLVLLHSVSSSLSSSELTELKFLCLGRVGKRKLERVQSGLDLFSMLLEQNDLEPGHT ELLRELLASLRRHDLLRRVDDFEAGAAAGAAPGEEDLCAAFNVICDNVGKDWRRLARQ LKVSDTK (SEQ ID NO: 54), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the variant of the FADD polypeptide comprising amino acids 1-125 of a wild- type FADD protein comprises the amino acid sequence of MDPFLVLLHSVSSSLSSSELTELKFLCLGRVGKRKLERVQSGLDLFSMLLEQNDLEPGHT ELLRELLASLRRHDLLRRVDDFEAGAAAGAAPGEEDLCAAFNVICDNVGKDWRRLARQ LKVSDTK (SEQ ID NO: 54), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0277] In some embodiments, the cell suicide domain comprises a variant of the Fas polypeptide comprising amino acids 175-304 of a wild-type Fas protein.

[0278] In some embodiments, the variant of the Fas polypeptide comprising amino acids 175-304 of a wild-type Fas protein comprises the amino acid sequence of IHSGQCPAAIVGGINVLLKPNTSVQFLRLGMLSPEGTCKAFDTAGNGYCRSEGVVAVLL TKKSLARRVYATILNAGTNTDGFKEQGVTFPSGDIQEQLIRSLYQSAGVAPESFEYIEAH GTGTKVGDPQE (SEQ ID NO: 55), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the variant of the Fas polypeptide comprising amino acids 175-304 of a wild-type Fas protein comprises the amino acid sequence of51326495150V1Attorney Docket No: 243735.000473IHSGQCPAAIVGGINVLLKPNTSVQFLRLGMLSPEGTCKAFDTAGNGYCRSEGVVAVLL TKKSLARRVYATILNAGTNTDGFKEQGVTFPSGDIQEQLIRSLYQSAGVAPESFEYIEAH GTGTKVGDPQE (SEQ ID NO: 55), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0279] In some embodiments, the mBax polypeptide comprises the amino acid sequence of MDGSGEQLGSGGPTSSEQIMKTGAFLLQGFIQDRAGRMAGETPELTLEQPPQDASTKKL SECLRRIGDELDSNMELQRMIADVDTDSPREVFFRVAADMFADGNFNWGRVVALFYFA SKLVLKALCTKVPELIRTIMGWTLDFLRERLLVWIQDQGGWEGLLSYFGTPTWQTVTIF VAGVLTASLTIWKKMG (SEQ ID NO: 56), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the mBax polypeptide comprises the amino acid sequence of MDGSGEQLGSGGPTSSEQIMKTGAFLLQGFIQDRAGRMAGETPELTLEQPPQDASTKKL SECLRRIGDELDSNMELQRMIADVDTDSPREVFFRVAADMFADGNFNWGRVVALFYFA SKLVLKALCTKVPELIRTIMGWTLDFLRERLLVWIQDQGGWEGLLSYFGTPTWQTVTIF VAGVLTASLTIWKKMG (SEQ ID NO: 56), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.

[0280] In some embodiments, the ligand binding domain fused to a cell suicide domain via a linker.

[0281] In some embodiments, the linker comprises the amino acid sequence of GGSGGGSGGG (SEQ ID NO: 12), GGSGG (SEQ ID NO: 47), SGGGSG (SEQ ID NO: 48), (SGGGS)n (SEQ ID NO: 20), wherein n is 1, 2, or 3. (GGGGS)n (SEQ ID NO: 21), wherein n is 1, 2, or 3, GGGGGG (SEQ ID NO: 16), GGGGGGGG (SEQ ID NO: 17), GSAGSAAGSGEF (SEQ ID NO: 18), or EGKSSGSGSESKST (SEQ ID NO: 19). In one embodiment, the linker comprises the amino acid sequence of SGGGS (SEQ ID NO: 9).

[0282] In some embodiments, the inducible cell suicide protein further comprises an HA tag at the C-terminus.52326495150V1Attorney Docket No: 243735.000473

[0283] In some embodiments, the HA tag comprises the amino acid sequence of YPYDVPDYALD (SEQ ID NO: 6).

[0284] In some embodiments, the inducible cell suicide protein comprise the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGF GDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSS LHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGT DGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEP DATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWA HSEDLQSLLLRV AN AVSVKGIYKQMPGCFNFLRKKLFFKTSVD YPYDVPDYALD (SEQ ID NO: 7), or an amino acid sequence having at least about 90% sequence identity thereof. In some embodiments, the inducible cell suicide protein comprise the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGF GDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSS LHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVYGT DGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEP DATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWA HSEDLQSLLLRV ANAVSVKGIYKQMPGCFNFLRKKLFFKTSVD YPYDVPDYALD (SEQ ID NO: 7), or an amino acid sequence having at least about 80%, at least about 85%, at least about 90%. at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity thereof.Recombinant Vectors

[0285] In an additional aspect, provided herein is a recombinant vector comprising the polynucleotide as described herein.

[0286] In some embodiments, the vector is a viral vector.

[0287] In some embodiments, the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, a baculoviral vector, or a vaccinia virus vector.53326495150V1Attorney Docket No: 243735.000473

[0288] In some embodiments, the vector is a non-viral vector.

[0289] In some embodiments, the non-viral vector is a minicircle plasmid, a Sleeping Beauty transposon, a PiggyBac transposon, or a single or double stranded DNA molecule that is used as a template for homology directed repair (HDR)-based gene editing.Cells

[0290] In a further aspect, provided herein is a cell comprising the polynucleotide as described herein or the recombinant vector as described herein.EXAMPLES

[0291] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.Example 1. iCADD Design and Initial Testing.

[0292] The MSCV vector backbone was used to clone several target POIs, such as Ikaros ZF23, Kras, Skpl, Cyclin DI, Skp2, cMyc, and Ringol in fusion with the iCasp9M gene with an HA-tag at the C-terminal via an SGGGS (SEQ ID NO: 9) linker (Figure 4A). The iCasp9M gene encodes the FKBP12 F36V-ACaspase9M (iCasp9M) fusion protein (SEQ ID NO: 5). The sequence of the FKBP12 F36V-ACaspase9M (iCasp9M)-HA fusion protein is SEQ ID NO: 7. The sequence of the POI Ikaros ZF23 domain is SEQ ID NO: 8. The resulting fusion constructs were then stably expressed in HeLa cells via viral transduction. Notably, the expression levels of high molecular weight proteins were lower compared to those of smaller molecular weight proteins. In addition, HeLa stable cell lines were prepared that only expressed either iCasp9M or empty vector (EV) as controls.

[0293] Treatment with 10 nM API 903 induced robust and efficient cell death in HeLa cells expressing iCasp9M-HA alone as well as in those cells expressing target POI-iCasp9M fusion constructs. Notably, the cells expressing POI-iCasp9M fusion proteins underwent nearly 100% cell death within 2-6 hours. In contrast, AP1903 treatment did not affect the viability of HeLa-EV cells. Interestingly, as the molecular weight of the target POI increased (e.g., Cyclin DI, Skp2, cMyc), the onset of cell death was progressively delayed (Figures 4B-4C). Optimization experiments revealed that increasing the linker length between the target POI and iCasp9M gene may further enhance killing efficiency in case of large molecular constructs. It was hypothesized54326495150V1Attorney Docket No: 243735.000473that large molecular weight proteins might create structural hindrance to AP1903-induced dimerization of iCasp9M-HA, thereby compromising or delaying its killing efficiency. To address this, the linker length was extended between the target POI and iCasp9M-HA genes by two- or three-fold, particularly for large molecular weight proteins such as Skp2 and cMyc. As hypothesized, the longer linker markedly enhanced the killing efficiency of API 903 in the 96-well plate format (Figure 4D).Example 2. Characterization of iCADD.

[0294] To demonstrate that the specific elimination of a target POI results in a survival phenotype, a genetic knockdown experiment was performed in HeLa-KrasWT-iCasp9M-HA cells by using specific (si-Kras) and non-specific (si-Skpl) si-RNAs. Knockdown of Kras (via pretreatment with si-Kras) in HeLa-KrasWT-iCasp9M-HA cells efficiently rescued API 903-induced cell death, while non-specific knockdown (via pretreatment with the non-specific si-Skpl) could not reverse cell death. Conversely, the pretreatment with both si-Kras and si-Skpl could not rescue AP1903-induced cell death in HeLa-iCasp9M-HA cells which lack POI (e.g., Kras) (Figure 5A). These findings provided proof-of-concept validation for the initial rationale of developing the iCADD system, demonstrating that co-depletion of iCasp9M and the target POI resulted in a survival phenotype upon API 903 treatment.

[0295] For further characterization, the iCADD system was tested with specific small molecule protein degraders and a translational inhibitor cycloheximide (CHX) to assess whether smallmolecule-induced co-degradation of iCasp9M with the target POI or inhibition of its protein synthesis results in a survival phenotype. The pretreatment of HeLa-Ikaros-ZF23-iCasp9M-HA cells with Ikaros degraders (e.g., Iberdomide, Pomalidomide, and Lenalidomide) rescued the AP1903-induced cell death in a dose-dependent manner, resulting in a survival phenotype.Interestingly, the iCADD assay effectively distinguished the potency differences among the degraders. For example. Iberdomide (the most potent Ikaros degrader) exhibited better rescue effects at 0.1 pM, 1 pM, and 5 pM compared to Pomalidomide and Lenalidomide, which are less potent degraders. Likewise, the translation inhibitor CHX also rescued AP1903-induced cell death. However, the rescue effect was not observed for AP1903-induced killing in HeLa-iCasp9M-HA cells, which lack the target POI (e.g., Ikaros ZF23), upon pretreatment with Ikaros degraders, even at concentrations up to 10 pM. Parallel western blot analyses confirmed that55326495150V1Attorney Docket No: 243735.000473Ikaros degraders (Iberdomide and Pomalidomide) induced dose-dependent degradation of Ikaros-ZF23-iCasp9M-HA within 6 hours in Hela cells (Figure 5B).

[0296] To further validate the specificity and utility of the iCADD platform for degrader discovery, the same rescue experiment was performed using the PROTAC degrader LC-2.Pretreatment with PROTAC degrader LC-2, which specifically degrades Kras (G72C) mutant, dose-dependently rescued AP1903-induced cell death in HeLa-Kras-G72C-iCasp9M-HA cells, while it could not rescue cell death in HeLa-KrasWT-iCasp9M-HA cells, used as a negative control. Consistently, western blot analysis confirmed that LC-2 induced Kras degradation only in Hela-Kras-G72C-iCasp9M-HA cells, with no detectable effect in Hela-KrasWT-iCasp9M-HA cells (Figure 5C). These findings reinforce the specificity of the iCADD system in detecting degrader molecules targeting a particular POL such as Kras (G72C).Example 3. iCADD Validation.

[0297] To evaluate the feasibility of the iCADD system as a high-throughput screening platform, a small-scale test screen was performed using an FDA-approved library comprising -3800 compounds. For this purpose, the iCADD system was adapted using K562 suspension cells and a stable expressing K562-Ikaros-ZF23-iCasp9M-HA cell line was successfully prepared. It was tested if the iCADD system was able to recognize Ikaros degraders included in the compound library as top hits (Figure 6A). To ensure the iCADD platform's robustness in rapidly identifying survival phenotypes, the total assay time was optimized to approximately 15-16 hours.

[0298] The screen identified different Ikaros degraders amongst the top hits when tested at both the concentrations of 1 pM and 10 pM. At a 1 pM concentration, the screen identified CC-92480 / Mezigdomide, the most potent Ikaros degrader reported to date, as the second top hit, causing >60% inhibition of API 903 activity. Additionally, Iberdomide and Pomalidomide were identified, each causing 41% inhibition at a 1 pM concentration. At 10 pM, the screen again highlighted Iberdomide, CC-92480 / Mezigdomide, and Pomalidomide among the top hits, all causing >60% inhibition of API 903 activity. Furthermore, Avadomide, another Ikaros degrader, was identified, causing 57% inhibition. In addition to degraders, the screen identified compounds with alternative mechanisms of action, including pro-caspase inhibitors (e.g., Emricasan and Q-VD-Oph), FKBP12 binders (e.g.. Rapamycin), and compounds that disrupt transcription or translation, all which inhibit the iCADD system by means different from the degradation of56326495150V1Attorney Docket No: 243735.000473Ikaros-ZF23 (Figure 6B). These “false positives” can be excluded through a counter- screen using cells expressing only iCasp9M without the target POI. Further small-scale pilot screens with other target POIs (e.g.. cMyc, Cyclin DI, and Kras) identified specific hits (Figure 6C). Overall, these small-scale screens in a 384-well plate format demonstrate the iCADD system's capability to efficiently identify protein degraders from large chemical libraries, underscoring its feasibility as a high-throughput screening platform.

[0299] Below are the methods used in the Examples described above.Amplification and cloning of the iCasp9M-HA fusion protein with target POIs (Figure 4A)

[0300] The FKBP12 F36V-ACaspase9M (iCasp9M)-HA fusion protein was amplified from the Addgene plasmid pMSCV-F-del Casp9.IRES.GFP and cloned along with various target POIs, including Ikaros ZF23, Kras, Skpl, Cyclin DI, Skp2, and cMyc, into the retroviral vector MSCV-puro. The POIs were positioned at the N-terminus of iCasp9M, separated by an SGGGS (SEQ ID NO: 9) linker. These fusion constructs were co-transfected into HEK293T cells along with viral packaging (gag-pol) and envelope (VSV-G) plasmids. After 72 hours, the viruscontaining supernatant was collected, filtered through a 0.45 pm filter, added to 8 pg / ml Polybrene, and used to transduce HeLa cells. Following overnight incubation at 37 °C, the viral medium was replaced with fresh medium, and the cells were allowed to recover for 24 hours. The transduced cells were then selected using 1-2 pg / ml puromycin. Stable cell lines expressing the desired POI-iCasp9M fusion proteins were confirmed via western blotting. Additionally, HeLa stable cell lines expressing either iCasp9M alone or an empty vector (EV) were generated as controls.Testing of cell lines expressing target P01-iCasp9M-HA in 96-well plate format (Figure 4C)

[0301] Testing of HeLa stable cell lines expressing either target POLiCasp9M-HA or iCasp9M / empty vector (EV) as controls was conducted in a 96-well plate format. For each stable cell line, 20,000 cells in 50 pL of media per well were seeded into a solid white, tissue culture-treated, 96-well microplate and allowed to adhere overnight at 37 °C with 5% CO2. The following day, cells were treated with either DMSO or 10 nM AP1903 at various time intervals ranging from 0 to 24 hours. After treatment, the plate was equilibrated to room temperature for 30 minutes, followed by the addition of 50 pL of CellTiter-Glo 2.0 reagent per well. The plate was shaken for two minutes to induce cell lysis and incubated at room temperature for 10 minutes to stabilize the luminescent signal before measurement.57326495150V1Attorney Docket No: 243735.000473Genetic knockdown experiments (Figure 5A)

[0302] Genetic knockdown experiments were performed in HeLa-KrasWT-iCasp9M-HA and HeLa-iCasp9M-HA (control) cells. Each cell line was seeded at 35,000 cells in 100 pL of media per well in a solid, white, tissue culture-treated 96-well microplate and allowed to attach overnight at 37 °C with 5% CO2. The following day, cells were transfected with either specific (si-Kras). non-specific (si-Skpl), or control (si-NT) siRNAs and incubated for 48 hours. Media was then replaced with 100 pL of fresh media containing either DMSO or AP1903, followed by a 24-hour incubation. Subsequently, the plate was equilibrated to room temperature for 30 minutes before the addition of 100 pL of CellTiter-Glo 2.0 reagent per well. The plate was shaken for two minutes to induce cell lysis and incubated at room temperature for 10 minutes to stabilize the luminescent signal before measurement.Rescue experiments (Figure 5B)

[0303] A rescue experiment was conducted using Hela-Ikaros-ZF23-iCasp9M-HA cells, with Hela-iCasp9M-HA cells serving as a control. For this experiment. 35.000 cells in 100 pL of media were seeded per well in a solid, white, 96-well, tissue culture-treated microplate and incubated overnight at 37 °C with 5% CO2. The following day, cells were treated with increasing concentrations of Ikaros molecular-glue degraders (Iberdomide, Pomalidomide, and Lenalidomide) or the translational inhibitor cycloheximide (CHX) for 6 hours. The media was then replaced with 100 pL of fresh media containing either DMSO or API 903. and cells were incubated for an additional 12 hours. Subsequently, the plate was equilibrated to room temperature for 30 minutes before the addition of 100 pL of CellTiter-Glo 2.0 per well. After two minutes of shaking to induce cell lysis and a 10-minute incubation to stabilize the luminescent signal, the luminescence was measured.Small-scale high-throughput test screen (Figure 6A)

[0304] A small-scale test screen was conducted in a 384-well plate format. For this study, an FDA-approved compound library was used, comprising -3,800 compounds including known Ikaros degraders. The iCADD system was adapted to K562 suspension cells, and a stably expressing K562-Ikaros-ZF23-iCasp9M-HA cell line was successfully established. Cells were seeded at a density of 1,000 cells per well in 20 pL of media. Subsequently, 5 pL of media containing each compound was added to achieve a final concentration of 1 pM or 10 pM in a total volume of 25 pL. After a 3-hour incubation, 5 pL of media containing API 903 was added58326495150V1Attorney Docket No: 243735.000473to reach a final concentration of 20 nM in a total volume of 30 pL. Following an overnight (~12 hour) incubation, 30 pL of CellTiter-Glo 2.0 was added per well to induce cell lysis. Plates were incubated at room temperature, and luminescence was measured at 1-hour and 3-hour intervals. The data were analyzed as the percentage inhibition of AP1903-induced activity.List of SequencesSEQ ID NO: 1 ACaspase9 with Q221 marked in bold GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRF SSLHFMVEVKGDLTAKKMVLALLELAQQDHGALDCCVVVILSHGCQASHLQFPGAVY GTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNP EPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQ WAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSSEQ ID NO: 2 ACaspase9 with Q221R mutation marked in bold (ACaspase9M) GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRF SSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFPGAVY GTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNP EPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQ WAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSSEQ ID NO: 3 FKBP12 F36V (also known as DmrB or dTAG domain) GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESEQ ID NO: 4 FKBP12 F36 -ACaspase9 (iCasp9) GYOYETISPGDGRTFPKRGOTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAOMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGF GDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRERTGSNIDCEKLRRRFSSLHFM VEVKGDLTAKKMVLALLELAQQDHGALDCCWVILSHGCQASHLQFPGAVYGTDGCPVSVE KIVNIFNGTSCPSLGGKPKEFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLR59326495150vlAttorney Docket No: 243735.000473TFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVA NA VSVKGIYKQMPGCFNFLRKKLFFKTSSEQ ID NO: 5 FKBP12 36N- Casvase9M (iCasp9M) GYOYETISPGDGRTFPKRGOTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRG WEEGVAOMSVGORAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGF GDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFM VEVKGDLTAKKMVLALLELAR QDHGALDCCWVILSHGCQASHLQFPGA VYGTDGCPVSVE KIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLR TFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVA NA VSVKGIYKQMPGCFNFLRKKLFFKTSSEQ ID NO: 6 hemagglutinin (HA) tagYPYDVPDYALDSEQ ID NO: 7 FKBP12 F36N-ACaspase9M (iCasp9M)-HA GVOVETISPGDGRTFPKRGOTCVVHYTGMLEDGKKYDSSRDRNKPFKFMLGKQEVIRG WEEGVAOMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSGVDGF GDVGALESLRGNADLAY1LSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFM VEVKGDLTAKKMVLALLELAR QDHGALDCCWVILSHGCQASHLQFPGA VYGTDGCPVSVE KIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLR TFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVA NAVSVKGIYKQMPGCFNFLRKKLFFKTSN YPYDVPDYALDSEQ ID NO: 8 Ikaros ZF23 domain VHKRSHTGERPFQCNQCGASFTQKGNLLRHIKLHSGEKPFKCHLCNYACRRRDALTGH LRTHSSEQ ID NO: 9 LI linkerSGGGS60326495150vlAttorney Docket No: 243735.000473 SEQ ID NO: 10 L2 linkerSGGGSSGGGS SEQ ID NO: 11 L3 linkerSGGGS SGGGSSGGGS SEQ ID NO: 12 LinkerGGSGGGSGGG SEQ ID NO: 13 LinkerGGGGS SEQ ID NO: 14 LinkerGGGGSGGGGS SEQ ID NO: 15 LinkerGGGGSGGGGSGGGGS SEQ ID NO: 16 LinkerGGGGGG SEQ ID NO: 17 LinkerGGGGGGGG SEQ ID NO: 18 LinkerGSAGSAAGSGEF SEQ ID NO: 19 LinkerEGKSSGSGSESKST SEQ ID NO: 20 Linker61326495150V1Attorney Docket No: 243735.000473(SGGGS)n, n = 1, 2, or 3SEQ ID NO: 21 Linker(GGGGS)n, n = 1, 2, or 3SEQ ID NO: 22 FAM96B MVGGGGVGGGLLENANPLIYQRSGERPVTAGEEDEQVPDSIDAREIFDLIRSINDPEHPLT LEELNVVEQVRVQVSDPESTVAVAFTPTIPHCSMATLIGLSIKVKLLRSLPQRFKMDVHI TPGTHASEHAVNKQLADKERVAAALENTHLLEVVNQCLSARSSEQ ID NO: 23 PDCD1 MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSN TSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRN DSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTP EPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPLSEQ ID NO: 24 SEC61A1 MAIKFLEVIKPFCVILPEIQKPERKIQFKEKVLWTAITLFIFLVCCQIPLFGIMSSDSADPFY WMRVILASNRGTLMELGISPIVTSGLIMQLLAGAKIIEVGDTPKDRALFNGAQKLFGMIIT IGQSIVYVMTGMYGDPSEMGAGICLLITIQLFVAGLIVLLLDELLQKGYGLGSGISLFIAT NICETIVWKAFSPTTVNTGRGMEFEGAIIALFHLLATRTDKVRALREAFYRQNLPNLMNL IATIFVFAVVIYFQGFRVDLPIKSARYRGQYNTYPIKLFYTSNIPIILQSALVSNLYVISQML SARFSGNLLVSLLGTWSDTSSGGPARAYPVGGLCYYLSPPESFGSVLEDPVHAVVYIVF MLGSCAFFSKTWIEVSGSSAKDVAKQLKEQQMVMRGHRETSMVHELNRYIPTAAAFG GLCIGALSVLADFLGAIGSGTGILLAVTIIYQYFEIFVKEQSEVGSMGALLFSEQ ID NO: 25 HMGCS1 MPGSLPLNAEACWPKDVGIVALEIYFPSQYVDQAELEKYDGVDAGKYTIGLGQARMGF CTDRED1NSLCLTVVQNLMERNSLSYDCIGRLEVGTETIIDKSKSVKSNLMQLFEESGNT DIEGIDTTN AC YGGTAA VFNA VNWIES S S WDGRYALVVAGDIAIYATGN ARPTGGVGA62326495150vlAttorney Docket No: 243735.000473VALLIGPNAPLTFDRGLRGTHMQHAYDFYKPDMLSEYPIVDGKLSIQCYLSALDRCYSV YRKKIRAQWQKEGNDNDFTLNDFGFMISHSPYCKLVQKSLARMFLNDFLNDQNRDKNS IYSGLEAFGDVKLEDTYFDRDVEKAFMKASSELFNQKTKASLLVSNQNGNMYTSSVYG SLASVLAQYSPQQLAGKRIGVFSYGSGLAATLYSLKVTQDATPGSALDKVTASLCDLKS RLDSRTCVAPDVFAENMKLREDTHHLANYIPQCSIDSLFEGTWYLVRVDEKHRRTYAR RPSTNDHNLGDGVGLVHSNTATEHIPSPAKKVPRLPATAAESESAVISNGEHSEQ ID NO: 26 ANAPC10 MTTPNKTPPGADPKQLERTGTVREIGSQAVWSLSSCKPGFGVDQLRDDNLETYWQSDG SQPHLVNIQFRRKTTVKTLCIYADYKSDESYTPSKISVRVGNNFHNLQEIRQLELVEPSG WIHVPLTDNHKKPTRTFMIQIAVLANHQNGRDTHMRQIKIYTPVEESSIGKFPRCTTIDF MMYRSIRSEQ ID NO: 27 IRF4 MNLEGGGRGGEFGMSAVSCGNGKLRQWLIDQIDSGKYPGLVWENEEKSIFRIPWKHAG KQDYNREEDAALFKAWALFKGKFREGIDKPDPPTWKTRLRCALNKSNDFEELVERSQL DISDPYKVYRIVPEGAKKGAKQLTLEDPQMSMSHPYTMTTPYPSLPAQVHNYMMPPLD RSWRDYVPDQPHPEIPYQCPMTFGPRGHHWQGPACENGCQVTGTFYACAPPESQAPGV PTEPSIRSAEALAFSDCRLHICLYYREILVKELTTSSPEGCRISHGHTYDASNLDQVLFPYP EDNGQRKNIEKLLSHLERGVVLWMAPDGLYAKRLCQSRIYWDGPLALCNDRPNKLERD QTCKLFDTQQFLSELQAFAHHGRSLPRFQVTLCFGEEFPDPQRQRKLITAHVLRSLASEQ ID NO: 28 CCND2 MELLCHEVDPVRRAVRDRNLLRDDRVLQNLLTIEERYLPQCSYFKCVQKDIQPYMRRM VATWMLEVCEEQKCEEEVFPLAMNYLDRFLAGVPTPKSHLQLLGAVCMFLASKLKETS PLTAEKLCIYTDNSIKPQELLEWELVVLGKLKWNLAAVTPHDFIEHILRKLPQQREKLSLI RKHAQTFIALCATDFKFAMYPPSMIATGSVGAAICGLQQDEEVSSLTCDALTELLAKITN TDVDCLKACQEQIEAVLLNSLQQYRQDQRDGSKSEDELDQASTPTDVRDIDLSEQ ID NO: 29 N-myc DDEDDEEEDEEEEIDVVTVEKRRSSSNTKAVTTFTITVRPKNAALGPGRAQSSELILKRCL63326495150vlAttorney Docket No: 243735.000473PIHQQHNYAAPSPYVESEDAPPQKKIKSEASPRPLKSVIPPKAKSLSPRNSDSEDSERRRN HNILERQRRNDLRSSFLTLRDHVPELVKNEKAAKVVILKKATEYVHSLQAEEHQLLLEK EKLQARQQQLLKKIEHARTCSEQ ID NO: 30 SOXIO MAEEQDLSEVELSPVGSEEPRCLSPGSAPSLGPDGGGGGSGLRASPGPGELGKVKKEQQ DGEADDDKFPVCIREAVSQVLSGYDWTLVPMPVRVNGASKSKPHVKRPMNAFMVWA QAARRKLADQYPHLHNAELSKTLGKLWRLLNESDKRPFIEEAERLRMQHKKDHPDYKY QPRRRKNGKAAQGEAECPGGEAEQGGTAAIQAHYKSAHLDHRHPGEGSPMSDGNPEH PSGQSHGPPTPPTTPKTELQSGKADPKRDGRSMGEGGKPHIDFGNVDIGEISHEVMSNME TFDVAELDQYLPPNGHPGHVSSYSAAGYGLGSALAVASGHSAWISKPPGVALPTVSPPG VDAKAQVKTETAGPQGPPHYTDQPSTSQIAYTSLSLPHYGSAFPSISRPQFDYSDHQPSG PYYGHSGQASGLYSAFSYMGPSQRPLYTAISDPSPSGPQSHSPTHWEQPVYTTLSRPSEQ ID NO: 31 SEH1L MFVARSIAADHKDLIHDVSFDFHGRRMATCSSDQSVKVWDKSESGDWHCTASWKTHS GSVWRVTWAHPEFGQVLASCSFDRTAAVWEEIVGESNDKLRGQSHWVKRTTLVDSRT SVTDVKFAPKHMGLMLATCSADGIVRIYEAPDVMNLSQWSLQHEISCKLSCSCISWNPS SSRAHSPMIAVGSDDSSPNAMAKVQIFEYNENTRKYAKAETLMTVTDPVHDIAFAPNLG RSFHILAIATKDVRIFTLKPVRKELTSSGGPTKFEIHIVAQFDNHNSQVWRVSWNITGTVL ASSGDDGCVRLWKANYMDNWKCTGILKGNGSPVNGSSQQGTSNPSLGSTIPSLQNSLN GSSAGRKHSSEQ ID NO: 32 TBCB MEVTGVSAPTVTVFISSSLNTFRSEKRYSRSLTIAEFKCKLELLVGSPASCMELELYGVD DKFYSKLDQEDALLGSYPVDDGCRIHVIDHSGARLGEYEDVSRVEKYTISQEAYDQRQD TVRSFLKRSKLGRYNEEERAQQEAEAAQRLAEEKAQASSIPVGSRCEVSQISSLATGLVS AMMSHWGKMMAVSEQ ID NO: 33 KLF4 MRQPPGESDMAVSDALLPSFSTFASGPAGREKTLRQAGAPNNRWREELSHMKRLPPVL64326495150vlAttorney Docket No: 243735.000473PGRPYDLAAATVATDLESGGAGAACGGSNLAPLPRRETEEFNDLLDLDFILSNSLTHPPE S VAATVSS S AS ASS S SSPS S SGPAS APSTCSFTYPIR AGNDPGVAPGGTGGGLLYGRES AP PPTAPFNLADINDVSPSGGFVAELLRPELDPVYIPPQQPQPPGGGLMGKFVLKASLSAPGS EYGSPSVISVSKGSPDGSHPVVVAPYNGGPPRTCPKIKQEAVSSCTHLGAGPPLSNGHRP AAHDFPLGRQLPSRTTPTLGLEEVLSSRDCHPALPLPPGFHPHPGPNYPSFLPDQMQPQV PPLHYQGQSRGFVARAGEPCVCWPHFGTHGMMLTPPSSPLELMPPGSCMPEEPKPKRG RRSWPRKRTATHTCDYAGCGKTYTKSSHLKAHLRTHTGEKPYHCDWDGCGWKFARS DELTRHYRKHTGHRPFQCQKCDRAFSRSDHLALHMKRHFSEQ ID NO: 34 CEBPA MHLQPGHPTPPPTPVPSPHPAPALGAAGLPGPGSALKGLGAAHPDLRASGGSGAGKAK KSVDKNSNEYRVRRERNNIAVRKSRDKAKQRNVETQQKVLELTSDNDRLRKRVEQLSR ELDTLRGIFRQLPES SLVKAMGNCASEQ ID NO: 35 RPP25L MEHYRKAGSVELPAPSPMPQLPPDTLEMRVRDGSKIRNLLGLALGRLEGGSARHVVFSG SGRAAGKAVSCAEIVKRRVPGLHQLTKLRFLQTEDSWVPASPDTGLDPLTVRRHVPAV WVLLSRDPLDPNECGYQPPGAPPGLGSMPSSSCGPRSRRRARDTRSSEQ ID NO: 36 POU2AF1 MLWQKPTAPEQAPAPARPYQGVRVKEPVKELLRRKRGHASSGAAPAPTAVVLPHQPLA TYTTVGPSCLDMEGSVSAVTEEAALCAGWLSQPTPATLQPLAPWTPYTEYVPHEAVSCP YSADMYVQPVCPSYTVVGPSSVLTYASPPLITNVTTRSSATPAVGPPLEGPEHQAPLTYF PWPQPLSTLPTSTLQYQPPAPALPGPQFVQLPISIPEPVLQDMEDPRRAASSLTIDKLLLEE EDSDAYALNHTLSVEGFSEQ ID NO: 37 Timl7-A MEEYAREPCPWRIVDDCGGAFTMGTIGGGIFQAIKGFRNSPVGVNHRLRGSLTAIKTRA PQLGGSFAVWGGLFSMIDCSMVQVRGKEDPWNSITSGALTGAILAARNGPVAMVGSAA MGGILLALIEGAGILLTRFASAQFPNGPQFAEDPSQLPSTQLPSSPFGDYRQYQ65326495150vlAttorney Docket No: 243735.000473SEQ ID NO: 38 SMARCB 1 MMMMALSKTFGQKPVKFQLEDDGEFYMIGSEVGNYLRMFRGSLYKRYPSLWRRLATV EERKKIVASSHGKKTKPNTKDHGYTTLATSVTLLKASEVEEILDGNDEKYKAVSISTEPP TYLREQKAKRNSQWVPTLPNSSHFILDAVPCSTTINRNRMGRDKKRTFPLCFDDHDPAVI HENASQPEVLVPIRLDMEIDGQKLRDAFTWNMNEKLMTPEMFSEILCDDLDLNPLTFVP AIASAIRQQIESYPTDSILEDQSDQRVIIKLNIHVGNISLVDQFEWDMSEKENSPEKFALKL CSELGLGGEFVTTIAYSIRGQLSWHQKTYAFSENPLPTVEIAIRNTGDADQWCPLLETLT DAEMEKKIRDQDRNTRRMRRLANTAPAWSEQ ID NO: 39 SOCS3 MVTHSKFPAAGMSRPLDTSLRLKTFSSKSEYQLVVNAVRKLQESGFYWSAVTGGEANL LLSAEPAGTFLIRDSSDQRHFFTLSVKTQSGTKNLRIQCEGGSFSLQSDPRSTQPVPRFDC VLKLVHHYMPPPGAPSFPSPPTEPSSEVPEQPSAQPLPGSPPRRAYYIYSGGEKIPLVLSRP LSSNVATLQHLCRKTVNGHLDSYEKVTQLPGPIREFLDQYDAPLSEQ ID NO: 40 CDC26 MLRRKPTRLELKLDDIEEFENIRKDLETRKKQKEDVEVVGGSDGEGAIGLSSDPKSREQ MINDRIGYKPQPKPNNRSSQFGSLEFSEQ ID NO: 41 FOXA l MLGTVKMEGHETSDWNSYYADTQEAYSSVPVSNMNSGLGSMNSMNTYMTMNTMTTS GNMTPASFNMSYANPGLGAGLSPGAVAGMPGGSAGAMNSMTAAGVTAMGTALSPSG MGAMGAQQAASMNGLGPYAAAMNPCMSPMAYAPSNLGRSRAGGGGDAKTFKRSYP HAKPPYSYISLITMAIQQAPSKMLTLSEIYQWIMDLFPYYRQNQQRWQNSIRHSLSFNDC FVKVARSPDKPGKGSYWTLHPDSGNMFENGCYLRRQKRFKCEKQPGAGGGGGSGSGG SGAKGGPESRKDPSGASNPSADSPLHRGVHGKTGQLEGAPAPGPAASPQTLDHSGATAT GGASELKTPASSTAPPISSGPGALASVPASHPAHGLAPHESQLHLKGDPHYSFNHPFSINN LMSSSEQQHKLDFKAYEQALQYSPYGSTLPASLPLGSASVTTRSPIEPSALEPAYYQGVY SRPVLNTS66326495150vlAttorney Docket No: 243735.000473SEQ ID NO: 42 LRR1 MKLHCEVEVISRHLPALGLRNRGKGVRAVLSLCQQTSRSQPPVRAFLLISTLKDKRGTR YELRENIEQFFTKFVDEGKATVRLKEPPVDICLSKAISSSLKGFLSAMRLAHRGCNVDTP VSTLTPVKTSEFENFKTKMVITSKKDYPLSKNFPYSLEHLQTSYCGLVRVDMRMLCLKS LRKLDLSHNHIKKLPATIGDLIHLQELNLNDNHLESFSVALCHSTLQKSLRSLDLSKNKIK ALPVQFCQLQELKNLKLDDNELIQFPCKIGQLINLRFLSAARNKLPFLPSEFRNLSLEYLD LFGNTFEQPKVLPVIKLQAPLTLLESSARTILHNRIPYGSHIIPFHLCQDLDTAKICVCGRF CLNSFIQGTTTMNLHSVAHTVVLVDNLGGTEAPIISYFCSLGCYVNSSDMLKSEQ ID NO: 43 FAU MQLFVRAQELHTFEVTGQETVAQIKAHVASLEGIAPEDQVVLLAGAPLEDEATLGQCG VEALTTLEVAGRMLGGKVHGSLARAGKVRGQTPKVAKQEKKKKKTGRAKRRMQYNR RFVNVVPTFGKKKGPNANSSEQ ID NO: 44 GATA3 MEVTADQPRWVSHHHPAVLNGQHPDTHHPGLSHSYMDAAQYPLPEEVDVLFNIDGQG NHVPPYYGNSVRATVQRYPPTHHGSQVCRPPLLHGSLPWLDGGKALGSHHTASPWNLS PFSKTSIHHGSPGPLSVYPPASSSSLSGGHASPHLFTFPPTPPKDVSPDPSLSTPGSAGSAR QDEKECLKYQ VPLPDSMKLES SHSRGSMTALGGAS S STHHPITTYPPYVPEYS SGLFPPS S LLGGSPTGFGCKSRPKARSSTGRECVNCGATSTPLWRRDGTGHYLCNACGLYHKMNG QNRPLIKPKRRLSAARRAGTSCANCQTTTTTLWRRNANGDPVCNACGLYYKLHNINRP LTMKKEGIQTRNRKMSSKSKKCKKVHDSLEDFPKNSSFNPAALSRHMSSLSHISPFSHSS HMLTTPTPMHPPSSLSFGPHHPSSMVTAMGSEQ ID NO: 45 SYF2 MAAIAASEVLVDSAEEGSLAAAAELAAQKREQRLRKFRELHLMRNEARKLNHQEVVEE DKRLKLPANWEAKKARLEWELKEEEKKKECAARGEDYEKVKLLEISAEDAERWERKK KRKNPDLGFSDYAAAQLRQYHRLTKQIKPDMETYERLREKHGEEFFPTSNSLLHGTHVP STEEIDRMVIDLEKQIEKRDKYSRRRPYNDDADIDYINERNAKFNKKAERFYGKYTAEIK QNLERGTAV67326495150vlAttorney Docket No: 243735.000473SEQ ID NO: 46 SOX2 MYNMMETELKPPGPQQTSGGGGGNSTAAAAGGNQKNSPDRVKRPMNAFMVWSRGQR RKMAQENPKMHNSEISKRLGAEWKLLSETEKRPFIDEAKRLRALHMKEHPDYKYRPRR KTKTLMKKDKYTLPGGLLAPGGNSMASGVGVGAGLGAGVNQRMDSYAHMNGWSNG SYSMMQDQLGYPQHPGLNAHGAAQMQPMHRYDVSALQYNSMTSSQTYMNGSPTYSM SYSQQGTPGMALGSMGSVVKSEASSSPPVVTSSSHSRAPCQAGDLRDMISMYLPGAEVP EPAAPSRLHMSQHYQSGPVPGTAINGTLPLSHMSEQ ID NO: 47 LinkerGGSGGSEQ ID NO: 48 LinkerSGGGSGSEQ ID NO: 49 FKBP12 G89P,I90K with G89P and WOK mutations marked in bold GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVIRG WEEGVAQMSVGQRAKLTISPDYAYGATGHPPKIPPHATLVFDVELLKLESEQ ID NO: 50 FRB1 T2098L with T2098L mutation marked in bold EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDLMEA QEWCRKYMKSGNVKDLLQAWDLYYHVFRRISKSEQ ID NO: 51 Caspase-8216-479 SESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKLHSIRDRNGTHLDAGALTTTFE ELHFEIKPHDDCTVEQIYEILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPIYELT SQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSEEQPYLEMDLSSPQTRYIPDEA DFLLGMATVNNCVSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNYEVSNKDDK KNMGKQMPQPTFTLRKKLVFPSDSEQ ID NO: 52 Caspase-168326495150vlAttorney Docket No: 243735.000473MADKVLKEKRKLFIRSMGEGTINGLLDELLQTRVLNKEEMEKVKRENATVMDKTRAL1 DSVIPKGAQACQICITYICEEDSYLAGTLGLSADQTSGNYLNMQDSQGVLSSFPAPQAVQ DNPAMPTSSGSEGNVKLCSLEEAQRIWKQKSAEIYPIMDKSSRTRLALIICNEEFDSIPRR TGAEVDITGMTMLLQNLGYSVDVKKNLTASDMTTELEAFAHRPEHKTSDSTFLVFMSH GIREGICGKKHSEQVPDILQLNAIFNMLNTKNCPSLKDKPKVIIIQACRGDSPGVVWFKD SVGVSGNLSLPTTEEFEDDAIKKAHIEKDFIAFCSSTPDNVSWRHPTMGSVFIGRLIEHMQ EYACSCDVEEIFRKVRFSFEQPDGRAQMPTTERVTLTRCFYLFPGHSEQ ID NO: 53 Caspase-3 MENTENSVDSKSIKNLEPKIIHGSESMDSGISLDNSYKMDYPEMGLCIIINNKNFHKSTG MTSRSGTDVDAANLRETFRNLKYEVRNKNDLTREEIVELMRDVSKEDHSKRSSFVCVL LSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLTGKPKLFIIQACRGTELDCGIETDSGVD DDMACHKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCAMLKQYADKLEFMHILT RVNRKVATEFESFSFDATFHAKKQIPCIVSMLTKELYFYHSEQ ID NO: 54 FAD 125 MDPFLVLLHSVSSSLSSSELTELKFLCLGRVGKRKLERVQSGLDLFSMLLEQNDLEPGHT ELLRELLASLRRHDLLRRVDDFEAGAAAGAAPGEEDLCAAFNVICDNVGKDWRRLARQ LKVSDTKSEQ ID NO: 55 Fas 175-304 IHSGQCPAAIVGGINVLLKPNTSVQFLRLGMLSPEGTCKAFDTAGNGYCRSEGVVAVLL TKKSLARRVYATILNAGTNTDGFKEQGVTFPSGDIQEQLIRSLYQSAGVAPESFEYIEAH GTGTKVGDPQESEQ ID NO: 56 mBax MDGSGEQLGSGGPTSSEQIMKTGAFLLQGFIQDRAGRMAGETPELTLEQPPQDASTKKL SECLRRIGDELDSNMELQRMIADVDTDSPREVFFRVAADMFADGNFNWGRVVALFYFA SKLVLKALCTKVPELIRTIMGWTLDFLRERLLVWIQDQGGWEGLLSYFGTPTWQTVTIF VAGVLTASLTIWKKMG69326495150vlAttorney Docket No: 243735.000473SEQ ID NO: 57 Kras (e.g., Kras.4b) MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTA GQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNK CDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQGVDDAFYTLVREIRKHKEKMSKDGKK KKKKSKTKCVIMSEQ ID NO: 58 Skpl MPSIKLQSSDGEIFEVDVEIAKQSVTIKTMLEDLGMDDEGDDDPVPLPNVNAAILKKVIQ WCTHHKDDPPPPEDDENKEKRTDDIPVWDQEFLKVDQGTLFELILAANYLDIKGLLDVT CKTVANMIKGKTPEEIRKTFNIKNDFTEEEEAQVRKENQWCEEKSEQ ID NO: 59 Cyclin DI MSEHQLLCCEVETIRRAYPDANLLNDRVLRAMLKAEETCAPSVSYFKCVQKEVLPSMR KIVATWMLEVCEEQKCEEEVFPLAMNYLDRFLSLEPVKKSRLQLLGATCMFVASKMKE TIPLTAEKLCIYTDNSIRPEELLQMELLLVNKLKWNLAAMTPHDFIEHFLSKMPEAEENK QIIRKHAQTFVALCATDVKFISNPPSMVAAGSVVAAVQGLNLRSPNNFLSYYRLTRFLSR VIKCDPDCLRACQEQIEALLESSLRQAQQNMDPKAAEEEEEEEEEVDLACTPTDVRDVDISEQ ID NO: 60 Skp2 MHRKHLQEIPDLSSNVATSFTWGWDSSKTSELLSGMGVSALEKEEPDSENIPQELLSNLG HPESPPRKRLKSKGSDKDFVIVRRPKLNRENFPGVSWDSLPDELLLGIFSCLCLPELLKVS GVCKRWYRLASDESLWQTLDLTGKNLHPDVTGRLLSQGVIAFRCPRSFMDQPLAEHFS PFRVQHMDLSNSVIEVSTLHGILSQCSKLQNLSLEGLRLSDPIVNTLAKNSNLVRLNLSG CSGFSEFALQTLLSSCSRLDELNLSWCFDFTEKHVQVAVAHVSETITQLNLSGYRKNLQ KSDLSTLVRRCPNLVHLDLSDSVMLKNDCFQEFFQLNYLQHLSLSRCYDIIPETLLELGEI PTLKTLQVFGIVPDGTLQLLKEALPHLQINCSHFTTIARPTIGNKKNQEIWGIKCRLTLQK PSCLSEQ ID NO: 61 cMyc MPLNVSFTNRNYDLDYDSVQPYFYCDEEENFYQQQQQSELQPPAPSEDIWKKFELLPTP PLSPSRRSGLCSPSYVAVTPFSLRGDNDGGGGSFSTADQLEMVTELLGGDMVNQSFICDP70326495150vlAttorney Docket No: 243735.000473DDETFIKNIIIQDCMWSGFSAAAKLVSEKLASYQAARKDSGSPNPARGHSVCSTSSLYLQ DLSAAASECIDPSVVFPYPLNDSSSPKSCASQDSSAFSPSSDSLLSSTESSPQGSPEPLVLHE ETPPTTSSDSEEEQEDEEEIDVVSVEKRQAPGKRSESGSPSAGGHSKPPHSPLVLKRCHVS THQHNYAAPPSTRKDYPAAKRVKLDSVRVLRQISNNRKCTSPRSSDTEENVKRRTHNVL ERQRRNELKRSFFALRDQIPELENNEKAPKVVILKKATAYILSVQAEEQKLISEEDLLRKR REQLKHKLEQLRNSCASEQ ID NO: 62 Ringo 1 MQKHYTVAWFLYSAPGVDPSPPCRSLGWKRKREWSDESEEEPEKELAPEPEETWVVET LCGLKMKLKQQRVSPILLEHHKDFNSQLAPGVDPSPPHRSFCWKRKMEWWDKSEESEE EPRKVLAPEPEEIWVAEMLCGLKMKLKRRRVSLVLPEHHEAFNRLLEDPVIKRFLAWDK DLRVSDKYLLAMVIAYFSRAGFPSWQYQRLHFFLALYLANDMEEDDEDSKQNIFHFLY GKNRSRIPLLRKRRFQLYRSMNPRARKNRSHIPLVRKRRFQLRRCMNPRARKNRSQIVL FQKRRFHFFCSMSCRAWVSPEELEEIQAYDPEHWVWARDRARLSSEQ ID NO: 63 hexahistidine tagHHHHHHReferencesAmara, J. F., Clackson, T., Rivera, V. M., Guo, T., Keenan, T., Natesan, S., Pollock, R., Yang, W., Courage, N. L., Holt, D. A., Gilman, M. A versatile synthetic dimerizer for the regulation of protein-protein interactions. Proc. Natl. Acad. Sci. U.S.A. 1997, 94(20): 10618-23. DOI: 10.1073 / pnas.94.20.10618; PMID: 9380684; PMCID: PMC23423. Amara, J. F., Courage. N. L., Gilman, M. Cell surface tagging and a suicide mechanism in a single chimeric human protein. Hum Gene Ther. 1999, 10(16): 2651-5. DOI:10.1089 / 10430349950016681; PMID: 10566892.Avrutsky, M. I. and Troy, C. M. Caspase-9: A Multimodal Therapeutic Target With Diverse Cellular Expression in Human Disease. Front. Pharmacol. 2021, 12: 701301. DOI: 10.3389 / fphar.2021.701301.71326495150vlAttorney Docket No: 243735.000473Carlotti, F., Zaldumbide, A., Martin, P., Boulukos, K. E., Hoeben, R. C., Pognonec, P.Development of an inducible suicide gene system based on human caspase 8. Cancer Gene Ther. 2005, 12(7): 627-39. 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A., Heslop, H. E., Spencer, D. M., Rooney, C. M., Brenner, M. K. Inducible apoptosis as a safety switch for adoptive cell therapy. N. Engl. J. Med. 2011, 365(18): 1673-83. DOI: 10.1056 / NEJMoal 106152; PMID: 22047558; PMCID:PMC3236370.Falcon, C., Smith, L., Al-Obaidi, M., Abu Zaanona, M., Purvis, K., Minagawa, K., Athar, M., Salzman, D., Bhatia, R., Goldman, F., Di Stasi, A. Combinatorial suicide gene strategies for the safety of cell therapies. Front. Immunol. 2022, 13: 975233. DOI:10.3389 / fimmu.2022.975233; PMID: 36189285; PMCID: PMC9515659.Fan, L., Freeman, K. W., Khan, T., Pham, E., Spencer, D. M. Improved artificial death switches based on caspases and FADD. Hum. Gene. Ther. 1999, 10(14): 2273-85. DOI:10.1089 / 10430349950016924; PMID: 10515447.Gargett, T., Brown, M. P. The inducible caspase-9 suicide gene system as a “safety switch” to limit on-target, off-tumor toxicides of chimeric antigen receptor T cells. Front.Pharmacol. 2014, 5: 235. DOI: 10.3389 / fphar.2014.00235; PMID: 25389405; PMCID: PMC4211380.Hanzl, A., Barone. E., Bauer, S., Yue, H., Nowak, R. P., Hahn, E., Pankevich, E. V., Koren, A., Kubicek, S., Fischer, E. S., Winter, G. E. E3-Specific Degrader Discovery by Dynamic72326495150V1Attorney Docket No: 243735.000473Tracing of Substrate Receptor Abundance. J. Am. Chem. Soc. 2023, 745(2): 1176-1184. DOI: 10.1021 / jacs.2cl0784; PMID: 36602777; PMCID: PMC9853857.Koduri, V., Duplaquet, L„ Lampson, B. L„ Wang, A. C., Sabet, A. H„ Ishoey, M., Paulk, J., Teng, M., Harris, I. S., Endress, J. E., Liu, X., Dasilva, E., Paulo, J. A., Briggs, K. J., Doench, J. G., Ott, C. J., Zhang, T., Donovan, K. A., Fischer, E. S., Gygi, S. P., Gray, N. S., Bradner, J., Medin, J. A., Buhrlage, S. J., Oser, M. G., Kaelin, W. G. Targeting oncoproteins with a positive selection assay for protein degraders. Sci. Adv. 2021, 7(6): eabd6263. DOI: 10.1126 / sciadv.abd6263; PMID: 33547076; PMCID: PMC7864573. Li, B., Desai, S. A., MacCorkle-Chosnek, R. A., Fan, L., Spencer, D. M. A novel conditional Akt 'survival switch' reversibly protects cells from apoptosis. Gene. Ther. 2002, 9(4): 233-44. DOI: 10.1038 / sj.gt.3301641; PMID: 11896462.Mallet, V. O., Mitchell, C., Guidotti, J. E., Jaffray, P., Fabre, M., Spencer, D., Arnoult, D., Kahn, A., Gilgenkrantz, H. Conditional cell ablation by tight control of caspase-3 dimerization in transgenic mice. Nat. Biotechnol. 2002, 20(12): 1234-9. DOI: 10.1038 / nbt762; PMID: 12434157.Pollock, R., Issner, R., Zoller, K., Natesan, S., Rivera, V. M., Clackson, T. Delivery of a stringent dimerizer-regulated gene expression system in a single retroviral vector. Proc. Natl. Acad. Sci. U.S.A. 2000, 97(24): 13221-6. DOI: 10.1073 / pnas.230446297; PMID: 11078518; PMCID: PMC27206.Shahi, P., Park, D., Pond, A. C., Seethammagari, M., Chiou, S. H., Cho, K., Carstens, J. L., Decker, W. K., McCrea, P. D., Ittmann, M. M., Rosen, J. M., Spencer, D. M. 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[0305] The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0306] All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present in this specification.74326495150V1

Claims

Attorney Docket No; 243735.000473Claims1. A method for identifying a degrader of a target protein, comprising:(a) contacting a population of cells with a test compound in a test cell culture, wherein the population of cells expresses an exogenous fusion protein, wherein the fusion protein comprises the target protein and an inducible cell suicide protein, and wherein the cell suicide protein induces the cells to undergo apoptosis in the presence of a suicide inducer;(b) incubating the test cell culture under conditions and for a time sufficient for degradation of the target protein to occur;(c) adding a suicide inducer to the test cell culture, and incubating the test cell culture under conditions and for a time sufficient for the cells to undergo apoptosis;(d) determining the level of cell viability using a chemical cell viability assay;(e) comparing the level of cell viability determined in step (d) to a control level of cell viability, wherein the control level of cell viability is a predetermined control cell viability level or the level of cell viability determined under the same conditions in a control cell culture; and (f) (i) determining the test compound is a degrader of the target protein when the level of cell viability determined in step (d) is higher than the control level of cell viability, or (ii) determining the test compound is not a degrader of the target protein when the level of cell viability determined in step (d) is not higher than the control level of cell viability.

2. The method of claim 1, wherein the target protein is positioned at the N-terminus relative to the inducible cell suicide protein.

3. The method of claim 1, wherein the target protein is positioned at the C-terminus relative to the inducible cell suicide protein.

4. The method of any one of claims 1-3, wherein the target protein is fused to the inducible cell suicide protein via a linker.

5. The method of any one of claims 1-4, wherein the linker comprises about 5-20 amino acids.

6. The method of any one of claims 1-5, wherein the linker comprises about 5-15 amino acids.75326495150V1Attorney Docket No: 243735.0004737. The method of any one of claims 1-6, wherein the linker comprises about 10-15 amino acids.

8. The method of any one of claims 4-7, wherein the linker comprises the amino acid sequence of GGSGGGSGGG (SEQ ID NO: 12). GGSGG (SEQ ID NO: 47), SGGGSG (SEQ ID NO: 48), (SGGGS)n (SEQ ID NO: 20), wherein n is 1, 2, or 3, (GGGGS)n (SEQ ID NO: 21), wherein n is 1, 2, or 3, GGGGGG (SEQ ID NO: 16), GGGGGGGG (SEQ ID NO: 17), GSAGSAAGSGEF (SEQ ID NO: 18). or EGKSSGSGSESKST (SEQ ID NO: 19).

9. The method of any one of claims 1-8, wherein the inducible cell suicide protein comprises a ligand binding domain fused to a cell suicide domain.

10. The method of claim 9, wherein the ligand binding domain comprises an FKBP12 polypeptide, an FRB1 polypeptide, or a variant, fragment, or combination thereof.

11. The method of claim 10, wherein the ligand binding domain comprises a variant of the FKBP12 polypeptide comprising an F36V substitution.

12. The method of claim 11, wherein the variant of the FKBP12 polypeptide comprising an F36V substitution comprises the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 3), or an amino acid sequence having at least about 90% sequence identity thereof.

13. The method of claim 11 or 12, wherein the ligand binding domain comprises two tandem copies of the variant of the FKBP12 polypeptide comprising an F36V substitution.

14. The method of claim 10, wherein the ligand binding domain comprises a variant of the FKBP12 polypeptide comprising G89P and I90K substitutions.

15. The method of claim 10, wherein the ligand binding domain comprises an FRB1 polypeptide, or a variant or fragment thereof.76326495150V1Attorney Docket No: 243735.00047316. The method of claim 15, wherein the ligand binding domain comprises a variant of the FRB1 polypeptide comprising a T2098L substitution.

17. The method of claim 10, wherein the ligand binding domain comprises a variant of the FRB1 polypeptide comprising a T2098L substitution and a variant of the FKBP12 polypeptide comprising G89P and I90K substitutions.

18. The method of claim 14 or 17, wherein the variant of the FKBP12 polypeptide comprising G89P and I90K substitutions comprises the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPPKIPPHATLVFDVELLKLE (SEQ ID NO: 49), or an amino acid sequence having at least about 90% sequence identity thereof.

19. The method of claim 16 or 17, wherein the variant of the FRB1 polypeptide comprising a T2098L substitution comprises the amino acid sequence of EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDL MEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK (SEQ ID NO: 50), or an amino acid sequence having at least about 90% sequence identity thereof.

20. The method of claim 9, wherein the cell suicide domain comprises a caspase-9 polypeptide, a caspase-8 polypeptide, a caspase- 1 polypeptide, a caspase-3 polypeptide, a FADD polypeptide, a Fas polypeptide, or a mBax polypeptide, or a variant, fragment, or combination thereof.

21. The method of claim 20, wherein the cell suicide domain comprises a variant of the caspase- 9 polypeptide comprising Q221R substitution.

22. The method of claim 21, wherein the variant of the caspase-9 polypeptide comprising Q221R substitution comprises the amino acid sequence of GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRR RFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFP GAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPED77326495150vlAttorney Docket No: 243735.000473ESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYV ETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 2), or an amino acid sequence having at least about 90% sequence identity thereof.

23. The method of claim 20, wherein the cell suicide domain comprises a variant of the caspase- 8 polypeptide comprising amino acids 216-479 of a wild-type caspase-8 protein.

24. The method of claim 23, wherein the variant of the caspase-8 polypeptide comprising amino acids 216-479 of a wild-type caspase-8 protein comprises the amino acid sequence of SESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKLHSIRDRNGTHLDAGALTTT FEELHFEIKPHDDCTVEQIYEILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPI YELTSQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSEEQPYLEMDLSSPQTR YIPDEADFLLGMATVNNCVSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNYE VSNKDDKKNMGKQMPQPTFTLRKKLVFPSD (SEQ ID NO: 51), or an amino acid sequence having at least about 90% sequence identity thereof.

25. The method of claim 20, wherein the caspase- 1 polypeptide comprises the amino acid sequence of MADKVLKEKRKLFIRSMGEGTINGLLDELLQTRVLNKEEMEKVKRENATVMDKTR ALIDSVIPKGAQACQICITYICEEDSYLAGTLGLSADQTSGNYLNMQDSQGVLSSFPA PQAVQDNPAMPTSSGSEGNVKLCSLEEAQRIWKQKSAEIYPIMDKSSRTRLALIICNE EFDSIPRRTGAEVDITGMTMLLQNLGYSVDVKKNLTASDMTTELEAFAHRPEHKTSD STFLVFMSHGIREGICGKKHSEQVPDILQLNAIFNMLNTKNCPSLKDKPKVIIIQACRG DSPGVVWFKDSVGVSGNLSLPTTEEFEDDAIKKAHIEKDFIAFCSSTPDNVSWRHPT MGSVFIGRLIEHMQEYACSCDVEEIFRKVRFSFEQPDGRAQMPTTERVTLTRCFYLFP GH (SEQ ID NO: 52), or an amino acid sequence having at least about 90% sequence identity thereof.

26. The method of claim 20, wherein the caspase-3 polypeptide comprises the amino acid sequence of78326495150V1Attorney Docket No: 243735.000473MENTENSVDSKSIKNLEPKIIHGSESMDSGISLDNSYKMDYPEMGLCIIINNKNFHKST GMTSRSGTDVDAANLRETFRNLKYEVRNKNDLTREEIVELMRDVSKEDHSKRSSFV CVLLSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLTGKPKLFIIQACRGTELDCGIET DSGVDDDMACHKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCAMLKQYADK LEFMHILTRVNRKVATEFESFSFDATFHAKKQIPCIVSMLTKELYFYH (SEQ ID NO: 53). or an amino acid sequence having at least about 90% sequence identity thereof.

27. The method of claim 20, wherein the cell suicide domain comprises a variant of the FADD polypeptide comprising amino acids 1-125 of a wild-type FADD protein.

28. The method of claim 27, wherein the variant of the FADD polypeptide comprising amino acids 1-125 of a wild-type FADD protein comprises the amino acid sequence of MDPFLVLLHS VS S SLS S SELTELKFLCLGRVGKRKLERVQSGLDLFSMLLEQNDLEPG HTELLRELLASLRRHDLLRRVDDFEAGAAAGAAPGEEDLCAAFNVICDNVGKDWR RLARQLKVSDTK (SEQ ID NO: 54), or an amino acid sequence having at least about 90% sequence identity thereof.

29. The method of claim 20, wherein the cell suicide domain comprises a variant of the Fas polypeptide comprising amino acids 175-304 of a wild-type Fas protein.

30. The method of claim 29, wherein the variant of the Fas polypeptide comprising amino acids 175-304 of a wild-type Fas protein comprises the amino acid sequence of IHSGQCPAAIVGGINVLLKPNTSVQFLRLGMLSPEGTCKAFDTAGNGYCRSEGVVAV LLTKKSLARRVYATILNAGTNTDGFKEQGVTFPSGDIQEQLIRSLYQSAGVAPESFEY IEAHGTGTKVGDPQE (SEQ ID NO: 55), or an amino acid sequence having at least about 90% sequence identity thereof.

31. The method of claim 20, wherein the mBax polypeptide comprises the amino acid sequence of MDGSGEQLGSGGPTSSEQIMKTGAFLLQGFIQDRAGRMAGETPELTLEQPPQDASTK KLSECLRRIGDELDSNMELQRMIADVDTDSPREVFFRVAADMFADGNFNWGRVVA79326495150vlAttorney Docket No: 243735.000473LFYFASKLVLKALCTKVPELIRTIMGWTLDFLRERLLVWIQDQGGWEGLLSYFGTPT WQTVTIFVAGVLTASLTIWKKMG (SEQ ID NO: 56), or an amino acid sequence having at least about 90% sequence identity thereof.

32. The method of any one of claims 9-31, wherein the ligand binding domain is fused to the cell suicide domain via a linker.

33. The method of claim 32, wherein the linker comprises the amino acid sequence of GGSGGGSGGG (SEQ ID NO: 12), GGSGG (SEQ ID NO: 47), SGGGSG (SEQ ID NO: 48), (SGGGS)n (SEQ ID NO: 20), wherein n is 1, 2, or 3, (GGGGS)n (SEQ ID NO: 21), wherein n is 1, 2, or 3. GGGGGG (SEQ ID NO: 16), GGGGGGGG (SEQ ID NO: 17), GSAGSAAGSGEF (SEQ ID NO: 18), or EGKSSGSGSESKST (SEQ ID NO: 19).

34. The method of claim 32, wherein the linker comprises the amino acid sequence of SGGGS (SEQ ID NO: 9).

35. The method of any one of claims 9-34, wherein the inducible cell suicide protein further comprises an HA tag at the C-terminus.

36. The method of claim 35, wherein the HA tag comprises the amino acid sequence of YPYDVPDYALD (SEQ ID NO: 6).

37. The method of any one of claims 9-36, wherein the inducible cell suicide protein comprise the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSG VDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKL RRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQ FPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSP EDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSW YVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSVD80326495150vlAttorney Docket No: 243735.000473YPYDVPDYALD (SEQ ID NO: 7), or an amino acid sequence having at least about 90% sequence identity thereof.

38. The method of any one of claims 9-37, wherein the suicide inducer is AP20187, AP1903, FK1012, AP1510, or AP22783.

39. The method of any one of claims 1-38, wherein the chemical cell viability assay is a luminescence-based cell viability assay.

40. The method of claim 39, the luminescence-based cell viability assay measures the number of viable cells by quantifying adenosine triphosphate (ATP).

41. The method of claim 39 or 40, the luminescence-based cell viability assay comprises a one- step reaction.

42. The method of claim 41, wherein the one-step reaction comprises addition of a recombinant luciferase reaction mix.

43. The method of claim 42, wherein the recombinant luciferase reaction mix comprises a recombinant luciferase, luciferin substrate, one or more components to lyse cell membranes, and one or more components to inhibit endogenous ATPases.

44. The method of claim 43, wherein the one or more components to lyse cell membranes comprises TCA (trichloroacetic acid), DMSA (dimercaptosuccinic acid), or CTAB (cetyl trimethylammonium bromide) .

45. The method of claim 43, wherein the one or more components to inhibit endogenous ATPases comprises NaF.

46. The method of any one of claims 1-38, wherein the chemical cell viability assay is an absorbance-based cell viability assay.81326495150V1Attorney Docket No: 243735.00047347. The method of claim 46, wherein the absorbance-based cell viability assay is a MTT (3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay or a MTS (3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay.

48. The method of any one of claims 1-38, wherein the chemical cell viability assay is a fluorescence-based cell viability assay.

49. The method of claim 48, wherein the fluorescence-based cell viability assay is a resazurin assay.

50. The method of any one of claims 1-49, wherein the control cell culture comprises a population of cells expressing the inducible cell suicide protein which is not fused to the target protein or which is fused to a protein that is not the target protein, and the control cell culture is subjected to the same test compound treatment.

51. The method of any one of claims 1-49, wherein the control cell culture comprises a population of cells expressing the same exogenous fusion protein as the test cell culture, but the control cell culture is not subjected to the test compound treatment.

52. The method of any one of claims 1-51, wherein in step (b), the test compound is incubated with the population of cells for about 3-4 hours.

53. The method of any one of claims 1-52, wherein in step (c). the test cell culture is incubated with the suicide inducer for about 12-16 hours.

54. The method of any one of claims 1-53, wherein the total time for conducting the method is about 15-20 hours.

55. The method of any one of claims 1-54, wherein the degrader is a small molecule, a nucleic acid molecule, a peptide, or a protein.82326495150V1Attorney Docket No: 243735.00047356. The method of any one of claims 1 -55, wherein the degrader is an exogenous degrader or an endogenous degrader.

57. The method of claim 56, wherein the exogenous degrader is a monovalent degrader, bifunctional degrader, or a destabilizer.

58. The method of claim 57, wherein the monovalent degrader is a molecular glue.

59. The method of claim 57, wherein the bifunctional degrader is a proteolysis targeting chimera (PROTAC).

60. The method of any one of claims 1-59, wherein the degrader comprises a library of compounds.

61. The method of any one of claims 1-60, wherein the target protein is a proto-oncoprotein.

62. The method of any one of claims 1-61, wherein the target protein is selected from Ikaros ZF23, Kras, Skpl, Cyclin DI, Skp2, cMyc, Ringol, FAM96B, PDCD1, SEC61A1, HMGCS1, ANAPC10, IRF4, CCND2, N-myc, SOXIO, SEH1L, TBCB, KLF4, CEBPA, RPP25L, POU2AF1, Timl7-A, SMARCB1, SOCS3, CDC26, FOXA1, LRR1, FAU, GATA3, SYF2, SOX2, and mutants or fragments thereof.

63. The method of claim 62, wherein the target protein comprises the amino acid sequence of Ikaros ZF23 (SEQ ID NO: 8), Kras (SEQ ID NO: 57), Skpl (SEQ ID NO: 58), Cyclin DI (SEQ ID NO: 59). Skp2 (SEQ ID NO: 60), cMyc (SEQ ID NO: 61), Ringol (SEQ ID NO: 62), FAM96B (SEQ ID NO: 22), PDCD1 (SEQ ID NO: 23), SEC61A1 (SEQ ID NO: 24), HMGCS1 (SEQ ID NO: 25), ANAPC10 (SEQ ID NO: 26), IRF4 (SEQ ID NO: 27), CCND2 (SEQ ID NO: 28), N-myc (SEQ ID NO: 29), SOXIO (SEQ ID NO: 30), SEH1L (SEQ ID NO: 31), TBCB (SEQ ID NO: 32), KLF4 (SEQ ID NO: 33), CEBPA (SEQ ID NO: 34), RPP25L (SEQ ID NO: 35), POU2AF1 (SEQ ID NO: 36), Timl7-A (SEQ ID NO: 37), SMARCB1 (SEQ ID NO: 38), SOCS3 (SEQ ID NO: 39), CDC26 (SEQ ID NO: 40), FOXA183326495150vlAttorney Docket No: 243735.000473(SEQ TD NO: 41), LRR 1 (SEQ TD NO: 42), FAU (SEQ ID NO: 43), GATA3 (SEQ ID NO: 44), SYF2 (SEQ ID NO: 45), or SOX2 (SEQ ID NO: 46).

64. The method of claim 62, wherein the target protein is a Kras (G12C) mutant.

65. The method of any one of claims 1-64, wherein the exogenous fusion protein is expressed from a transgene integrated into the chromosome of the cells.

66. The method of any one of claims 1-65, wherein the population of cells comprises mammalian cells.

67. The method of any one of claims 1-66, wherein the population of cells comprises adherent cells and / or suspension cells.

68. The method of claim 67, wherein the adherent cells comprise HEK293, CHO, MCF7, or HeLa cells.

69. The method of claim 67, wherein the suspension cells comprise Jurkat, Expi293F, K562 cells, or HEK293 suspension-adapted cells.

70. The method of any one of claims 1-69, wherein the method is conducted in a high-throughput format.

71. The method of claim 70, wherein the test compound represents a library of compounds.

72. A polynucleotide encoding a fusion protein comprising a target protein and an inducible cell suicide protein, wherein when the fusion protein is expressed in a cell, the cell suicide protein induces the cell to undergo apoptosis in the presence of a suicide inducer.

73. The polynucleotide of claim 72, wherein the fusion protein is expressed from a transgene integrated into the chromosome of the cell.84326495150V1Attorney Docket No: 243735.00047374. The polynucleotide of claim 72 or 73, wherein the target protein is positioned at the N- terminus relative to the inducible cell suicide protein.

75. The polynucleotide of claim 72 or 73, wherein the target protein is positioned at the C- terminus relative to the inducible cell suicide protein.

76. The polynucleotide of any one of claims 72-75, wherein the target protein is fused to the inducible cell suicide protein via a linker.

77. The polynucleotide of any one of claims 72-76, wherein the linker comprises about 5-20 amino acids.

78. The polynucleotide of any one of claims 72-77, wherein the linker comprises about 5-15 amino acids.

79. The polynucleotide of any one of claims 72-78, wherein the linker comprises about 10-15 amino acids.

80. The polynucleotide of any one of claims 76-78, wherein the linker comprises the amino acid sequence of GGSGGGSGGG (SEQ ID NO: 12), GGSGG (SEQ ID NO: 47), SGGGSG (SEQ ID NO: 48), (SGGGS)n (SEQ ID NO: 20), wherein n is 1, 2, or 3. (GGGGS)n (SEQ ID NO: 21), wherein n is 1, 2, or 3, GGGGGG (SEQ ID NO: 16), GGGGGGGG (SEQ ID NO: 17), GSAGSAAGSGEF (SEQ ID NO: 18), or EGKSSGSGSESKST (SEQ ID NO: 19).

81. The polynucleotide of any one of claims 72-80, wherein the inducible cell suicide protein comprises a ligand binding domain fused to a cell suicide domain.

82. The polynucleotide of claim 81, wherein the ligand binding domain comprises an FKBP12 polypeptide, an FRB 1 polypeptide, or a variant, fragment, or combination thereof.

83. The polynucleotide of claim 82, wherein the ligand binding domain comprises a variant of the FKBP12 polypeptide comprising an F36V substitution.85326495150V1Attorney Docket No: 243735.00047384. The polynucleotide of claim 83, wherein the variant of the FKBP12 polypeptide comprising an F36V substitution comprises the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLE (SEQ ID NO: 3), or an amino acid sequence having at least about 90% sequence identity thereof.

85. The polynucleotide of claim 83 or 84, wherein the ligand binding domain comprises two tandem copies of the variant of the FKBP12 polypeptide comprising an F36V substitution.

86. The polynucleotide of claim 82, wherein the ligand binding domain comprises a variant of the FKBP12 polypeptide comprising G89P and I90K substitutions.

87. The polynucleotide of claim 82, wherein the ligand binding domain comprises an FRB1 polypeptide, or a variant or fragment thereof.

88. The polynucleotide of claim 87, wherein the ligand binding domain comprises a variant of the FRB 1 polypeptide comprising a T2098L substitution.

89. The polynucleotide of claim 82, wherein the ligand binding domain comprises a variant of the FRB1 polypeptide comprising a T2098L substitution and a variant of the FKBP12 polypeptide comprising G89P and I90K substitutions.

90. The polynucleotide of claim 86 or 89, wherein the variant of the FKBP12 polypeptide comprising G89P and 190K substitutions comprises the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPPKIPPHATLVFDVELLKLE (SEQ ID NO: 49), or an amino acid sequence having at least about 90% sequence identity thereof.

91. The polynucleotide of claim 88 or 89, wherein the variant of the FRB1 polypeptide comprising a T2098L substitution comprises the amino acid sequence of EMWHEGLEEASRLYFGERNVKGMFEVLEPLHAMMERGPQTLKETSFNQAYGRDL86326495150vlAttorney Docket No: 243735.000473MEAQEWCRKYMKSGNVKDLLQAWDLYYHVFRRISK (SEQ ID NO: 50), or an amino acid sequence having at least about 90% sequence identity thereof.

92. The polynucleotide of claim 81, wherein the cell suicide domain comprises a caspase-9 polypeptide, a caspase-8 polypeptide, a caspase- 1 polypeptide, a caspase-3 polypeptide, a FADD polypeptide, a Fas polypeptide, or a mBax polypeptide, or a variant, fragment, or combination thereof.

93. The polynucleotide of claim 92, wherein the cell suicide domain comprises a variant of the caspase-9 polypeptide comprising Q221R substitution.

94. The polynucleotide of claim 93, wherein the variant of the caspase-9 polypeptide comprising Q221R substitution comprises the amino acid sequence of GFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRR RFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQFP GAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPED ESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYV ETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTS (SEQ ID NO: 2), or an amino acid sequence having at least about 90% sequence identity thereof.

95. The polynucleotide of claim 92, wherein the cell suicide domain comprises a variant of the caspase-8 polypeptide comprising amino acids 216-479 of a wild-type caspase-8 protein.

96. The polynucleotide of claim 95, wherein the variant of the caspase-8 polypeptide comprising amino acids 216-479 of a wild-type caspase-8 protein comprises the amino acid sequence of SESQTLDKVYQMKSKPRGYCLIINNHNFAKAREKVPKLHSIRDRNGTHLDAGALTTT FEELHFEIKPHDDCTVEQIYEILKIYQLMDHSNMDCFICCILSHGDKGIIYGTDGQEAPI YELTSQFTGLKCPSLAGKPKVFFIQACQGDNYQKGIPVETDSEEQPYLEMDLSSPQTR YIPDEADFLLGMATVNNCVSYRNPAEGTWYIQSLCQSLRERCPRGDDILTILTEVNYE VSNKDDKKNMGKQMPQPTFTLRKKLVFPSD (SEQ ID NO: 51), or an amino acid sequence having at least about 90% sequence identity thereof.87326495150V1Attorney Docket No: 243735.00047397. The polynucleotide of claim 92, wherein the caspase- 1 polypeptide comprises the amino acid sequence of MADKVLKEKRKLFIRSMGEGTINGLLDELLQTRVLNKEEMEKVKRENATVMDKTR ALIDSVIPKGAQACQICITYICEEDSYLAGTLGLSADQTSGNYLNMQDSQGVLSSFPA PQAVQDNPAMPTSSGSEGNVKLCSLEEAQRIWKQKSAEIYPIMDKSSRTRLALIICNE EFDSIPRRTGAEVDITGMTMLLQNLGYSVDVKKNLTASDMTTELEAFAHRPEHKTSD STFLVFMSHGIREGICGKKHSEQVPDILQLNAIFNMLNTKNCPSLKDKPKVIIIQACRG DSPGVVWFKDSVGVSGNLSLPTTEEFEDDAIKKAHIEKDFIAFCSSTPDNVSWRHPT MGSVFIGRLIEHMQEYACSCDVEEIFRKVRFSFEQPDGRAQMPTTERVTLTRCFYLFP GH (SEQ ID NO: 52), or an amino acid sequence having at least about 90% sequence identity thereof.

98. The polynucleotide of claim 92, wherein the caspase-3 polypeptide comprises the amino acid sequence of MENTENSVDSKSIKNLEPKIIHGSESMDSGISLDNSYKMDYPEMGLCIIINNKNFHKST GMTSRSGTDVDAANLRETFRNLKYEVRNKNDLTREEIVELMRDVSKEDHSKRSSFV CVLLSHGEEGIIFGTNGPVDLKKITNFFRGDRCRSLTGKPKLFIIQACRGTELDCGIET DSGVDDDMACHKIPVEADFLYAYSTAPGYYSWRNSKDGSWFIQSLCAMLKQYADK LEFMHILTRVNRKVATEFESFSFDATFHAKKQIPCIVSMLTKELYFYH (SEQ ID NO: 53), or an amino acid sequence having at least about 90% sequence identity thereof.

99. The polynucleotide of claim 92, wherein the cell suicide domain comprises a variant of the FADD polypeptide comprising amino acids 1-125 of a wild-type FADD protein.

100. The polynucleotide of claim 99, wherein the variant of the FADD polypeptide comprising amino acids 1-125 of a wild-type FADD protein comprises the amino acid sequence of MDPFLVLLHSVSSSLSSSELTELKFLCLGRVGKRKLERVQSGLDLFSMLLEQNDLEPG HTELLRELLASLRRHDLLRRVDDFEAGAAAGAAPGEEDLCAAFNVICDNVGKDWR88326495150vlAttorney Docket No: 243735.000473RLARQLKVSDTK (SEQ ID NO: 54), or an amino acid sequence having at least about 90% sequence identity thereof.

101. The polynucleotide of claim 92, wherein the cell suicide domain comprises a variant of the Fas polypeptide comprising amino acids 175-304 of a wild-type Fas protein.

102. The polynucleotide of claim 101, wherein the variant of the Fas polypeptide comprising amino acids 175-304 of a wild-type Fas protein comprises the amino acid sequence of IHSGQCPAAIVGGINVLLKPNTSVQFLRLGMLSPEGTCKAFDTAGNGYCRSEGVVAV LLTKKSLARRVYATILNAGTNTDGFKEQGVTFPSGDIQEQLIRSLYQSAGVAPESFEY IEAHGTGTKVGDPQE (SEQ ID NO: 55), or an amino acid sequence having at least about 90% sequence identity thereof.

103. The polynucleotide of claim 92, wherein the mBax polypeptide comprises the amino acid sequence of MDGSGEQLGSGGPTSSEQIMKTGAFLLQGFIQDRAGRMAGETPELTLEQPPQDASTK KLSECLRRIGDELDSNMELQRMIADVDTDSPREVFFRVAADMFADGNFNWGRVVA LFYFASKLVLKALCTKVPELIRTIMGWTLDFLRERLLVWIQDQGGWEGLLSYFGTPT WQTVTIFVAGVLTASLTIWKKMG (SEQ ID NO: 56), or an amino acid sequence having at least about 90% sequence identity thereof.

104. The polynucleotide of any one of claims 81-103, wherein the ligand binding domain fused to a cell suicide domain via a linker.

105. The polynucleotide of claim 104, wherein the linker comprises the amino acid sequence of GGSGGGSGGG (SEQ ID NO: 12), GGSGG (SEQ ID NO: 47), SGGGSG (SEQ ID NO: 48), (SGGGS)n (SEQ ID NO: 20), wherein n is 1, 2, or 3, (GGGGS)n (SEQ ID NO: 21), wherein n is 1, 2, or 3, GGGGGG (SEQ ID NO: 16), GGGGGGGG (SEQ ID NO: 17), GSAGSAAGSGEF (SEQ ID NO: 18), or EGKSSGSGSESKST (SEQ ID NO: 19).89326495150vlAttorney Docket No: 243735.000473106. The polynucleotide of claim 105, wherein the linker comprises the amino acid sequence of SGGGS (SEQ ID NO: 9).

107. The polynucleotide of any one of claims 81-106, wherein the inducible cell suicide protein further comprises an HA tag at the C-terminus.

108. The polynucleotide of claim 107, wherein the HA tag comprises the amino acid sequence of YPYDVPDYALD (SEQ ID NO: 6).

109. The polynucleotide of any one of claims 81-108, wherein the inducible cell suicide protein comprise the amino acid sequence of GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVI RGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLESGGGSG VDGFGDVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKL RRRFSSLHFMVEVKGDLTAKKMVLALLELARQDHGALDCCVVVILSHGCQASHLQ FPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSP EDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSW YVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSVD YPYDVPDYALD (SEQ ID NO: 7), or an amino acid sequence having at least about 90% sequence identity thereof.

110. A recombinant vector comprising the polynucleotide of any one of claims 72-109.

111. The recombinant vector of claim 110, wherein the vector is a viral vector.

112. The recombinant vector of claim 111, wherein the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, a baculoviral vector, or a vaccinia virus vector.

113. The recombinant vector of claim 110, wherein the vector is a non-viral vector.90326495150V1Attorney Docket No: 243735.000473114. The recombinant vector of claim 113, wherein the non-viral vector is a minicircle plasmid, a Sleeping Beauty transposon, a PiggyBac transposon, or a single or double stranded DNA molecule that is used as a template for homology directed repair (HDR)-based gene editing.

115. A cell comprising the polynucleotide of claim 72 or the recombinant vector of any one of claims 110-114.91326495150V1

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