APAF1 targeting therapies for treatment of cancer

Abstract

The present disclosure provides methods for treating cancer using one or more therapeutic agents that target APAF1. In particular, the present disclosure relates to administering a therapeutically effective amount of at least one agent to reduce the expression of APAF1 to a subject diagnosed with, or at risk for cancer.

Description

Atty. Dkt. No.: 115872-3388APAF1 TARGETING THERAPIES FOR TREATMENT OF CANCER CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U. S. Provisional Patent Application No. 63 / 739,031, filed December 26, 2024, the entire contents of which are incorporated herein by reference.TECHNICAL FIELD

[0002] The present disclosure relates to methods for treating cancer using one or more therapeutic agents that target APAF 1.GOVERNMENT SUPPORT CLAUSE

[0003] This invention was made with government support under CA008748, CA252658, and CA274492 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0004] The following description of the background of the present technology is provided simply as an aid in understanding the present technology and is not admitted to describe or constitute prior art to the present technology.

[0005] The approval of immune checkpoint blockade (ICB) therapy across multiple cancer types has revolutionized cancer care. However, only a small fraction of patients achieve a durable complete response, making it crucial to identify therapeutic strategies that can enhance ICB-based therapies. One promising approach is the induction of immunogenic cell death (ICD). ICD is defined as any form of cell death capable of activating an adaptive immune response and inducing the formation of immunological memory. It was initially described as being activated by certain chemotherapeutic agents and is characterized by ATP secretion, calreticulin exposure, IFN-β induction, and CXCL10 production. ICD provides robust immunostimulatory signals to dendritic cells (DCs), facilitating their maturation and functional licensing. Once mature, DCs can cross-present antigens from phagocytosed dead cells to cytotoxic T lymphocytes, thereby activating and-1- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388expanding these T cells. Recently characterized necrotic forms of programmed cell death (PCD), including necroptosis and pyroptosis, are pro-inflammatory and immunogenic.

[0006] Although baciptosis has been shown to trigger antitumor immunity, its capacity and the underlying mechanisms for inducing immunological memory against tumor recurrence remain unclear.SUMMARY OF THE PRESENT TECHNOLOGY

[0007] In one aspect, the present disclosure provides a method for treating cancer in a subject in need thereof comprising administering to the subject an effective amount of an Apafl inhibitor. In some embodiments, the method further comprises separately, sequentially or simultaneously administering at least one additional anti-cancer therapy to the subject. In another aspect, the present disclosure provides a method for enhancing efficacy of an anti-cancer therapy in a subject in need thereof comprising administering to the subject an effective amount of an Apafl inhibitor.

[0008] In any of the preceding embodiments of the methods disclosed herein, the Apafl inhibitor is a small molecule, or an inhibitory RNA that targets Apafl. Examples of Apafl -specific small molecule inhibitors include but are not limited to ZYZ-488, QM31 (SVT016426), UCN-01, SVT017686, SVT017923, SVT016448, andN-alkylglycine trimers. In some embodiments, the inhibitory RNA that targets Apafl may be a siRNA, a shRNA, an antisense oligonucleotide, a ribozyme, or a sgRNA as described herein. Additionally or alternatively, in some embodiments, the Apafl inhibitor is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, iontophoretically, transmucosally, or intramuscularly.

[0009] In any and all embodiments of the methods disclosed herein, the anti-cancer therapy comprises one or more of chemotherapy, radiotherapy, adoptive cell therapy, immune checkpoint blockade therapy or targeted therapy. The adoptive cell therapy may comprise one or more of CAR T-cell therapy, tumor-infiltrating lymphocyte (TIL) therapy, T-cell receptor (TCR) therapy, natural killer (NK) cell therapy, or dendritic cell therapy.-2- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388

[0010] In any of the above embodiments of the methods disclosed herein, the chemotherapy comprises one or more of alkylating agents, topoisomerase inhibitors, endoplasmic reticulum stress inducing agents, antimetabolites, mitotic inhibitors, nitrogen mustards, nitrosoureas, alkyl sulfonates, platinum agents, taxanes, vinca agents, antiestrogen drugs, aromatase inhibitors, ovarian suppression agents, cytostatic alkaloids, cytotoxic antibiotics, endocrine / hormonal agents, or bisphosphonate therapy agents.Examples of chemotherapeutic agents include but are not limited to cyclophosphamide, fluorouracil (or 5 -fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserlin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, tykerb, anthracyclines (e.g., daunorubicin and doxorubicin), cladribine, midostaurin, bevacizumab, oxaliplatin, melphalan, etoposide, mechlorethamine, bleomycin, microtubule poisons, annonaceous acetogenins, chlorambucil, ifosfamide, streptozocin, carmustine, lomustine, busulfan, dacarbazine, temozolomide, altretamine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, hydroxyurea, pemetrexed, epirubicin, idarubicin, SN-38, ARC, NPC, campothecin, 9-nitrocamptothecin, 9-aminocamptothecin, rubifen, gimatecan, diflomotecan, BN80927, DX-8951f, MAG-CPT, amsacrine, etoposide phosphate, teniposide, azacitidine (Vidaza), decitabine, accatin III, 10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, cephalomannine, 10-deacetyl-7-epitaxol, 7-epitaxol, 10-deacetylbaccatin III, 10-deacetyl cephalomannine, streptozotocin, nimustine, ranimustine, bendamustine, uramustine, estramustine, mannosulfan, camptothecin, exatecan, lurtotecan, lamellarin D9-aminocamptothecin, ellipticines, aurintricarboxylic acid, HU-331, or combinations thereof.

[0011] Additionally or alternatively, in some embodiments of the methods disclosed herein, the immune checkpoint blockade therapy comprises one or more of an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-4- IBB antibody, an anti-CD73 antibody, an anti-GITR antibody, or an anti-LAG-3 antibody. Examples of immune checkpoint blockade therapy -3- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388include, but are not limited to, cemiplimab, tremelimumab, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, envafolimab, atezolizumab, avelumab, durvalumab, dostarlimab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, AMP-224, MDX-1105, arelumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS (inducible T-cell costimulatory), DLBCL (diffuse large B-cell lymphoma) inhibitors, BTLA (B and T lymphocyte attenuator), PDR001, or any combination thereof.

[0012] Additionally or alternatively, in some embodiments of the methods disclosed herein, the targeted therapy comprises one or more of VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, BCL-2 inhibitors, or CDK9 inhibitors. Examples of targeted therapy include but are not limited to bevacizumab, nimotuzumab, buparlisib, pilaralisib, sonolisib, paxalisib, dactolisib, voxtalisib, PQR309, AMG232, venetoclax, dinaciclib, ribociclib, dasatinib, imatinib, or rindopepimut.

[0013] In any and all embodiments of the methods disclosed herein, the cancer is selected from among adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, leukemias, liver cancers, lymph node cancers, lymphomas, lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non- Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, and metastases thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIGs. 1A-1I. BAK exists in three protein complexes with distinct death-regulatory activity. FIG. 1A: Mitochondria isolated from Bax- / -Bak- / -mouse embryonic fibroblasts (MEFs) reconstituted with the N-terminal Protein C (PrC)-tagged BAK ± the BIM BH3 peptide were sequentially extracted with 1% and 2% CHAPS. The 1% CHAPS -4- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388insoluble, 2% CHAPS soluble mitochondrial subfraction was subjected to anti-PrC affinity chromatography and analyzed by polyacrylamide gel electrophoresis and silver staining. The silver-stained bands were subjected to tryptic digestion and liquid chromatographytandem mass spectrometry analysis. FIG. IB: Bax- / -Bak- / -MEFs or Bax ' 'Bak ' MEFs reconstituted with the N-terminal PrC -tagged BAK were subjected to anti-PrC immunoprecipitation as described in (FIG. 1A), followed by immunoblot analyses. FIG.1C: The 1% CHAPS insoluble, 2% CHAPS soluble mitochondrial subfraction isolated from wild-type (WT) MEFs was subjected to Superdex 200 (HR10 / 30) gel filtration chromatography and immunoblot analyses. FIGs. 1D-1E: The 1% CHAPS insoluble, 2% CHAPS soluble mitochondrial subfraction isolated from either Bax- / -Bak- / -MEFs or Bax~~ Bak~ MEFs reconstituted with the N-terminal PrC -tagged BAK was subjected to Superdex 200 (HR10 / 30) gel filtration chromatography. The fractions collected around 600 kDa were subjected to anti-PrC affinity chromatography and analyzed by polyacrylamide gel electrophoresis and silver staining (FIG. ID) or the indicated immunoblots (FIG. IE). FIG. IF: Mitochondria isolated from WT MEFs ± recombinant tBID protein were sequentially extracted with 1% and 2% CHAPS to obtain 1% CHAPS soluble or 1% CHAPS insoluble, 2% CHAPS soluble subfraction, followed by Superdex 200 (HR10 / 30) gel filtration chromatography and immunoblot analyses. FIG. 1G: The 1% CHAPS soluble mitochondrial subfraction isolated from Bax- / -Bak- / -MEFs or Bax- / -Bak- / -MEFs reconstituted with the N-terminal PrC -tagged BAK was analyzed by Superdex 200 (HR10 / 30) gel filtration chromatography. The fractions eluted around 160 kDa were subjected to anti-PrC affinity chromatography and analyzed by polyacrylamide gel electrophoresis and silver staining. FIGs. 1H-1I: The 1% CHAPS insoluble, 2% CHAPS soluble mitochondrial subfraction isolated from WT MEFs was analyzed by Superdex 200 (HR10 / 30) gel filtration chromatography. The fractions eluted at the indicated molecular weight were untreated or treated with the BMH protein crosslinker and analyzed by an anti-BAK immunoblot (H), or were incubated with Bax 'Bak" mitochondria to assess cytochrome c releasing activity using ELISA assays (I). Data shown are mean ± SD (n = 3 independent experiments). *** P < 0.001 (Student’s / -test). See also FIG. 7.

[0015] FIGs. 2A-2F. Loss of VDAC1 or VDAC2 inhibits BAK / BAX-dependent caspase-independent but not caspase-dependent cell death. FIG. 2A: The 1% CHAPS -5- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388insoluble, 2% CHAPS soluble mitochondrial subfraction isolated from tBID-inducible Apaf1- / -MEFs ± doxycycline (1 pg / ml) for 6 hours was analyzed by Superdex 200 (Increase 10 / 300 GL) gel filtration chromatography and the indicated immunoblots. FIGs. 2B-2C: tBID-inducible Apaf1- / -or WT MEFs transduced with Cas9 and the indicated sgRNAs were analyzed by the indicated immunoblots and treated with doxycycline to induce tBID or the combination of ABT-263 (2 μM) and S63845 (10 μM). Cell death was quantified by FACS analysis following annexin-V staining at 6 hours (WT) or propidium iodide (PI) staining at 48 hours (Apaf1- / -. FIG. 2D: tBID-inducible ApafP' or Apaf1- / -Vdac P / ~Vdac2 / ~ MEFs were treated as in (FIG. 2B) and cell death was quantified at 48 hours. FIG. 2E: tBID-inducible Apaf1- / -or Apaf1- / -Vdac1- / -Vdac2- / -MEFs ± doxycycline for the indicated times were subjected to subcellular fractionation and analyzed by the indicated immunoblots. FIG. 2F: tBID-inducible Apaf1- / -or Apaf1- / -Vdac1- / -Vdac2- / -MEFs ± doxycycline for 6 hours were treated with the BMH crosslinker and analyzed by the indicated immunoblots. Asterisk denotes an intramolecular crosslinked BAK monomer. Data shown in (FIGs. 2B-2D) are mean ± SD (n = 3 independent experiments). * P < 0.05; *** P < 0.001; **** P < 0.0001; n.s., not significant (Student’s / -test). See also FIG. 7.

[0016] FIGs. 3A-3J. Loss of VDAC1, VDAC2, or prohibitins inhibits BAK / BAX-dependent mtDNA efflux and caspase-independent cell death. FIG. 3A: tBID-inducible Apaf1- / -MEFs transduced with Cas9 and the indicated sgRNAs ± doxycycline for 6 hours were analyzed by the indicated immunoblots. Cell death was quantified at 48 hours.Cytosolic mtDNA was assessed by quantitative PCR (qPCR) for D-loop following subcellular fractionation of the indicated cells ± doxycycline for 6 hours or ABT-263 plus S63845 for 4 hours. FIGs. 3B-3C: tBID-inducible ApafP' or Apaf1- / -Vdac / ~Vdac2 / ~ MEFs were treated with doxycycline to induce tBID (FIG. 3B) or the combination of ABT-263 and S63845 (FIG. 3C). Cytosolic mtDNA was assessed by qPCR at 6 hours (FIG. 3B) or 4 hours (FIG. 3C). FIG. 3D: tBID-inducible Apaf1- / -Vdac1- / -Vdac2- / -MEFs transduced with control retrovirus or retrovirus expressing VDAC1 / 2 / 3 ± doxycycline for 6 hours were analyzed by the indicated immunoblots. Cell death was quantified at 48 hours. Cytosolic mtDNA was assessed at 6 hours. FIG. 3E: The 1% CHAPS insoluble, 2% CHAPS soluble mitochondrial subfraction isolated from tBID-inducible Apaf1- / -Vdac1- / -Vdac2- / -MEFs ± doxycycline for 6 hours was analyzed by Superdex 200 (Increase 10 / 300 GL) gel filtration -6- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388chromatography and the indicated immunoblots. FIGs. 3F-3J: tBID-inducible Apaf1- / -MEFs transduced with Cas9 and the indicated sgRNAs were treated with doxycycline or the combination of ABT-263 and S63845. Cell death was quantified at 48 hours and immunoblots analyses were performed at 6 hours. Cytosolic mtDNA was assessed by qPCR at 6 hours (FIGs. 3G and 3J) or 4 hours (FIG.3H). Data shown in FIGs. 3A-3D and 3F-3J are mean ± SD (n = 3-4 independent experiments). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; n.s., not significant (Student’s / -test). See also FIG. 8.

[0017] FIGs. 4A-4I. Loss of VDAC1, VDAC2, or prohibitins impairs BAK / BAX activation-induced mtDNA efflux and cell death in apoptosome-deficient human cancers. FIG. 4A: Caspase activities were quantified by Caspase-Glo 3 / 7 assays in A498, PC9 and MDA-MB-231 cells ± ABT-263 plus S63845 (AS) at the indicated times. FIG. 4B: A498 cells ± ABT-263 plus S63845 for the indicated times were subjected to subcellular fractionation and analyzed by the indicated immunoblots. FIG. 4C: The 1% CHAPS insoluble, 2% CHAPS soluble mitochondrial subfraction isolated from A498 cells ± ABT-263 plus S63845 (AS) for 6 hours was analyzed by Superdex 200 (Increase 10 / 300 GL) gel filtration chromatography and the indicated immunoblots. FIG. 4D: The indicated A498 cells ± ABT-263 plus S63845 were subjected to cytosolic mtDNA quantification by qPCR after 12 hours and cell death quantification by FACS after 3 days. FIG. 4E: The indicated PC9 cells ± ABT-263 plus S63845 were subjected to cytosolic mtDNA quantification by qPCR after 6 hours and cell death quantification by FACS after 5 days. FIG. 4F: A498 cells transduced with the indicated shRNAs ± ABT-263 plus S63845 were subjected to cytosolic mtDNA quantification by qPCR after 3 hours. FIG. 4G: PC9 cells transduced with the indicated shRNAs ± ABT-263 plus S63845 were subjected to cytosolic mtDNA quantification by qPCR after 3 hours. FIG. 4H: The indicated MDA-MB-231 cells ± ABT-263 plus S63845 were subjected to cell death quantification by FACS after 24 hours. FIG. 41: A498 cells reconstituted with APAF1 and transduced with the indicated sgRNAs ± ABT-263 plus S63845 were subjected to cell death quantification by FACS after 24 hours. Data shown in FIGs. 4D-4I are mean ± SD (n = 3-4 independent experiments). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001; n.s., not significant (Student’s t-test). See also FIG. 9.-7- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388

[0018] FIGs. 5A-5G. The ability of VD AC 1 / 2 to control BAK / B AX activation-induced mitochondrial calcium influx and mtDNA efflux is impaired by calcium-binding site mutations. FIGs. 5A-5B: The indicated tBID-inducible Apaf1- / -MEFs transduced with the mitochondria-targeted fluorescent protein-based calcium indicator (mito-G-GECO1.2) ± doxycycline for 4 hours were analyzed by confocal fluorescence microscopy. Two independent clones were analyzed in FIG. 5B. Fluorescence intensity was quantified by ImageJ (see Methods). Each dot represents the relative fluorescence intensity of a cell. FIG. 5C: The indicated Apaf1- / -MEFs transduced with mito-G-GECOl.2 ± ABT-263 plus S63845 for 4 hours were analyzed by confocal fluorescence microscopy. Two independent clones were analyzed. FIG. 5D:Apaf1- / -Vdac1- / -Vdac2- / -MEFs transduced with wild-type VDAC1 / 2 (VDAC1 / 2WT) or the calcium-binding mutants of VDAC1 / 2 (VDAC1 / 2MT) plus mito-G-GECO1.2 ± doxycycline for 4 hours were analyzed by confocal fluorescence microscopy as in FIG. 5B. FIG. 5E: tBID-inducible Apaf1- / -VdacP / ~Vdac2~ / ~ MEFs transduced with VDAC1 / 2WTor VDAC1 / 2MT± doxycycline were subjected to immunoblot analyses at 6 hours, cell death quantification at 48 hours, and cytosolic mtDNA quantification at 6 hours. FIG. 5F: tBID-inducible 4 / / / / A ’dctcl / _Vdac2~ / ~ MEFs transduced with VDAC1 / 2WTor VDAC1 / 2MT± doxycycline at the indicated times were subjected to subcellular fractionation followed by immunoblot analyses. FIG.5G: tBID-inducible Apaf1- / -Vdac1- / -Vdac2- / -MEFs transduced with VDAC1WTor the indicated VDAC1 mutants ± doxycycline were subjected to immunoblot analyses at 6 hours, cell death quantification at 48 hours, and cytosolic mtDNA quantification at 6 hours. Statistics in FIGs. 5A-5D were calculated using Student’s / -test to test whether the average relative fluorescence intensity from one sample is greater than 150% of that from the other sample. Data shown in FIGs. 5E and 5G are mean ± SD (n = 3 independent experiments). ** P < 0.01; *** P < 0.001; **** P < 0.0001; n.s., not significant (Student’s / -test). See also FIGs. 10-11

[0019] FIGs. 6A-6D. The 600 kDa BAK complex containing tBID can permeabilize mitoplasts deficient for BAX, BAK, VDAC1, and VDAC2 to induce mtDNA efflux. FIG. 6A: Mitochondria isolated from tBID-inducible Apaf1- / -MEFs ± doxycycline for 6 hours were analyzed by Superdex 200 (Increase 10 / 300 GL) gel filtration chromatography as shown in FIG. 2A. The fractions eluted at the indicated molecular weight were incubated -8- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388with mitoplasts isolated from Bax- / -Bak- / -Vdac1- / -Vdac2- / -Apaf1- / -MEFs to assess mtDNA releasing activity using qPCR. Data shown are mean ± SD (n = 3 independent experiments). FIG. 6B: Electron microscopy imaging of tBID-inducible Apaf1- / -and Apaf1- / -Vdac1- / -Vdac2- / -MEFs treated with doxycycline for 3.5 hours to visualize mitochondrial inner membrane herniation (red arrowheads). Insets are enlarged images from areas indicated by red arrowheads. Scale bars, 1 pm. FIG. 6C: tBID-inducible Apaf1- / -and Apaf1- / -Vdac1- / -Vdac2- / -MEFs stably expressing the MOM protein TOMM20-GFP and the MIM protein TIMM50-mCherry were untreated or treated with doxycycline for 2.5 hours and subjected to immunofluorescence imaging using anti-TOMM20 and anti-mCherry antibodies (see also FIG. 12A). Data presented are the Manders’ colocalization coefficient of TOMM20-GFP and TIMM50-mCherry (n = 22 for Apaf1- / -control, n = 38 for Apaf1- / -tBID, n = 24 for Apaf1- / -Vdac1- / -Vdac2- / -control, and n = 33 for Apaf1- / -Vdac1- / -Vdac2- / -tBID). FIG. 6D: A schematic summarizing the regulation of caspase-dependent and caspase-independent cell death by BAK and BAX. * P < 0.05; ** P < 0.01; **** p < 0.0001; n.s., not significant (Student’s / -test). See also FIG. 12.

[0020] FIGs. 7A-7I. Characterization of BAX / BAK activation-induced caspaseindependent cell death (baciptosis), related to FIGs. 1 and 2. FIG. 7A: Mitochondria isolated from wild-type MEFs were subjected to hypotonic buffer treatment, sonication to generate sub-mitochondrial vesicles, and fractionation using continuous sucrose gradient to separate into outer membrane, inner membrane, and contact site fractions. The protein lysates from each fraction were analyzed by the indicated immunoblots. FIG. 7B: Time course of cell death of Apaf1+ / +and Apaf1- / -MEFs induced with tBID or treated with ABT-263 plus S63845. Cell death was quantified by IncuCyte following SYTOX-Green staining.FIG. 7C: SV40-transformed WT or Bid / Bim / Puma / Noxa / AAEEs were treated with the combination of ABT-263 (1 μM) and S63845 (5 μM). Cell death was quantified by FACS analysis following annexin-V staining at 48 hours. FIG. 7D: Apaf1- / -and Apaf1- / -Vdac1- / -Vdac2- / -MEFs were infected with retrovirus expressing GFP, tBID, BIM, or PUMA. Cell death was quantified by FACS analysis following propidium iodide (PI) staining at 48 hours. FIG. 7E: Apaf1- / -MEFs transduced with Cas9 and the indicated sgRNAs were infected with retrovirus expressing GFP or tBID-IRES-GFP. Cell death was quantified by propidium iodide (PI) staining at 48 hours. FIG. 7F: tBID-inducible- MEFs treated -9- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388with the indicated agents for 48 hours were subjected to cell death quantification by FACS.FIGs. 7G-7H: tBID-inducible- MEFs transduced with Cas9 and sgPolg or cultured in the presence of ethidium bromide were subjected to qPCR-based mtDNA quantification. The indicated cells were treated with doxycycline and cell death was quantified by FACS at 48 hours. FIG. 7I: Electron microscopy imaging of tBID-inducible WT or Apaf1- / -MEFs before or after doxycycline treatment for 3 hours (WT) or 40 hours (Apafl- / -').

[0021] FIGs. 8A-8G. Characterization of the 600 kDa BAK complex and BAX / BAK-dependent mtDNA efflux, related to FIG. 3. FIG. 8A: tBID-inducible Apaf1- / -MEFs transduced with Cas9 and the indicated sgRNAs were treated with doxycycline to induce tBID. Cell death was quantified at 48 hours. FIG. 8B: The 1% CHAPS insoluble, 2% CHAPS soluble mitochondrial subfraction isolated from tBID-inducible Apaf1- / -MEFs ± doxycycline (1 μg / ml) for 6 hours were analyzed by Superdex 200 (Increase 10 / 300 GL) gel filtration chromatography and the indicated immunoblots. FIG. 8C: tBID-inducible Apaf1- / -Bak- / -MEFs transduced with HA-tagged BAK ± doxycycline for 6 hours were subjected to anti -HA immunoprecipitation followed by immunoblot analyses. FIG. 8D: tBID-inducible Apaf1- / -MEFs transduced with Cas9 and the indicated sgRNAs were treated with doxycycline to induce tBID. Cell death was quantified at 48 hours. Cytosolic mtDNA was assessed by quantitative PCR (qPCR) for D-loop following subcellular fractionation at 6 hours. FIG. 8E: tBID-inducible Apaf1- / -B16-F10 cells transduced with Cas9 and the indicated sgRNAs were treated with doxycycline to induce tBID. Cytosolic mtDNA was assessed 8 hours post-treatment, cell death was quantified 72 hours post-treatment, and immunoblot analysis was performed 6 hours post-treatment. FIG. 8F: tBID-inducible Apaf1- / -B16-F10 cells transduced with Cas9 and the indicated sgRNAs were treated with doxycycline to induce tBID. Cell death was quantified 18 hours post-treatment and immunoblot analysis was performed 3 hours post-treatment. FIG. 8G: tBID-inducible Apaf1- / -B16-F10 cells transduced with the indicated shRNAs were treated with doxycycline to induce tBID and analyzed by the indicated immunoblots at 6 hours. Cytosolic mtDNA was assessed at 12 hours and cell death was quantified at 48 hours.

[0022] FIGs. 9A-9H. Loss of VDAC1, VDAC2, or prohibitins impedes BAX / BAK activation-induced mtDNA efflux and cell death in apoptosome-deficient human-10- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388cancers, related to FIG. 4. FIG. 9A: Immunoblot analyses of MDA-MB-231, A498, PC9 cells. FIG. 9B: The indicated cells ± ABT-263 plus S63845 for the indicated times were subjected to subcellular fractionation and analyzed by the indicated immunoblots. FIG. 9C: The indicated cells ± ABT-263 plus S63845 for the indicated times were subjected to cell death quantification by FACS following annexin-V staining. FIGs. 9D-9F: A498, PC9, or MDA-MD-231 cells transduced with the indicated sgRNAs or shRNAs were analyzed by the indicated immunoblots. FIG. 9G: A498 cells infected with retrovirus expressing APAF1 were analyzed by the indicated immunoblots. Caspase activities were quantified by Caspase-Glo 3 / 7 assays. FIG. 9H: A498 cells stably expressing APAF1 were transduced with the indicated sgRNAs and subsequently analyzed by the indicated immunoblots.

[0023] FIGs. 10A-10F. Activator BH3-only molecules inhibit VDAC-mediated ADP import into the mitochondria, related to FIG. 5. FIG. 10A: A schematic representation of the BH3-inducible system. GFP flanked by loxP sites is placed upstream of the indicated BH3-only molecule to prevent the expression of the BH3-only molecule driven by the LTR promoter. 4-hydroxytamoxifen (4-OHT) treatment activates Cre-ERT2 to delete the floxed GFP cassette and allow for juxtaposition of the LTR promoter and the BH3-only molecule, leading to the expression of the BH3-only molecule. FIGs. 10B-10C: Apafl - MEV expressing Cre-ERT2 and the indicated inducible BH3-only molecule were treated with 500 nM 4-OHT and analyzed by the indicated immunoblots at 6h. Cell death was quantified by FACS analyses following PI staining at 60 hours. FIGs. 10D-10E: Mitochondria or mitoplasts isolated from the indicated BH3-inducible Apafl- / - MEFs ± 4-hydroxytamoxifen for 17 hours were incubated with 14C-ADP with or without a preincubation with ANT inhibitor atractyloside to determine ANT-dependent uptake of ADP. FIG. 10F: Alignment of three VDAC isoforms. The red rectangles indicate the calcium-binding sites.

[0024] FIGs. 11A-11D. The ability of VDAC1 / 2 to control BAX / BAK activation-induced mitochondrial calcium influx is impaired by calcium-binding site mutations, related to FIG. 5. FIGs. 11A-11B: The indicated tBID-inducible 4 / / 7 - MEFs transduced with the mitochondria-targeted fluorescent protein-based calcium indicator (mito-G-GECO1.2) ± doxycycline for 4 hours were analyzed by confocal fluorescence -11- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388microscopy. Representative images from three independent experiments are shown. FIG.11C: The indicated Apafl- MEV transduced with mito-G-GECO1.2 ± ABT-263 plus S63845 for 4 hours were analyzed by confocal fluorescence microscopy. Representative images from three independent experiments are shown. FIG. 11D: tBID-inducible Apafl - / -Vdac 1 - / -Vdac2- / -MEF s transduced with wild-type VDAC1 / 2 (VDAC1 / 2WT) or the calcium-binding mutant of VD AC 1 / 2 (VDAC1 / 2MT) plus mito-G-GECO1.2 ± doxycycline for 4 hours were analyzed by confocal fluorescence microscopy. Representative images from three independent experiments are shown.

[0025] FIGs. 12A-12C. Loss of VDAC1 and VDAC2 neither prevents mitochondrial inner membrane herniation nor confer clonogenic survival in Apafl-deficient cells following BAX / BAK activation, related to FIG. 6. FIG. 12A:Immunofluorescence imaging of tBID-inducible Apafl- / - and Apafl - / -Vdac 1 - / -Vdac2- / -'MEP s stably expressing TOMM20-GFP and TIMM50-mCherry treated with doxycycline for 2.5 hours. Cells were stained with anti-TOMM20 (green) and anti-mCherry (red) antibodies. Insets show enlarged views of regions marked by dashed lines. Scale bars, 5 pm. FIG. 12B: Time course of cell death of tBID-inducible Apafl- / - and Apafl - / -Vdacl- / -Vdac2- / -'MEEs treated with doxycycline to induce tBID. Cell death was quantified by IncuCyte following SYTOX-Green staining. Data shown are mean ± SD (n = 3 independent experiments). ***, P < 0.001; ****, P <0.0001 (two-way ANOVA). FIG. 12C: The indicated tBID-inducible MEFs were treated with doxycycline to induce tBID. Colonies were stained with crystal violet after 10 days. Representative images from two independent experiments are shown.

[0026] FIGs. 13A-13J. Baciptosis is the most immunogenic cell death type that confers immunological memory against tumor rechallenge. FIG. 13A: Schematic of molecular pathways of BAK / B AX-dependent cell death. FIG. 13B: A tetracycline-inducible system was engineered to express tBID, CASP3CAM, or CO-RIPK3 in B16-F10 cells. CRISPR / Cas9-mediated KO of Apafl was performed in tet-tBID B16 cells. Cell death of the indicated cells ± doxycycline (1 pg / ml) was quantified by propidium iodide (PI) staining. FIG. 13C: Immunoblot analyses of the indicated cells ± doxycycline at the indicated times. FIG. 13D: Cytosolic mtDNA was assessed by quantitative PCR (qPCR) for D-loop following subcellular fractionation of B16 cells ± doxycycline for 4 hours.-12- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388Cellular levels of 2’, 3’-cGAMP was assessed by ELISA in B16 cells ± doxycycline for 8 hours. IFN-P was assessed by ELISA in culture media of B16 cells ± doxycycline for 12 hours. FIG. 13E: Fold increase of cytosolic mtDNA and IFN-P mRNA in tet-tBID Apaf1- / -B16 cells ± doxycycline at the indicated times. IFN-P mRNA was assessed by qRT-PCR and normalized against fi-Actin. FIG. 13F: The indicated tet-tBID B16 cells ± doxycycline were evaluated for cell death (72 hours), cytosolic mtDNA (12 hours), 2’, 3’-cGAMP (8 hours), and IFN-P mRNA (12 hours). FIG. 13G: Immunoblot analyses of the indicated B16 cells ± doxycycline at the indicated times. FIG. 13H: C57BL / 6J mice bearing the indicated B16 intradermal tumors that reached 300 mm3were randomized and given doxycycline. Tumor volume was determined by caliper measurements. FIG. 131:C57BL / 6J mice bearing the indicated B16 intradermal tumors that reached 200 mm3were given doxycycline for 2-3 weeks to induce tumor regression and maintained on doxycycline for 2 more weeks. Two weeks after doxycycline withdrawal, mice were rechallenged with the same B16 cells on opposite flanks (n = 7-8 mice for each cell line). B16 cells were also inoculated in naive mice (n = 6-8 mice for each cell line). Survival of mice is shown. **P < 0.01; ***P < 0.001 (log-rank test). FIG. 13J: C57BL / 6J mice at 6 months after rechallenge with tet-tBID ApafP' B16 cells as described in (I) were subjected to the second rechallenge. Survival of mice is shown (n = 8). Data shown in (B) and (D)-(F) are mean ± SD from three independent experiments. **P < 0.01; ***P < 0.001;0.0001 (unpaired Student t-test). See also FIGs.19 and 20.

[0027] FIGs. 14A-14F. Intratumoral induction of baciptosis alters tumor microenvironment and activates CD8 T cells. FIG. 14A: Heatmap of intratumoral cytokines in the indicated B16 tumors ± doxycycline for 24 hours measured by ELISA (IFN-P) or multiplex Luminex (all the other cytokines) assays (n = 3 tumors per group). FIG. 14B: Cytokine levels in the indicated B16 tumors ± doxycycline for 24 hours. Data are presented as mean ± SD (n = 3 tumors per group). FIGs. 14C-14D: Flow cytometric quantification of the indicated immune cells in the indicated B 16 tumors ± doxycycline for 48 hours. Data are presented as mean ± SD (n = 4-7 tumors per group). FIG. 14E:C57BL / 6J mice bearing intradermal tumors derived from H2-Kb-SIINFEKL (SEQ ID NO: 53)-mCherry-transdued tet-tBID Apafl+ / +or ApafP / ' B16 cells were given doxycycline and-13- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388the tumor-draining lymph nodes were subjected to flow cytometric analyses at the indicated times. Data are presented as mean ± SD (n = 4-7 lymph nodes per group). FIG. 14F:C57BL / 6J mice bearing intradermal tumors derived from tet-tBID Apafl+ / +or Apaf1- / -B16 cells were given doxycycline for 14 days and the tumor-draining lymph nodes were subjected to flow cytometric analyses. Data are presented as mean ± SD (n = 5-11 lymph nodes per group). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant (unpaired Student t-test). See also FIG. 21.

[0028] FIGs. 15A-15H. Single-cell transcriptomic profiling of B16 melanoma undergoing baciptosis versus apoptosis. FIG. 15A: Uniform manifold approximation and projection (UMAP) of T cell clusters from tet-tBID Apafl+ / +and ApafP / ' B16 tumors at 0 and 48 hours following tBID induction. Stacked barplots show the proportion of each T cell cluster in the indicated B16 tumors. FIG. 15B: Stacked barplots show the number of cells in each CD8 T cell cluster (left) or the proportion of each CD8 T cell cluster (right) in the indicated B16 tumors. FIG. 15C: Topic modeling using latent Dirichlet allocation (LDA) with 10 topics, with topic posterior probabilities across single cells grouped by T cell clusters (left) and by sample (right). FIG. 15D: Kaplan-Meier analysis of overall survival in cutaneous melanoma patients from TCGA based on the expression of the baciptosis signature of CD8 T cells. The top 50% highly expressed are shown in red and the bottom 50% are shown in blue. FIG. 15E: UMAP of myeloid clusters as in (A). Stacked barplots show the proportion of each myeloid cluster in the indicated B 16 tumors. FIG. 15F:Stacked barplots show the number of cells in each myeloid cluster (left) or the proportion of each myeloid cell cluster (right) in the indicated B 16 tumors. FIGs. 15G-15H: Kaplan-Meier analysis of overall survival in cutaneous melanoma patients from TCGA based on the expression of the baciptosis signature of Clq-high monocytes (FIG. 15G) or cancer cells (FIG. 15H). The top 50% highly expressed are shown in red and the bottom 50% are shown in blue. P values in FIGs. 15D, 15G, 15H are determined by Mantel-Cox test. See also FIG. 22

[0029] FIGs. 16A-16H. Activation of cGAS-STING-IFN-P signaling contributes significantly yet incompletely to baciptosis-induced immunological memory and MHC-I upregulation. FIG. 16A: C57BL / 6J mice were injected intradermally with tet-tBID ApafP' B16 cells or PBS (n = 7 for each group). One week later, mice were injected with -14- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388parental B16 cells on opposite flanks. One more week later, mice were given doxycycline and monitored for tumor growth and survival. Survival of mice is shown. P = 0.0015 (log-rank test). FIGs. 16B-16C: C57BL / 6J mice with regressed tet-tBID Apafl ^ B 16 tumors were treated with the indicated antibodies starting one day prior to rechallenge as in Fig. II (n = 8-10 tumors per group). Antibody-mediated depletion of specific immune cells was confirmed by flow cytometric analyses of peripheral blood in (FIG. 16B). Kaplan-Meier survival curves of mice are shown in (FIG. 16C) FIG. 16D: Survival curves of Rag2~ / ~ mice bearing regressed tet-tBID Apaf1- / -B16 tumors (n = 8) or naive Rag2~ / ~ mice (n = 6) following tumor implantation. FIG. 16E: Survival curves of C57BL / 6J mice bearing regressed tet-tBID Apafl ^ B16 tumors (n = 8) or tet-tBID Cgas^Apafl ^ B16 tumors (n = 10) as well as Sting"'"' mice bearing regressed tet-tBID Apaf1- / -B16 tumors (n = 9) following rechallenge. FIG. 16F: Intratumoral levels of 2’, 3’-cGAMP and IFN-0 were assessed by ELISA in the indicated tumors ± doxycycline for 24 hours. FIGs. 16G-16H:Flow cytometric quantification of MHC-I in the indicated B 16 cells ± doxycycline for the indicated times. Data are presented as mean ± SD (n = 3). P values in FIGs. 16A, 16C, 16D, and 16E are determined by log-rank test. P values in FIGs. 16F-16H, are determined by unpaired Student t-test. * < 0.05; **P < 0.01; *** < 0.001; ****P < 0.0001; ns, not significant. See also FIG. 23.

[0030] FIGs. 17A-17E. Baciptosis enhances MHC-I cross-dressing of antigen-presenting cells (APCs) to confer antitumor memory. FIGs. 17A-17B: B2m~ ~ mice bearing tet-tBID ApafP' B16 (FIG. 17A) or tet-tBID Apafl+ / +(FIG. 17B) tumors were mock treated and treated with doxycycline water to induce tBID. The indicated B 16 tumors ± doxycycline for 48 hours were subjected to flow cytometric analyses. Representative histograms depicting the H-2Kbexpression on tumor cells and the indicated immune subsets from 3 independent experiments. FMO, Fluorescence Minus One. FIGs. 17C-17D: B2m~ ~ mice bearing tet-tBID ApafP / ' B 16 tumors were treated as described above. Representative immunofluorescence images depicting the H-2Kbexpression on tumor-infiltrating CD1 lc+APCs (FIG. 17C) or F4 / 80+APCs (FIG. 17D) in the indicated B16 tumors with the quantification shown in the right panel. Yellow arrowheads indicate CD1 lc+or F4 / 80+APCs that are positive for H-2Kb, while white arrowheads denote cells that are singly positive for CD11c, F4 / 80, or H-2Kb. Data are presented as mean ± SD (n = 8-10 per group -15- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388from 2 independent experiments). ***P < 0.001; ****P < 0.0001 (unpaired Student t-test).FIG. 17E: C57BL / 6J mice bearing tet-tBID ApafP / ~B2m~ / ~ or tet-tBID Apaf1- / -B16 tumors were given doxycycline to induce tumor regression as in FIG. II. Four weeks after tumor regression, mice were rechallenged with tet-tBID ApafP B 16 cells on the opposite flank. Kaplan-Meier survival curve of mice is shown. P = 0.0220 (log-rank test). See also FIG.23.

[0031] FIGs. 18A-18G: Pharmacological activation of baciptosis sensitizes apoptosome-defective melanomas to anti-PD-1 and facilitates cancer eradication. FIG.18A: Cell death oiApafl+ / +orApaf1- / -B16-F10 cells treated with the indicated agents for 24 hours was assessed by annexin-V staining. Data are presented as mean ± SD (n = 3). FIG. 18B: IFN-0 mRNA of Apafl+ / +or ApafP' B16-F10 cells treated with the indicated agents for 12 hours was assessed by qRT-PCR and normalized against p-Actin. Data are presented as mean ± SD (n = 4). FIG. 18C: Flow cytometric quantification of the indicated immune cells in the indicated B16 tumors ± venetoclax and dinaciclib. Data are presented as mean ± SD (n = 6-7 tumors per group). FIGs. 18D-18E: C57BL / 6J mice were injected subcutaneously with parental or ApafP B16-F10 cells. One week later, mice were randomized and treated with the indicated agents for up to 7 weeks. Survival of mice is shown. FIGs. 18F-18G: C57BL / 6J mice were injected subcutaneously with parental or Apaf YUMM1.7 cells. Once tumors were palpable, mice were randomized and treated with the indicated agents for up to 9 weeks. Survival of mice is shown. P values in FIGs.18A-18C are determined by unpaired Student t-test. P values in (D)-(G) are determined by log-rank test. * < 0.05; **P < 0.01; *** < 0.001; ****P < 0.0001; ns, not significant. See also FIG.24.

[0032] FIGs 19A-19B. Ultrastructural characterization of BAX / BAK activation-induced caspase independent cell death (baciptosis), related to Figure 13. FIGs. 19A-19B: Electron microscopy imaging of tBID-inducible 4 / / / 7 (FIG. 19A) or^ / ? / _B16-F10 (FIG. 19B) before or after doxycycline treatment for 6 hours (Apafl+ / +) or 48 hours (ApafP /

[0033] FIGs 20A-20I. Baciptosis is the most immunogenic type of cell death that confers immunological memory against tumor rechallenge, related to Figure 13. FIGs -16- 4920-5495-9747.1Atty. Dkt. No.: 115872-338820A-20B: Extracellular LDH levels and cell death of the indicated B16 cells ± doxycycline (1 pg / ml) at the indicated times were measured using the LDH-Glo™ cytotoxicity assay and annexin-V staining, respectively. Data are presented as mean ± SD (n = 6 for LDH assays and n = 3 for viability assays). Immunoblot analyses of the indicated cells ± doxycycline for 6 hours. FIG. 20C: Cell death of the indicated cell lines treated with the indicated agents was assessed by propidium iodide staining at the indicated times. Data are presented as mean ± SD (n = 3). FIG. 20D: Immunoblot analyses of the indicated cells ± doxycycline for 6 hours. FIG. 20E: Extracellular ATP levels of the indicated B16 cells ± doxycycline at the indicated times were measured using the RealTime-Glo extracellular ATP assay (n = 4).FIG. 20F: Surface calreticulin exposure of the indicated B16 cells ± doxycycline at the indicated times was determined by FACS. The geometric mean fluorescence intensity of surface calreticulin on live cells is shown. Data are presented as mean ± SD (n = 4). FIG.20G: C57BL / 6J mice bearing the indicated B16 intradermal tumors that reached 200 mm3 were given doxycycline for 2-3 weeks to induce tumor regression and maintained on doxycycline for 2 more weeks. Two weeks after doxycycline withdrawal, mice were rechallenged with the same B 16 cells on opposite flanks (n = 7-8 mice for each cell line). B16 cells were also inoculated in naive mice to serve as controls (n = 6-8 mice for each cell line). Tumor growth curves are shown. FIG. 20H: C57BL / 6J mice at 6 months after rechallenge with tet-tBID Apafl- / - B16 cells as described in FIG. 20G were subjected to the second rechallenge. Tumor growth curves are shown (n = 8). FIG. 201: C57BL / 6J mice bearing the indicated B16 intradermal tumors were given doxycycline to induce tumor regression followed by rechallenge as in FIG. 20G (n = 10-11 mice for each cell line). B16 cells were also inoculated in naive mice (n = 5-8 mice for each cell line). Survival of mice is shown. P values in FIGs. 20A, 20C, and 20F are determined by unpaired Student / -test. P values in FIG. 201 are determined by log-rank test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, < 0.0001.

[0034] FIGs. 21A-21D. Characterization of tumor-infiltrating CD8+T cells and antigen-specific CD8+T cells in the tumor-draining lymph nodes, related to Figure 14. FIG. 21 A: Representative flow cytometry plots of CD8+ T cells from the indicated B 16 tumors ± doxycycline for 2 days. FIG. 21B: mCherry expression as measured by flow -17- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388cytometry in the indicated B16 cells following retroviral transduction with H2-Kb-SIINFEKL (SEQ ID NO: 53) -mCherry and flow sorting for uniform expression. FIG. 21C: Surface H2-Kb-SIINFEKL (SEQ ID NO: 53) expression at baseline (left) and 24 hours following treatment with 10 ng / mL IFN-y (right) in cell lines from FIG. 21B. FIG. 21D:Representative flow cytometry plots of H2-Kb-SIINFEKL (SEQ ID NO: 53) tetramer+ CD8+ T cells in the tumor draining lymph nodes from C57BL / 6J mice bearing intradermal tumors derived from H2-Kb-SIINFEKL (SEQ ID NO: 53) -mCherry-transduced, tet-tBID Apafl+ / +or Apaf / ~ 6 cells following doxycycline administration at the indicated times.

[0035] FIGs. 22A-22H. Characterization of tumor-infiltrating leukocytes by single-cell RNA-seq in B16 melanoma, related to Figure 15. FIG. 22A: Uniform manifold approximation and projection (UMAP) embedding of transcriptional profiles of single cells from tet-tBID Apafl+ / +and tet-tBID Apafl ^ B 16 tumors at 0 and 48 hours post-tBID induction (n = 39,360). Each dot represents a single cell, colored by inferred cell type clusters. Stacked barplots illustrate the number of cells in each cell type in the indicated B16 tumors. FIG. 22B: Stacked barplots show the number of cells in each T cell cluster within the indicated B16 tumors. FIG. 22C: Stacked barplots show the number of cells in each T cell cluster (left) or the proportion of each T cell cluster (right) within the indicated B16 tumors. FIG. 22D: Violin plots depict the expression levels of specified genes across different T cell clusters. FIG. 22E: Stacked barplots show the number of cells in each myeloid cluster in the indicated B16 tumors. FIG. 22F: Violin plots display the expression levels of specified genes across different myeloid clusters. FIG. 22G: Kaplan-Meier analysis of overall survival in cutaneous melanoma patients from TCGA based on the expression of the baciptosis signature of CCR7+ dendritic cells. The top 50% highly expressed are shown in red and the bottom 50% are shown in blue. The P value is determined by Mantel-Cox test. FIG. 22H: Scatter plot demonstrating a negative correlation between the shared immune signature and APAF1 mRNA expression in the TCGA cutaneous melanoma cohort. The Pearson correlation coefficient and its associated P value are calculated using a two-sided test.

[0036] FIGs. 23A-23F. Molecular characterization of baciptosis-induced antitumor immunity, related to Figures 16 and 17. FIG. 23A: C57BL / 6J mice were injected intradermally with tet-tBID ApafP / ' B 16 cells or PBS (n = 7 for each group). One week -18- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388later, parental B16 cells were injected on the opposite flank. One more week later, mice were given doxycycline and monitored for tumor growth and survival. Tumor growth curves are shown. FIG. 23B: C57BL / 6J mice bearing regressed tet-tBID Apafl^ B16 tumors were treated with the indicated antibodies one day prior to rechallenge (n = 8-10 tumors / group). Tumor growth curves are shown. FIG. 23C: Rag2 / ' mice bearing tet-tBID Apafl / _B16 intradermal tumors that reached 200 mm3were randomized and given doxycycline. Tumor volume was determined by caliper measurements. FIG. 23D: Tumor growth curves of Rag2 / ' mice bearing regressed tet-tBID Apafl^ B 16 tumors (n = 8) or naive Rag2~ / ~ mice (n = 6) following tumor implantation. FIG. 23E: Tumor growth curves of C57BL / 6J mice bearing regressed tet-tBID Apafl B16 tumors (n = 8) or tet-tBID Cgasr / ~ Apafl^Xb tumors (n = 10) as well as Stinggt / gtmice bearing regressed tet-tBID Apafl / _B16 tumors (n = 9) following rechallenge. FIG. 23F: Tumor growth curves of C57BL / 6J mice bearing regressed tet-tBID Apafl~ / ~B2m~ / ~ or tet-tBID Apafl / _B16 tumors following rechallenge with tet-tBID Apafl ^ B16 cells.

[0037] FIGs. 24A-24F. Baciptosis can be exploited as an anticancer strategy to eradicates cancer, related to Figure 18. FIG.24A: C57BL / 6J mice were injected subcutaneously with parental B16-F10 cells. One week later, mice were randomized and treated with the indicated agents. Tumor growth curves are shown from the time of B16 injection. FIG. 24B: C57BL / 6J mice were injected subcutaneously with Apafl ^ B16-F10 cells. One week later, mice were randomized and treated with the indicated agents. Tumor growth curves are shown from the time of B16 injection. FIG. 24C: C57BL / 6J mice were injected subcutaneously with parental YUMM1.7 cells. Once tumors were palpable, mice were randomized and treated with the indicated agents. Tumor growth curves are shown from the time of treatment. FIG. 24D: C57BL / 6J mice were injected subcutaneously with Apafl ^ YUMM1.7 cells. Once tumors were palpable, mice were randomized and treated with the indicated agents. Tumor growth curves are shown from the time of treatment. FIG. 24E: Flow cytometric quantification of the indicated immune cells nApafl ^ YUMM1.7 tumors ± venetoclax and dinaciclib. Data are presented as mean ± SD (n = 6 tumors for each group). FIG. 24F: C57BL / 6J mice were injected subcutaneously with parental B16-F10 cells. One week later, mice were randomized and treated with the indicated agents. Survival of mice is shown. P values in FIG. 24E are determined by -19- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388unpaired Student / -test. P values in FIG. 24F are determined by log-rank test, ns, not significant; *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

[0038] FIG. 25. Schematic illustration depicting the dual mechanisms through which baciptosis enhances robust antitumor CD8 T cell responses.

[0039] FIG. 26. Schematic of cancer therapies converging on BAX / BAK activation.DETAILED DESCRIPTION

[0040] It is to be appreciated that certain aspects, modes, embodiments, variations and features of the present methods are described below in various levels of detail in order to provide a substantial understanding of the present technology.

[0041] In practicing the present methods, many conventional techniques in molecular biology, protein biochemistry, cell biology, immunology, microbiology and recombinant DNA are used. See, e.g., Sambrook and Russell eds. (2001) Molecular Cloning: A Laboratory Manual, 3rd edition; the series Ausubel etal. eds. (2007) Current Protocols in Molecular Biology, the series Methods in Enzymology (Academic Press, Inc., N. Y.);MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach,' Harlow and Lane eds. ( \999 Antibodies, A Laboratory Manual,' Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th edition; Gait ed. (1984) Oligonucleotide Synthesis,' U. S. Patent No. 4,683,195; Hames and Higgins eds. (1984) Nucleic Acid Hybridization,' Anderson (1999) Nucleic Acid Hybridization,' Hames and Higgins eds. (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; Miller and Calos eds. (1987) Gene Transfer Vectors for Mammalian Cells (Cold Spring Harbor Laboratory); Makrides ed. (2003) Gene Transfer and Expression in Mammalian Cells; Mayer and Walker eds. (1987) Immunochemical Methods in Cell and Molecular Biology (Academic Press, London); and Herzenberg et al. eds (1996) Weir ’s Handbook of Experimental Immunology. Methods to detect and measure levels of polypeptide gene expression products (i.e., gene translation level) are well-known in the art and include the use of polypeptide detection methods such as antibody detection and quantification techniques. (See also, Strachan & Read, Human Molecular Genetics, Second Edition. (John Wiley and Sons, Inc., NY, 1999)).-20- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388

[0042] Mitochondria play a pivotal role in mammalian cell death because various apoptotic signals converge to trigger the release of cytochrome c from the mitochondrial intermembrane space to activate APAF1 for the assembly of a heptameric complex known as the apoptosome, coupling this organelle to caspase activation.1,2Multidomain proapoptotic BAK and BAX are essential effectors responsible for the permeabilization of the mitochondrial outer membrane (MOM), whereas antiapoptotic BCL-2, BCL-XL, and MCL-1 preserve mitochondrial integrity.3'6The third subfamily of BCL-2 proteins, BH3-only molecules (BH3s), integrate upstream apoptotic signals to promote apoptosis by either activating BAK / B AX directly ^‘activator” BH3s) or inactivating BCL-2 / BCL-XL / MCL-1 (“inactivator" or “sensitizer” BH3s).4-16In response to apoptotic signals, activator BH3s bind directly to BAK / B AX to induce the homo-oligomerization of BAK / B AX, leading to mitochondrial outer membrane permeabilization (MOMP).4,6,9-16Conversely, antiapoptotic BCL-2, BCL-XL, and MCL-1 sequester either activator BH3s to prevent the initiation of BAK / B AX activation or “BH3 -exposed” BAK / B AX monomers to inhibit their homooligomerization.4,9,15,17Research on BCL-2 family-regulated apoptosis has not only illuminated its critical role in normal physiological and disease processes but also led to the development of the first anti-cancer drug targeting protein-protein interactions.18-21The BCL-2 selective inhibitor venetoclax (ABT- 199) has been approved by the FDA for the treatment of relapsed chronic lymphocytic leukemia since 2016.20Clinical trials have also demonstrated the efficacy of ABT-263 (navitoclax), a dual inhibitor of BCL-2 and BCL-XL. Additionally, several MCL-1 inhibitors are currently undergoing clinical trials.21,22

[0043] Activation of BAK / B AX not only triggers APAF1 -mediated caspase activation but also initiates caspase-independent mitochondrial dysfunction and cellular demise4Recent findings have shown that BAK / B AX activation in the absence of caspases leads to sustained release of mitochondrial DNA (mtDNA) into the cytosol, activating the cGAS / STING pathway and type I interferon (IFN) response.23-26State-of-the-art imaging analyses revealed that cytochrome c exits from BAK / B AX pores that are too small to be resolved by super-resolution microscopy.25After cytochrome c efflux, BAK / B AX coalesces to form macropores in the MOM through which the mitochondrial inner membrane (MIM) herniates, resulting in “mitochondrial inner membrane permeabilization” (MIMP) and mtDNA efflux.25,26Intriguingly, only a minority of herniated MIM loses membrane-21- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388integrity and most TF AM-bound mtDNA remains inside the mitochondria during this process.25In the presence of caspases, mtDNA efflux occurs transiently on the verge of cell death due to the rapid kinetics of caspase-mediated apoptosis.25,26Even when a small amount of mtDNA is released during apoptosis, mtDNA-induced cGAS-STING-type I IFN signaling is abrogated by caspase-3 -mediated cleavage of cGAS and IRF3, rendering apoptosis immunologically silent.23,24,27,28

[0044] The discovery of BAK / BAX activation-induced mtDNA efflux and subsequent activation of immunogenic cell death opens exciting new avenues for cancer cell death research, particularly given that impaired apoptosome function is common in human cancers.29However, the molecular and biochemical basis of mtDNA efflux remains elusive. Specifically, it remains unclear whether mtDNA efflux is simply a consequence of MOMP and MOMP-associated mitochondria inner membrane herniation, or whether it involves yet-to-be-identified active biochemical processes. Furthermore, it is unknown whether MIMP in general, and mtDNA release specifically, contribute to caspase-independent cell death. Given that neurons and myocardium express little APAF1,30,31MIMP and mtDNA efflux may contribute to the pathogenesis of diseases associated with neuronal and myocardial cell death. The present disclosure identifies and characterizes the macromolecular BAK- and B AX-associated complexes that regulate cytochrome c versus mtDNA efflux.Definitions

[0045] Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. For example, reference to “a cell” includes a combination of two or more cells, and the like. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, analytical chemistry and nucleic acid chemistry and hybridization described below are those well-known and commonly employed in the art.

[0046] As used herein, the term “about” in reference to a number is generally taken to include numbers that fall within a range of 1%, 5%, or 10% in either direction (greater than -22- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388or less than) of the number unless otherwise stated or otherwise evident from the context (except where such number would be less than 0% or exceed 100% of a possible value).

[0047] As used herein, the “administration” of an agent or drug to a subject includes any route of introducing or delivering to a subject a compound to perform its intended function. Administration can be carried out by any suitable route, including but not limited to, orally, intranasally, intrathecally, parenterally (intravenously, intramuscularly, intraperitoneally, or subcutaneously), rectally, intrathecally, intraocularly, intradermally, transmucosally, iontophoretically, or topically. Administration includes self-administration and the administration by another.

[0048] As used herein, a "control" is an alternative sample used in an experiment for comparison purpose. A control can be "positive" or "negative." For example, where the purpose of the experiment is to determine a correlation of the efficacy of a therapeutic agent for the treatment for a particular type of disease, a positive control (a compound or composition known to exhibit the desired therapeutic effect) and a negative control (a subject or a sample that does not receive the therapy or receives a placebo) are typically employed.

[0049] As used herein, the term “effective amount” refers to a quantity sufficient to achieve a desired therapeutic and / or prophylactic effect, e.g., an amount which results in the prevention of, or a decrease in a disease or condition described herein or one or more signs or symptoms associated with a disease or condition described herein. In the context of therapeutic or prophylactic applications, the amount of a composition administered to the subject will vary depending on the composition, the degree, type, and severity of the disease and on the characteristics of the individual, such as general health, age, sex, body weight and tolerance to drugs. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. The compositions can also be administered in combination with one or more additional therapeutic compounds. In the methods described herein, the therapeutic compositions may be administered to a subject having one or more signs or symptoms of a disease or condition described herein. As used herein, a "therapeutically effective amount" of a composition refers to composition levels in which-23- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388the physiological effects of a disease or condition are ameliorated or eliminated. A therapeutically effective amount can be given in one or more administrations.

[0050] As used herein, “expression” includes one or more of the following: transcription of the gene into precursor mRNA; splicing and other processing of the precursor mRNA to produce mature mRNA; mRNA stability; translation of the mature mRNA into protein (including codon usage and tRNA availability); and glycosylation and / or other modifications of the translation product, if required for proper expression and function.

[0051] As used herein, the term “gene” means a segment of DNA that contains all the information for the regulated biosynthesis of an RNA product, including promoters, exons, introns, and other untranslated regions that control expression.

[0052] As used herein, the term “pharmaceutically-acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal compounds, isotonic and absorption delaying compounds, and the like, compatible with pharmaceutical administration. Pharmaceutically-acceptable carriers and their formulations are known to one skilled in the art and are described, for example, in Remington's Pharmaceutical Sciences (20thedition, ed. A. Gennaro, 2000, Lippincott, Williams & Wilkins, Philadelphia, Pa.).

[0053] As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to mean a polymer comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, / .<., peptide isosteres. Polypeptide refers to both short chains, commonly referred to as peptides, glycopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids. Polypeptides include amino acid sequences modified either by natural processes, such as post-translational processing, or by chemical modification techniques that are well known in the art.

[0054] As used herein, “prevention,” “prevent,” or “preventing” of a disorder or condition refers to one or more compounds that, in a statistical sample, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of one or more symptoms of the disorder or condition relative to -24- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388the untreated control sample. As used herein, prevention includes preventing or delaying the initiation of symptoms of a disease or condition described herein and / or preventing a recurrence of one or more signs or symptoms of a disease or condition described herein.

[0055] As used herein, a “sample” or “biological sample” refers to a body fluid or a tissue sample isolated from a subject. In some cases, a biological sample may consist of or comprise whole blood, platelets, red blood cells, white blood cells, plasma, sera, urine, feces, epidermal sample, vaginal sample, skin sample, cheek swab, sperm, amniotic fluid, cultured cells, bone marrow sample, tumor biopsies, aspirate and / or chorionic villi, cultured cells, endothelial cells, synovial fluid, lymphatic fluid, ascites fluid, interstitial or extracellular fluid and the like. The term "sample" may also encompass the fluid in spaces between cells, including gingival crevicular fluid, bone marrow, cerebrospinal fluid (CSF), saliva, mucus, sputum, semen, sweat, urine, or any other bodily fluids. Samples can be obtained from a subject by any means including, but not limited to, venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage, scraping, surgical incision, or intervention or other means known in the art. A blood sample can be whole blood or any fraction thereof, including blood cells (red blood cells, white blood cells or leukocytes, and platelets), serum and plasma.

[0056] As used herein, the terms “subject”, “patient”, or “individual” are used interchangeably, and can be an individual organism, a vertebrate, a mammal, or a human. In some embodiments, the subject, patient or individual is a human.

[0057] “Treating” or “treatment” as used herein covers the treatment of a disease or disorder described herein, in a subject, such as a human, and includes: (i) inhibiting a disease or disorder, z.e., arresting its development; (ii) relieving a disease or disorder, z.e., causing regression of the disorder; (iii) slowing progression of the disorder; and / or (iv) inhibiting, relieving, or slowing progression of one or more symptoms of the disease or disorder. In some embodiments, treatment means that the symptoms associated with the disease are, e.g., alleviated, reduced, cured, or placed in a state of remission.

[0058] It is also to be appreciated that the various modes of treatment of disorders as described herein are intended to mean “substantial,” which includes total but also less than total treatment, and wherein some biologically or medically relevant result is achieved. The -25- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388treatment may be a continuous prolonged treatment for a chronic disease or a single, or few time administrations for the treatment of an acute condition.Compositions Including APAF1 Inhibitors of the Present Technology

[0059] The present disclosure provides therapeutic agents that inhibit the activity or expression of Apafl. In some embodiments, the Apafl inhibitor is a small molecule, an inhibitory nucleic acid (e.g., siRNA, antisense nucleic acid, shRNA, sgRNA, ribozymes), or an antibody (e.g., a neutralizing antibody). Examples of Apafl -specific small molecule inhibitors include, but are not limited to ZYZ-488, QM31 (SVT016426), UCN-01, SVT017686, SVT017923, SVT016448, or N-alkylglycine trimers. Examples oiApafl-specific small molecule inhibitors are known in the art and are described in Wang et al., Scientific Reports 6: 29820 (2016), Malet et al., Cell Death & Differentiation volume 13, pagesl 523-1532 (2006), the contents of which are incorporated herein by reference in their entirety.

[0060] Exemplary mRNA sequences of Apafl are provided below, represented by SEQ IDNOs: 54-58.NM 001160.3 Homo sapiens apoptotic peptidase activating factor 1 (APAF1 ), transcript variant 2, mRNA ( SEQ ID NO: 54 ) AGAGGCGGAGAAGAAGAGGTAGCGAGTGGACGTGACTGCTCTATCCCGGGCAAAAGGGATAGAACCAGAG GTGGGGAGTCTGGGCAGTCGGCGACCCGCGAAGACTTGAGGTGCCGCAGCGGCATCCGGAGTAGCGCCGG GCTCCCTCCGGGGTGCAGCCGCCGTCGGGGGAAGGGCGCCACAGGCCGGGAAGACCTCCTCCCTTTGTGT CCAGTAGTGGGGTCCACCGGAGGGCGGCCCGTGGGCCGGGCCTCACCGCGGCGCTCCGGGACTGTGGGGT CAGGCTGCGTTGGGTGGACGCCCACCTCGCCAACCTTCGGAGGTCCCTGGGGGTCTTCGTGCGCCCCGGG GCTGCAGAGATCCAGGGGAGGCGCCTGTGAGGCCCGGACCTGCCCCGGGGCGAAGGGTATGTGGCGAGAC AGAGCCCTGCACCCCTAATTCCCGGTGGAAAACTCCTGTTGCCGTTTCCCTCCACCGGCCTGGAGTCTCC CAGTCTTGTCCCGGCAGTGCCGCCCTCCCCACTAAGACCTAGGCGCAAAGGCTTGGCTCATGGTTGACAG CTCAGAGAGAGAAAGATCTGAGGGAAGATGGATGCAAAAGCTCGAAATTGTTTGCTTCAACATAGAGAAG CTCTGGAAAAGGACATCAAGACATCCTACATCATGGATCACATGATTAGTGATGGATTTTTAACAATATC AGAAGAGGAAAAAGTAAGAAATGAGCCCACTCAACAGCAAAGAGCAGCTATGCTGATTAAAATGATACTT AAAAAAGATAATGATTCCTACGTATCATTCTACAATGCTCTACTACATGAAGGATATAAAGATCTTGCTG CCCTTCTCCATGATGGCATTCCTGTTGTCTCTTCTTCCAGTGTAAGGACAGTCCTGTGTGAAGGTGGAGT ACCACAGAGGCCAGTTGTTTTTGTCACAAGGAAGAAGCTGGTGAATGCAATTCAGCAGAAGCTCTCCAAA TTGAAAGGTGAACCAGGATGGGTCACCATACATGGAATGGCAGGCTGTGGGAAGTCTGTATTAGCTGCAG AAGCTGTTAGAGATCATTCCCTTTTAGAAGGTTGTTTCCCAGGGGGAGTGCATTGGGTTTCAGTTGGGAA ACAAGACAAATCTGGGCTTCTGATGAAACTGCAGAATCTTTGCACACGGTTGGATCAGGATGAGAGTTTT TCCCAGAGGCTTCCACTTAATATTGAAGAGGCTAAAGACCGTCTCCGCATTCTGATGCTTCGCAAACACC CAAGGTCTCTCTTGATCTTGGATGATGTTTGGGACTCTTGGGTGTTGAAAGCTTTTGACAGTCAGTGTCA GATTCTTCTTACAACCAGAGACAAGAGTGTTACAGATTCAGTAATGGGTCCTAAATATGTAGTCCCTGTG GAGAGTTCCTTAGGAAAGGAAAAAGGACTTGAAATTTTATCCCTTTTTGTTAATATGAAGAAGGCAGATT TGCCAGAACAAGCTCATAGTATTATAAAAGAATGTAAAGGCTCTCCCCTTGTAGTATCTTTAATTGGTGC ACTTTTACGTGATTTTCCCAATCGCTGGGAGTACTACCTCAAACAGCTTCAGAATAAGCAGTTTAAGAGA ATAAGGAAATCTTCGTCTTATGATTATGAGGCTCTAGATGAAGCCATGTCTATAAGTGTTGAAATGCTCA-26- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388 GAGAAGACATCAAAGATTATTACACAGATCTTTCCATCCTTCAGAAGGACGTTAAGGTGCCTACAAAGGT GTTATGTATTCTCTGGGACATGGAAACTGAAGAAGTTGAAGACATACTGCAGGAGTTTGTAAATAAGTCT CTTTTATTCTGTGATCGGAATGGAAAGTCGTTTCGTTATTATTTACATGATCTTCAAGTAGATTTTCTTA CAGAGAAGAATTGCAGCCAGCTTCAGGATCTACATAAGAAGATAATCACTCAGTTTCAGAGATATCACCA GCCGCATACTCTTTCACCAGATCAGGAAGACTGTATGTATTGGTACAACTTTCTGGCCTATCACATGGCC AGTGCCAAGATGCACAAGGAACTTTGTGCTTTAATGTTTTCCCTGGATTGGATTAAAGCAAAAACAGAAC TTGTAGGCCCTGCTCATCTGATTCATGAATTTGTGGAATACAGACATATACTAGATGAAAAGGATTGTGC AGTCAGTGAGAATTTTCAGGAGTTTTTATCTTTAAATGGACACCTTCTTGGACGACAGCCATTTCCTAAT ATTGTACAACTGGGTCTCTGTGAGCCGGAAACTTCAGAAGTTTATCAGCAAGCTAAGCTGCAGGCCAAGC AGGAGGTCGATAATGGAATGCTTTACCTGGAATGGATAAACAAAAAAAACATCACGAATCTTTCCCGCTT AGTTGTCCGCCCCCACACAGATGCTGTTTACCATGCCTGCTTTTCTGAGGATGGTCAGAGAATAGCTTCT T GT GGAGCT GATAAAACCTTACAGGT GTT CAAAGCT GAAACAGGAGAGAAACTT CTAGAAAT CAAGGCT C ATGAGGATGAAGTGCTTTGTTGTGCATTCTCTACAGATGACAGATTTATAGCAACCTGCTCAGTGGATAA AAAAGT GAAGATTT GGAATT CTAT GACT GGGGAACTAGTACACACCTAT GAT GAGCACT CAGAGCAAGT C AATTGCTGCCATTTCACCAACAGTAGTCATCATCTTCTCTTAGCCACTGGGTCAAGTGACTGCTTCCTCA AACTTTGGGATTTGAATCAAAAAGAATGTCGAAATACCATGTTTGGTCATACAAATTCAGTCAATCACTG CAGATTTTCACCAGATGATAAGCTTTTGGCTAGTTGTTCAGCTGATGGAACCTTAAAGCTTTGGGATGCG ACATCAGCAAATGAGAGGAAAAGCATTAATGTGAAACAGTTCTTCCTAAATTTGGAGGACCCTCAAGAGG ATAT GGAAGT GATAGT GAAGT GTT GTT CGT GGT CT GCT GAT GGT GCAAGGATAAT GGT GGCAGCAAAAAA TAAAATCTTTTTGTGGAATACAGACTCACGTTCAAAGGTGGCTGATTGCAGAGGACATTTAAGTTGGGTT CATGGTGTGATGTTTTCTCCTGATGGATCATCATTTTTGACATCTTCTGATGACCAGACAATCAGGCTCT GGGAGACAAAGAAAGTAT GTAAGAACT CT GCT GTAAT GTTAAAGCAAGAAGTAGAT GTT GT GTTT CAAGA AAATGAAGTGATGGTCCTTGCAGTTGACCATATAAGACGTCTGCAACTCATTAATGGAAGAACAGGTCAG ATTGATTATCTGACTGAAGCTCAAGTTAGCTGCTGTTGCTTAAGTCCACATCTTCAGTACATTGCATTTG GAGATGAAAATGGAGCCATTGAGATTTTAGAACTTGTAAACAATAGAATCTTCCAGTCCAGGTTTCAGCA CAAGAAAACTGTATGGCACATCCAGTTCACAGCCGATGAGAAGACTCTTATTTCAAGTTCTGATGATGCT GAAATTCAGGTATGGAATTGGCAATTGGACAAATGTATCTTTCTACGAGGCCATCAGGAAACAGTGAAAG ACTTTAGACTCTTGAAAAATTCAAGACTGCTTTCTTGGTCATTTGATGGAACAGTGAAGGTATGGAATAT TATTACTGGAAATAAAGAAAAAGACTTTGTCTGTCACCAGGGTACAGTACTTTCTTGTGACATTTCTCAC GATGCTACCAAGTTTTCATCTACCTCTGCTGACAAGACTGCAAAGATCTGGAGTTTTGATCTCCTTTTGC CACTTCATGAATTGAGGGGCCACAACGGCTGTGTGCGCTGCTCTGCCTTCTCTGTGGACAGTACCCTGCT GGCAACGGGAGATGACAATGGAGAAATCAGGATATGGAATGTCTCAAACGGTGAGCTTCTTCATTTGTGT GCTCCGCTTTCAGAAGAAGGAGCTGCTACCCATGGAGGCTGGGTGACTGACCTTTGCTTTTCTCCAGATG GCAAAATGCTTATCTCTGCTGGAGGATATATTAAGTGGTGGAACGTTGTCACTGGGGAATCCTCACAGAC CTTCTACACAAATGGAACCAATCTTAAGAAAATACACGTGTCCCCTGACTTCAAAACATATGTGACTGTG GATAATCTTGGTATTTTATATATTTTACAGACTTTAGAATAAAATAGTTAAGCATTAATGTAGTTGAACT TTTTAAATTTTTGAATTGGAAAAAAATTCTAATGAAACCCTGATATCAACTTTTTATAAAGCTCTTAATT GTTGTGCAGTATTGCATTCATTACAAAAGTGTTTGTGGTTGGATGAATAATATTAATGTAGCTTTTTCCC AAATGAACATACCTTTAATCTTGTTTTTCATGATCATCATTAACAGTTTGTCCTTAGGATGCAAATGAAA ATGTGAATACATACCTTGTTGTACTGTTGGTAAAATTCTGTCTTGATGCATTCAAAATGGTTGACATAAT TAATGAGAAGAATTTGGAAGAAATTGGTATTTTAATACTGTCTGTATTTATTACTGTTATGCAGGCTGTG CCTCAGGGTAGCAGTGGCCTGCTTTTTGAACCACACTTACCCCAAGGGGGTTTTGTTCTCCTAAATACAA TCTTAGAGGTTTTTTGCACTCTTTAAATTTGCTTTAAAAATATTGTGTCTGTGTGCATAGTCTGCAGCAT TTCCTTTAATTGACTCAATAAGTGAGTCTTGGATTTAGCAGGCCCCCCCACCTTTTTTTTTTGTTTTTGG AGACAGAGTCTTGCTTTGTTGCCAGGCTGGAGTGCAGTGGCGCGATCTCGGCTCACCACAATCGCTGCCT CCTGGGTTCAAGCAATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGTGTGCGCACATGCCAG GCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCACCATGTTGGCCGGGATGGTCTCGATCTCTTGACC TCATGATCTACCCGCCTTGGCCTCCCAAAGTGCTGAGATTACAGGCGTGAGCCACCGTGCCTGGCCAGGC CCCTTCTCTTTTAATGGAGACAGGGTCTTGCACTATCACCCAGGCTGGAGTGCAGTGGCATAATCATACC TCATTGCAGCCTCAGACTCCTGGGTTCAAGCAATCCTCCTGCCTCAGCCTCCCAAGTAGCTGAGACTACA GGCACGAGCCACCACACCCAGCTAATTTTTAAGTTTTCTTGTAGAGACAGGGTCTCACTATGTTGTCTAG GCTGGTCTTGAACTCTTGGCCTCAAGTAATCCTCCTGCCTCAGCCTCCCAAAGTGTTGGGATTGCAGATA TGAGCCACTGGCCTGGCCTTCAGCAGTTCTTTTTGTGAAGTAAAACTTGTATGTTGGAAAGAGTAGATTT TATTGGTCTACCCTTTTCTCACTGTAGCTGCTGGCAGCCCTGTGCCATATCTGGACTCTAGTTGTCAGTA TCTGAGTTGGACACTATTCCTGCTCCCTCTTGTTTCTTACATATCAGACTTCTTACTTGAATGAAACCTG ATCTTTCCTAATCCTCACTTTTTTCTTTTTTAAAAAGCAGTTTCTCCACTGCTAAATGTTAGTCATTGAG GTGGGGCCAATTTTAATCATAAGCCTTAATAAGATTTTTCTAAGAAATGTGAAATAGAACAATTTTCATC-27- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388 TAATTCCATTTACTTTTAGATGAATGGCATTGTGAATGCCATTCTTTTAATGAATTTCAAGAGAATTCTC TGGTTTTCTGTGTAATTCCAGATGAGTCACTGTAACTCTAGAAGATTAACCTTCCAGCCAACCTATTTTC CTTTCCCTTGTCTCTCTCATCCTCTTTTCCTTCCTTCTTTCCTTTCTCTTCTTTTATCTCCAAGGTTAAT CAGGAAAAATAGCTTTTGACAGGGGAAAAAACTCAATAACTAGCTATTTTTGACCTCCTGATCAGGAACT TTAGTTGAAGCGTAAATCTAAAGAAACATTTTCTCTGAAATATATTATTAAGGGCAATGGAGATAAATTA ATAGTAGATGTGGTTCCCAGAAAATATAATCAAAATTCAAAGATTTTTTTTGTTTCTGTAACTGGAACTA AATCAAATGATTACTAGTGTTAATAGTAGATAACTTGTTTTTATTGTTGGTGCATATTAGTATAACTGTG GGGTAGGTCGGGGAGAGGGTAAGGGAATAGATCACTCAGATGTATTTTAGATAAGCTATTTAGCCTTTGA TGGAATCATAAATACAGTGAATACAATCCTTTGCATTGTTAAGGAGGTTTTTTGTTTTTAAATGGTGGGT CAAGGAGCTAGTTTACAGGCTTACTGTGATTTAAGCAAATGTGAAAAGTGAAACCTTAATTTTATCAAAA GAAATTTCTGTAAATGGTATGTCTCCTTAGAATACCCAAATCATAATTTTATTTGTACACACTGTTAGGG GCTCATCTCATGTAGGCAGAGTATAAAGTATTACCTTTTGGAATTAAAAGCCACTGACTGTTATAAAGTA TAACAACACACATCAGGTTTTAAAAAGCCTTGAATGGCCCTTGTCTTAAAAAGAAATTAGGAGCCAGGTG CGGTGGCACGTGCCTGTAATCCCAGCTCCTTGGGAGGCTAAGACAGGAGGATTCCTTGAGCCCTGGAGTT TGAGTCCAGCCTGGGTGACATAGCAAGACCCTGTCTTAAAAGAAAAATGGGAAGAAAGACAAGGTAACAT GAAGAAAGAAGAGATACCTAGTATGATGGAGCTGCAAATTTCATGGCAGTTCATGCAGTCGGTCAAGAGG AGGATTTTGTTTTGTAGTTTGCAGATGAGCATTTCTAAAGCATTTTCCCTTGCTGTATTTTTTTGTATTA TAAATTACATTGGACTTCATATATATAATTTTTTTTTACATTATATGTCTCTTGTATGTTTTGAAACTCT TGTATTTATGATATAGCTTATATGATTTTTTTGCCTTGGTATACATTTTAAAATATGAATTTAAAAAATT T T T GT AAAAAT AAAAT T CACAAAAT T GT T T T GAAAAACA> NM 013229.3 Homo sapiens apoptotic peptidase activating factor 1 (APAF1 ), transcript variant 1, mRNA ( SEQ ID NO: 55 ) AGAGGCGGAGAAGAAGAGGTAGCGAGTGGACGTGACTGCTCTATCCCGGGCAAAAGGGATAGAACCAGAG GTGGGGAGTCTGGGCAGTCGGCGACCCGCGAAGACTTGAGGTGCCGCAGCGGCATCCGGAGTAGCGCCGG GCTCCCTCCGGGGTGCAGCCGCCGTCGGGGGAAGGGCGCCACAGGCCGGGAAGACCTCCTCCCTTTGTGT CCAGTAGTGGGGTCCACCGGAGGGCGGCCCGTGGGCCGGGCCTCACCGCGGCGCTCCGGGACTGTGGGGT CAGGCTGCGTTGGGTGGACGCCCACCTCGCCAACCTTCGGAGGTCCCTGGGGGTCTTCGTGCGCCCCGGG GCTGCAGAGATCCAGGGGAGGCGCCTGTGAGGCCCGGACCTGCCCCGGGGCGAAGGGTATGTGGCGAGAC AGAGCCCTGCACCCCTAATTCCCGGTGGAAAACTCCTGTTGCCGTTTCCCTCCACCGGCCTGGAGTCTCC CAGTCTTGTCCCGGCAGTGCCGCCCTCCCCACTAAGACCTAGGCGCAAAGGCTTGGCTCATGGTTGACAG CTCAGAGAGAGAAAGATCTGAGGGAAGATGGATGCAAAAGCTCGAAATTGTTTGCTTCAACATAGAGAAG CTCTGGAAAAGGACATCAAGACATCCTACATCATGGATCACATGATTAGTGATGGATTTTTAACAATATC AGAAGAGGAAAAAGTAAGAAATGAGCCCACTCAACAGCAAAGAGCAGCTATGCTGATTAAAATGATACTT AAAAAAGATAATGATTCCTACGTATCATTCTACAATGCTCTACTACATGAAGGATATAAAGATCTTGCTG CCCTTCTCCATGATGGCATTCCTGTTGTCTCTTCTTCCAGTGTAAGGACAGTCCTGTGTGAAGGTGGAGT ACCACAGAGGCCAGTTGTTTTTGTCACAAGGAAGAAGCTGGTGAATGCAATTCAGCAGAAGCTCTCCAAA TTGAAAGGTGAACCAGGATGGGTCACCATACATGGAATGGCAGGCTGTGGGAAGTCTGTATTAGCTGCAG AAGCTGTTAGAGATCATTCCCTTTTAGAAGGTTGTTTCCCAGGGGGAGTGCATTGGGTTTCAGTTGGGAA ACAAGACAAATCTGGGCTTCTGATGAAACTGCAGAATCTTTGCACACGGTTGGATCAGGATGAGAGTTTT TCCCAGAGGCTTCCACTTAATATTGAAGAGGCTAAAGACCGTCTCCGCATTCTGATGCTTCGCAAACACC CAAGGTCTCTCTTGATCTTGGATGATGTTTGGGACTCTTGGGTGTTGAAAGCTTTTGACAGTCAGTGTCA GATTCTTCTTACAACCAGAGACAAGAGTGTTACAGATTCAGTAATGGGTCCTAAATATGTAGTCCCTGTG GAGAGTTCCTTAGGAAAGGAAAAAGGACTTGAAATTTTATCCCTTTTTGTTAATATGAAGAAGGCAGATT TGCCAGAACAAGCTCATAGTATTATAAAAGAATGTAAAGGCTCTCCCCTTGTAGTATCTTTAATTGGTGC ACTTTTACGTGATTTTCCCAATCGCTGGGAGTACTACCTCAAACAGCTTCAGAATAAGCAGTTTAAGAGA ATAAGGAAATCTTCGTCTTATGATTATGAGGCTCTAGATGAAGCCATGTCTATAAGTGTTGAAATGCTCA GAGAAGACATCAAAGATTATTACACAGATCTTTCCATCCTTCAGAAGGACGTTAAGGTGCCTACAAAGGT GTTATGTATTCTCTGGGACATGGAAACTGAAGAAGTTGAAGACATACTGCAGGAGTTTGTAAATAAGTCT CTTTTATTCTGTGATCGGAATGGAAAGTCGTTTCGTTATTATTTACATGATCTTCAAGTAGATTTTCTTA CAGAGAAGAATTGCAGCCAGCTTCAGGATCTACATAAGAAGATAATCACTCAGTTTCAGAGATATCACCA GCCGCATACTCTTTCACCAGATCAGGAAGACTGTATGTATTGGTACAACTTTCTGGCCTATCACATGGCC AGTGCCAAGATGCACAAGGAACTTTGTGCTTTAATGTTTTCCCTGGATTGGATTAAAGCAAAAACAGAAC-28- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388 TTGTAGGCCCTGCTCATCTGATTCATGAATTTGTGGAATACAGACATATACTAGATGAAAAGGATTGTGC AGTCAGTGAGAATTTTCAGGAGTTTTTATCTTTAAATGGACACCTTCTTGGACGACAGCCATTTCCTAAT ATTGTACAACTGGGTCTCTGTGAGCCGGAAACTTCAGAAGTTTATCAGCAAGCTAAGCTGCAGGCCAAGC AGGAGGTCGATAATGGAATGCTTTACCTGGAATGGATAAACAAAAAAAACATCACGAATCTTTCCCGCTT AGTTGTCCGCCCCCACACAGATGCTGTTTACCATGCCTGCTTTTCTGAGGATGGTCAGAGAATAGCTTCT T GT GGAGCT GATAAAACCTTACAGGT GTT CAAAGCT GAAACAGGAGAGAAACTT CTAGAAAT CAAGGCT C ATGAGGATGAAGTGCTTTGTTGTGCATTCTCTACAGATGACAGATTTATAGCAACCTGCTCAGTGGATAA AAAAGT GAAGATTT GGAATT CTAT GACT GGGGAACTAGTACACACCTAT GAT GAGCACT CAGAGCAAGT C AATTGCTGCCATTTCACCAACAGTAGTCATCATCTTCTCTTAGCCACTGGGTCAAGTGACTGCTTCCTCA AACTTTGGGATTTGAATCAAAAAGAATGTCGAAATACCATGTTTGGTCATACAAATTCAGTCAATCACTG CAGATTTTCACCAGATGATAAGCTTTTGGCTAGTTGTTCAGCTGATGGAACCTTAAAGCTTTGGGATGCG ACATCAGCAAATGAGAGGAAAAGCATTAATGTGAAACAGTTCTTCCTAAATTTGGAGGACCCTCAAGAGG ATAT GGAAGT GATAGT GAAGT GTT GTT CGT GGT CT GCT GAT GGT GCAAGGATAAT GGT GGCAGCAAAAAA TAAAATCTTTCTTTTTGACATTCATACTAGTGGCCTATTGGGAGAAATCCACACGGGCCATCACAGCACC ATCCAGTACTGTGACTTCTCCCCACAAAACCATTTGGCAGTGGTTGCTTTGTCCCAGTACTGTGTAGAGT TGTGGAATACAGACTCACGTTCAAAGGTGGCTGATTGCAGAGGACATTTAAGTTGGGTTCATGGTGTGAT GTTTTCTCCTGATGGATCATCATTTTTGACATCTTCTGATGACCAGACAATCAGGCTCTGGGAGACAAAG AAAGTAT GTAAGAACT CT GCT GTAAT GTTAAAGCAAGAAGTAGAT GTT GT GTTT CAAGAAAAT GAAGT GA TGGTCCTTGCAGTTGACCATATAAGACGTCTGCAACTCATTAATGGAAGAACAGGTCAGATTGATTATCT GACTGAAGCTCAAGTTAGCTGCTGTTGCTTAAGTCCACATCTTCAGTACATTGCATTTGGAGATGAAAAT GGAGCCATTGAGATTTTAGAACTTGTAAACAATAGAATCTTCCAGTCCAGGTTTCAGCACAAGAAAACTG TATGGCACATCCAGTTCACAGCCGATGAGAAGACTCTTATTTCAAGTTCTGATGATGCTGAAATTCAGGT ATGGAATTGGCAATTGGACAAATGTATCTTTCTACGAGGCCATCAGGAAACAGTGAAAGACTTTAGACTC TTGAAAAATTCAAGACTGCTTTCTTGGTCATTTGATGGAACAGTGAAGGTATGGAATATTATTACTGGAA ATAAAGAAAAAGACTTTGTCTGTCACCAGGGTACAGTACTTTCTTGTGACATTTCTCACGATGCTACCAA GTTTTCATCTACCTCTGCTGACAAGACTGCAAAGATCTGGAGTTTTGATCTCCTTTTGCCACTTCATGAA TTGAGGGGCCACAACGGCTGTGTGCGCTGCTCTGCCTTCTCTGTGGACAGTACCCTGCTGGCAACGGGAG ATGACAATGGAGAAATCAGGATATGGAATGTCTCAAACGGTGAGCTTCTTCATTTGTGTGCTCCGCTTTC AGAAGAAGGAGCTGCTACCCATGGAGGCTGGGTGACTGACCTTTGCTTTTCTCCAGATGGCAAAATGCTT ATCTCTGCTGGAGGATATATTAAGTGGTGGAACGTTGTCACTGGGGAATCCTCACAGACCTTCTACACAA ATGGAACCAATCTTAAGAAAATACACGTGTCCCCTGACTTCAAAACATATGTGACTGTGGATAATCTTGG T AT T T T AT AT AT T T T AC AGAC T T T AGAAT AAAAT AGT T AAG CAT T AAT GT AGT T GAAC T T T T T AAAT T T T TGAATTGGAAAAAAATTCTAATGAAACCCTGATATCAACTTTTTATAAAGCTCTTAATTGTTGTGCAGTA TTGCATTCATTACAAAAGTGTTTGTGGTTGGATGAATAATATTAATGTAGCTTTTTCCCAAATGAACATA CCTTTAATCTTGTTTTTCATGATCATCATTAACAGTTTGTCCTTAGGATGCAAATGAAAATGTGAATACA TACCTTGTTGTACTGTTGGTAAAATTCTGTCTTGATGCATTCAAAATGGTTGACATAATTAATGAGAAGA ATTTGGAAGAAATTGGTATTTTAATACTGTCTGTATTTATTACTGTTATGCAGGCTGTGCCTCAGGGTAG CAGTGGCCTGCTTTTTGAACCACACTTACCCCAAGGGGGTTTTGTTCTCCTAAATACAATCTTAGAGGTT TTTTGCACTCTTTAAATTTGCTTTAAAAATATTGTGTCTGTGTGCATAGTCTGCAGCATTTCCTTTAATT GACTCAATAAGTGAGTCTTGGATTTAGCAGGCCCCCCCACCTTTTTTTTTTGTTTTTGGAGACAGAGTCT TGCTTTGTTGCCAGGCTGGAGTGCAGTGGCGCGATCTCGGCTCACCACAATCGCTGCCTCCTGGGTTCAA GCAATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGACTACAGGTGTGCGCACATGCCAGGCTAATTTTTG TATTTTTAGTAGAGACGGGGTTTCACCATGTTGGCCGGGATGGTCTCGATCTCTTGACCTCATGATCTAC CCGCCTTGGCCTCCCAAAGTGCTGAGATTACAGGCGTGAGCCACCGTGCCTGGCCAGGCCCCTTCTCTTT TAATGGAGACAGGGTCTTGCACTATCACCCAGGCTGGAGTGCAGTGGCATAATCATACCTCATTGCAGCC TCAGACTCCTGGGTTCAAGCAATCCTCCTGCCTCAGCCTCCCAAGTAGCTGAGACTACAGGCACGAGCCA CCACACCCAGCTAATTTTTAAGTTTTCTTGTAGAGACAGGGTCTCACTATGTTGTCTAGGCTGGTCTTGA ACTCTTGGCCTCAAGTAATCCTCCTGCCTCAGCCTCCCAAAGTGTTGGGATTGCAGATATGAGCCACTGG CCTGGCCTTCAGCAGTTCTTTTTGTGAAGTAAAACTTGTATGTTGGAAAGAGTAGATTTTATTGGTCTAC CCTTTTCTCACTGTAGCTGCTGGCAGCCCTGTGCCATATCTGGACTCTAGTTGTCAGTATCTGAGTTGGA CACTATTCCTGCTCCCTCTTGTTTCTTACATATCAGACTTCTTACTTGAATGAAACCTGATCTTTCCTAA TCCTCACTTTTTTCTTTTTTAAAAAGCAGTTTCTCCACTGCTAAATGTTAGTCATTGAGGTGGGGCCAAT T T T AAT CAT AAG C C T T AAT AAGAT T T T T C T AAGAAAT GT GAAAT AGAAC AAT T T T CAT C T AAT T C CAT T T ACTTTTAGATGAATGGCATTGTGAATGCCATTCTTTTAATGAATTTCAAGAGAATTCTCTGGTTTTCTGT GTAATTCCAGATGAGTCACTGTAACTCTAGAAGATTAACCTTCCAGCCAACCTATTTTCCTTTCCCTTGT CTCTCTCATCCTCTTTTCCTTCCTTCTTTCCTTTCTCTTCTTTTATCTCCAAGGTTAATCAGGAAAAATA GCTTTTGACAGGGGAAAAAACTCAATAACTAGCTATTTTTGACCTCCTGATCAGGAACTTTAGTTGAAGC-29- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388 GTAAATCTAAAGAAACATTTTCTCTGAAATATATTATTAAGGGCAATGGAGATAAATTAATAGTAGATGT GGTTCCCAGAAAATATAATCAAAATTCAAAGATTTTTTTTGTTTCTGTAACTGGAACTAAATCAAATGAT TACTAGTGTTAATAGTAGATAACTTGTTTTTATTGTTGGTGCATATTAGTATAACTGTGGGGTAGGTCGG GGAGAGGGTAAGGGAATAGATCACTCAGATGTATTTTAGATAAGCTATTTAGCCTTTGATGGAATCATAA ATACAGTGAATACAATCCTTTGCATTGTTAAGGAGGTTTTTTGTTTTTAAATGGTGGGTCAAGGAGCTAG TTTACAGGCTTACTGTGATTTAAGCAAATGTGAAAAGTGAAACCTTAATTTTATCAAAAGAAATTTCTGT AAATGGTATGTCTCCTTAGAATACCCAAATCATAATTTTATTTGTACACACTGTTAGGGGCTCATCTCAT GTAGGCAGAGTATAAAGTATTACCTTTTGGAATTAAAAGCCACTGACTGTTATAAAGTATAACAACACAC ATCAGGTTTTAAAAAGCCTTGAATGGCCCTTGTCTTAAAAAGAAATTAGGAGCCAGGTGCGGTGGCACGT GCCTGTAATCCCAGCTCCTTGGGAGGCTAAGACAGGAGGATTCCTTGAGCCCTGGAGTTTGAGTCCAGCC T GGGT GACATAGCAAGACCCT GT CTTAAAAGAAAAAT GGGAAGAAAGACAAGGTAACAT GAAGAAAGAAG AGATACCTAGTATGATGGAGCTGCAAATTTCATGGCAGTTCATGCAGTCGGTCAAGAGGAGGATTTTGTT TTGTAGTTTGCAGATGAGCATTTCTAAAGCATTTTCCCTTGCTGTATTTTTTTGTATTATAAATTACATT GGACTTCATATATATAATTTTTTTTTACATTATATGTCTCTTGTATGTTTTGAAACTCTTGTATTTATGA TATAGCTTATATGATTTTTTTGCCTTGGTATACATTTTAAAATATGAATTTAAAAAATTTTTGTAAAAAT AAAAT T CACAAAAT T GT T T T GAAAAACA> NM 181861.2 Homo sapiens apoptotic peptidase activating factor 1 (APAF1 ), transcript variant 3, mRNA ( SEQ ID NO: 56) AGAGGCGGAGAAGAAGAGGTAGCGAGTGGACGTGACTGCTCTATCCCGGGCAAAAGGGATAGAACCAGAG GTGGGGAGTCTGGGCAGTCGGCGACCCGCGAAGACTTGAGGTGCCGCAGCGGCATCCGGAGTAGCGCCGG GCTCCCTCCGGGGTGCAGCCGCCGTCGGGGGAAGGGCGCCACAGGCCGGGAAGACCTCCTCCCTTTGTGT CCAGTAGTGGGGTCCACCGGAGGGCGGCCCGTGGGCCGGGCCTCACCGCGGCGCTCCGGGACTGTGGGGT CAGGCTGCGTTGGGTGGACGCCCACCTCGCCAACCTTCGGAGGTCCCTGGGGGTCTTCGTGCGCCCCGGG GCTGCAGAGATCCAGGGGAGGCGCCTGTGAGGCCCGGACCTGCCCCGGGGCGAAGGGTATGTGGCGAGAC AGAGCCCTGCACCCCTAATTCCCGGTGGAAAACTCCTGTTGCCGTTTCCCTCCACCGGCCTGGAGTCTCC CAGTCTTGTCCCGGCAGTGCCGCCCTCCCCACTAAGACCTAGGCGCAAAGGCTTGGCTCATGGTTGACAG CTCAGAGAGAGAAAGATCTGAGGGAAGATGGATGCAAAAGCTCGAAATTGTTTGCTTCAACATAGAGAAG CTCTGGAAAAGGACATCAAGACATCCTACATCATGGATCACATGATTAGTGATGGATTTTTAACAATATC AGAAGAGGAAAAAGTAAGAAATGAGCCCACTCAACAGCAAAGAGCAGCTATGCTGATTAAAATGATACTT AAAAAAGATAATGATTCCTACGTATCATTCTACAATGCTCTACTACATGAAGGATATAAAGATCTTGCTG CCCTTCTCCATGATGGCATTCCTGTTGTCTCTTCTTCCAGTGGTAAAGATTCAGTTAGTGGAATAACTTC GTATGTAAGGACAGTCCTGTGTGAAGGTGGAGTACCACAGAGGCCAGTTGTTTTTGTCACAAGGAAGAAG CT GGT GAAT GCAATT CAGCAGAAGCT CT CCAAATT GAAAGGT GAACCAGGAT GGGT GAG CATACAT GGAA TGGCAGGCTGTGGGAAGTCTGTATTAGCTGCAGAAGCTGTTAGAGATCATTCCCTTTTAGAAGGTTGTTT CCCAGGGGGAGTGCATTGGGTTTCAGTTGGGAAACAAGACAAATCTGGGCTTCTGATGAAACTGCAGAAT CTTTGCACACGGTTGGATCAGGATGAGAGTTTTTCCCAGAGGCTTCCACTTAATATTGAAGAGGCTAAAG ACCGTCTCCGCATTCTGATGCTTCGCAAACACCCAAGGTCTCTCTTGATCTTGGATGATGTTTGGGACTC TTGGGTGTTGAAAGCTTTTGACAGTCAGTGTCAGATTCTTCTTACAACCAGAGACAAGAGTGTTACAGAT TCAGTAATGGGTCCTAAATATGTAGTCCCTGTGGAGAGTTCCTTAGGAAAGGAAAAAGGACTTGAAATTT TATCCCTTTTTGTTAATATGAAGAAGGCAGATTTGCCAGAACAAGCTCATAGTATTATAAAAGAATGTAA AGGCTCTCCCCTTGTAGTATCTTTAATTGGTGCACTTTTACGTGATTTTCCCAATCGCTGGGAGTACTAC CTCAAACAGCTTCAGAATAAGCAGTTTAAGAGAATAAGGAAATCTTCGTCTTATGATTATGAGGCTCTAG ATGAAGCCATGTCTATAAGTGTTGAAATGCTCAGAGAAGACATCAAAGATTATTACACAGATCTTTCCAT CCTTCAGAAGGACGTTAAGGTGCCTACAAAGGTGTTATGTATTCTCTGGGACATGGAAACTGAAGAAGTT GAAGACATACTGCAGGAGTTTGTAAATAAGTCTCTTTTATTCTGTGATCGGAATGGAAAGTCGTTTCGTT ATTATTTACATGATCTTCAAGTAGATTTTCTTACAGAGAAGAATTGCAGCCAGCTTCAGGATCTACATAA GAAGATAATCACTCAGTTTCAGAGATATCACCAGCCGCATACTCTTTCACCAGATCAGGAAGACTGTATG TATTGGTACAACTTTCTGGCCTATCACATGGCCAGTGCCAAGATGCACAAGGAACTTTGTGCTTTAATGT TTTCCCTGGATTGGATTAAAGCAAAAACAGAACTTGTAGGCCCTGCTCATCTGATTCATGAATTTGTGGA ATACAGACATATACTAGATGAAAAGGATTGTGCAGTCAGTGAGAATTTTCAGGAGTTTTTATCTTTAAAT GGACACCTTCTTGGACGACAGCCATTTCCTAATATTGTACAACTGGGTCTCTGTGAGCCGGAAACTTCAG AAGTTTATCAGCAAGCTAAGCTGCAGGCCAAGCAGGAGGTCGATAATGGAATGCTTTACCTGGAATGGAT AAACAAAAAAAACATCACGAATCTTTCCCGCTTAGTTGTCCGCCCCCACACAGATGCTGTTTACCATGCC TGCTTTTCTGAGGATGGTCAGAGAATAGCTTCTTGTGGAGCTGATAAAACCTTACAGGTGTTCAAAGCTG AAACAGGAGAGAAACTTCTAGAAATCAAGGCTCATGAGGATGAAGTGCTTTGTTGTGCATTCTCTACAGA-30- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388 TGACAGATTTATAGCAACCTGCTCAGTGGATAAAAAAGTGAAGATTTGGAATTCTATGACTGGGGAACTA GTACACACCTATGATGAGCACTCAGAGCAAGTCAATTGCTGCCATTTCACCAACAGTAGTCATCATCTTC TCTTAGCCACTGGGTCAAGTGACTGCTTCCTCAAACTTTGGGATTTGAATCAAAAAGAATGTCGAAATAC CATGTTTGGTCATACAAATTCAGTCAATCACTGCAGATTTTCACCAGATGATAAGCTTTTGGCTAGTTGT TCAGCTGATGGAACCTTAAAGCTTTGGGATGCGACATCAGCAAATGAGAGGAAAAGCATTAATGTGAAAC AGTTCTTCCTAAATTTGGAGGACCCTCAAGAGGATATGGAAGTGATAGTGAAGTGTTGTTCGTGGTCTGC TGATGGTGCAAGGATAATGGTGGCAGCAAAAAATAAAATCTTTCTTTTTGACATTCATACTAGTGGCCTA TTGGGAGAAATCCACACGGGCCATCACAGCACCATCCAGTACTGTGACTTCTCCCCACAAAACCATTTGG CAGTGGTTGCTTTGTCCCAGTACTGTGTAGAGTTGTGGAATACAGACTCACGTTCAAAGGTGGCTGATTG CAGAGGACATTTAAGTTGGGTTCATGGTGTGATGTTTTCTCCTGATGGATCATCATTTTTGACATCTTCT GAT GACCAGACAAT CAGGCT CT GGGAGACAAAGAAAGTAT GTAAGAACT CT GCT GTAAT GTTAAAGCAAG AAGTAGATGTTGTGTTTCAAGAAAATGAAGTGATGGTCCTTGCAGTTGACCATATAAGACGTCTGCAACT CATTAATGGAAGAACAGGTCAGATTGATTATCTGACTGAAGCTCAAGTTAGCTGCTGTTGCTTAAGTCCA CATCTTCAGTACATTGCATTTGGAGATGAAAATGGAGCCATTGAGATTTTAGAACTTGTAAACAATAGAA TCTTCCAGTCCAGGTTTCAGCACAAGAAAACTGTATGGCACATCCAGTTCACAGCCGATGAGAAGACTCT TATTTCAAGTTCTGATGATGCTGAAATTCAGGTATGGAATTGGCAATTGGACAAATGTATCTTTCTACGA GGCCATCAGGAAACAGTGAAAGACTTTAGACTCTTGAAAAATTCAAGACTGCTTTCTTGGTCATTTGATG GAACAGT GAAGGTAT GGAATATTATTACT GGAAATAAAGAAAAAGACTTT GT CT GT CAC CAGGGTACAGT ACTTTCTTGTGACATTTCTCACGATGCTACCAAGTTTTCATCTACCTCTGCTGACAAGACTGCAAAGATC TGGAGTTTTGATCTCCTTTTGCCACTTCATGAATTGAGGGGCCACAACGGCTGTGTGCGCTGCTCTGCCT T CT CT GT GGACAGTACCCT GCT GGCAACGGGAGAT GACAAT GGAGAAAT CAGGATAT GGAAT GT CT CAAA CGGTGAGCTTCTTCATTTGTGTGCTCCGCTTTCAGAAGAAGGAGCTGCTACCCATGGAGGCTGGGTGACT GACCTTTGCTTTTCTCCAGATGGCAAAATGCTTATCTCTGCTGGAGGATATATTAAGTGGTGGAACGTTG TCACTGGGGAATCCTCACAGACCTTCTACACAAATGGAACCAATCTTAAGAAAATACACGTGTCCCCTGA CTTCAAAACATATGTGACTGTGGATAATCTTGGTATTTTATATATTTTACAGACTTTAGAATAAAATAGT TAAGCATTAATGTAGTTGAACTTTTTAAATTTTTGAATTGGAAAAAAATTCTAATGAAACCCTGATATCA ACTTTTTATAAAGCTCTTAATTGTTGTGCAGTATTGCATTCATTACAAAAGTGTTTGTGGTTGGATGAAT AAT AT T AAT GTAGCTTTTTCC C AAAT GAAC AT AC C T T T AAT CTTGTTTTT CAT GAT CAT CAT T AAC AGT T TGTCCTTAGGATGCAAATGAAAATGTGAATACATACCTTGTTGTACTGTTGGTAAAATTCTGTCTTGATG CATTCAAAATGGTTGACATAATTAATGAGAAGAATTTGGAAGAAATTGGTATTTTAATACTGTCTGTATT TATTACTGTTATGCAGGCTGTGCCTCAGGGTAGCAGTGGCCTGCTTTTTGAACCACACTTACCCCAAGGG GGTTTTGTTCTCCTAAATACAATCTTAGAGGTTTTTTGCACTCTTTAAATTTGCTTTAAAAATATTGTGT CTGTGTGCATAGTCTGCAGCATTTCCTTTAATTGACTCAATAAGTGAGTCTTGGATTTAGCAGGCCCCCC CACCTTTTTTTTTTGTTTTTGGAGACAGAGTCTTGCTTTGTTGCCAGGCTGGAGTGCAGTGGCGCGATCT CGGCTCACCACAATCGCTGCCTCCTGGGTTCAAGCAATTCTCCTGCCTCAGCCTCCCGAGTAGCTGGGAC TACAGGTGTGCGCACATGCCAGGCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCACCATGTTGGCCG GGATGGTCTCGATCTCTTGACCTCATGATCTACCCGCCTTGGCCTCCCAAAGTGCTGAGATTACAGGCGT GAGCCACCGTGCCTGGCCAGGCCCCTTCTCTTTTAATGGAGACAGGGTCTTGCACTATCACCCAGGCTGG AGTGCAGTGGCATAATCATACCTCATTGCAGCCTCAGACTCCTGGGTTCAAGCAATCCTCCTGCCTCAGC CTCCCAAGTAGCTGAGACTACAGGCACGAGCCACCACACCCAGCTAATTTTTAAGTTTTCTTGTAGAGAC AGGGTCTCACTATGTTGTCTAGGCTGGTCTTGAACTCTTGGCCTCAAGTAATCCTCCTGCCTCAGCCTCC CAAAGTGTTGGGATTGCAGATATGAGCCACTGGCCTGGCCTTCAGCAGTTCTTTTTGTGAAGTAAAACTT GTATGTTGGAAAGAGTAGATTTTATTGGTCTACCCTTTTCTCACTGTAGCTGCTGGCAGCCCTGTGCCAT ATCTGGACTCTAGTTGTCAGTATCTGAGTTGGACACTATTCCTGCTCCCTCTTGTTTCTTACATATCAGA CTTCTTACTTGAATGAAACCTGATCTTTCCTAATCCTCACTTTTTTCTTTTTTAAAAAGCAGTTTCTCCA CTGCTAAATGTTAGTCATTGAGGTGGGGCCAATTTTAATCATAAGCCTTAATAAGATTTTTCTAAGAAAT GTGAAATAGAACAATTTTCATCTAATTCCATTTACTTTTAGATGAATGGCATTGTGAATGCCATTCTTTT AATGAATTTCAAGAGAATTCTCTGGTTTTCTGTGTAATTCCAGATGAGTCACTGTAACTCTAGAAGATTA ACCTTCCAGCCAACCTATTTTCCTTTCCCTTGTCTCTCTCATCCTCTTTTCCTTCCTTCTTTCCTTTCTC TTCTTTTATCTCCAAGGTTAATCAGGAAAAATAGCTTTTGACAGGGGAAAAAACTCAATAACTAGCTATT TTTGACCTCCTGATCAGGAACTTTAGTTGAAGCGTAAATCTAAAGAAACATTTTCTCTGAAATATATTAT TAAGGGCAATGGAGATAAATTAATAGTAGATGTGGTTCCCAGAAAATATAATCAAAATTCAAAGATTTTT TTTGTTTCTGTAACTGGAACTAAATCAAATGATTACTAGTGTTAATAGTAGATAACTTGTTTTTATTGTT GGTGCATATTAGTATAACTGTGGGGTAGGTCGGGGAGAGGGTAAGGGAATAGATCACTCAGATGTATTTT AGATAAGCTATTTAGCCTTTGATGGAATCATAAATACAGTGAATACAATCCTTTGCATTGTTAAGGAGGT TTTTTGTTTTTAAATGGTGGGTCAAGGAGCTAGTTTACAGGCTTACTGTGATTTAAGCAAATGTGAAAAG TGAAACCTTAATTTTATCAAAAGAAATTTCTGTAAATGGTATGTCTCCTTAGAATACCCAAATCATAATT-31- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388 TTATTTGTACACACTGTTAGGGGCTCATCTCATGTAGGCAGAGTATAAAGTATTACCTTTTGGAATTAAA AGCCACTGACTGTTATAAAGTATAACAACACACATCAGGTTTTAAAAAGCCTTGAATGGCCCTTGTCTTA AAAAGAAATTAGGAGCCAGGTGCGGTGGCACGTGCCTGTAATCCCAGCTCCTTGGGAGGCTAAGACAGGA GGATTCCTTGAGCCCTGGAGTTTGAGTCCAGCCTGGGTGACATAGCAAGACCCTGTCTTAAAAGAAAAAT GGGAAGAAAGACAAGGTAACAT GAAGAAAGAAGAGATACCTAGTAT GAT GGAGCT GCAAATTT CAT GGCA GTTCATGCAGTCGGTCAAGAGGAGGATTTTGTTTTGTAGTTTGCAGATGAGCATTTCTAAAGCATTTTCC CTTGCTGTATTTTTTTGTATTATAAATTACATTGGACTTCATATATATAATTTTTTTTTACATTATATGT CTCTTGTATGTTTTGAAACTCTTGTATTTATGATATAGCTTATATGATTTTTTTGCCTTGGTATACATTT T AAAAT AT GAAT T T AAAAAAT T T T T GT AAAAAT AAAAT T CACAAAAT T GT T T T GAAAAACA> NM 181868.2 Homo sapiens apoptotic peptidase activating factor 1 (APAF1 ), transcript variant 4, mRNA ( SEQ ID NO: 57 ) AGAGGCGGAGAAGAAGAGGTAGCGAGTGGACGTGACTGCTCTATCCCGGGCAAAAGGGATAGAACCAGAG GTGGGGAGTCTGGGCAGTCGGCGACCCGCGAAGACTTGAGGTGCCGCAGCGGCATCCGGAGTAGCGCCGG GCTCCCTCCGGGGTGCAGCCGCCGTCGGGGGAAGGGCGCCACAGGCCGGGAAGACCTCCTCCCTTTGTGT CCAGTAGTGGGGTCCACCGGAGGGCGGCCCGTGGGCCGGGCCTCACCGCGGCGCTCCGGGACTGTGGGGT CAGGCTGCGTTGGGTGGACGCCCACCTCGCCAACCTTCGGAGGTCCCTGGGGGTCTTCGTGCGCCCCGGG GCTGCAGAGATCCAGGGGAGGCGCCTGTGAGGCCCGGACCTGCCCCGGGGCGAAGGGTATGTGGCGAGAC AGAGCCCTGCACCCCTAATTCCCGGTGGAAAACTCCTGTTGCCGTTTCCCTCCACCGGCCTGGAGTCTCC CAGTCTTGTCCCGGCAGTGCCGCCCTCCCCACTAAGACCTAGGCGCAAAGGCTTGGCTCATGGTTGACAG CTCAGAGAGAGAAAGATCTGAGGGAAGATGGATGCAAAAGCTCGAAATTGTTTGCTTCAACATAGAGAAG CTCTGGAAAAGGACATCAAGACATCCTACATCATGGATCACATGATTAGTGATGGATTTTTAACAATATC AGAAGAGGAAAAAGTAAGAAATGAGCCCACTCAACAGCAAAGAGCAGCTATGCTGATTAAAATGATACTT AAAAAAGATAATGATTCCTACGTATCATTCTACAATGCTCTACTACATGAAGGATATAAAGATCTTGCTG CCCTTCTCCATGATGGCATTCCTGTTGTCTCTTCTTCCAGTGGTAAAGATTCAGTTAGTGGAATAACTTC GTATGTAAGGACAGTCCTGTGTGAAGGTGGAGTACCACAGAGGCCAGTTGTTTTTGTCACAAGGAAGAAG CT GGT GAAT GCAATT CAGCAGAAGCT CT CCAAATT GAAAGGT GAACCAGGAT GGGT CAC CATACAT GGAA TGGCAGGCTGTGGGAAGTCTGTATTAGCTGCAGAAGCTGTTAGAGATCATTCCCTTTTAGAAGGTTGTTT CCCAGGGGGAGTGCATTGGGTTTCAGTTGGGAAACAAGACAAATCTGGGCTTCTGATGAAACTGCAGAAT CTTTGCACACGGTTGGATCAGGATGAGAGTTTTTCCCAGAGGCTTCCACTTAATATTGAAGAGGCTAAAG ACCGTCTCCGCATTCTGATGCTTCGCAAACACCCAAGGTCTCTCTTGATCTTGGATGATGTTTGGGACTC TTGGGTGTTGAAAGCTTTTGACAGTCAGTGTCAGATTCTTCTTACAACCAGAGACAAGAGTGTTACAGAT TCAGTAATGGGTCCTAAATATGTAGTCCCTGTGGAGAGTTCCTTAGGAAAGGAAAAAGGACTTGAAATTT TATCCCTTTTTGTTAATATGAAGAAGGCAGATTTGCCAGAACAAGCTCATAGTATTATAAAAGAATGTAA AGGCTCTCCCCTTGTAGTATCTTTAATTGGTGCACTTTTACGTGATTTTCCCAATCGCTGGGAGTACTAC CTCAAACAGCTTCAGAATAAGCAGTTTAAGAGAATAAGGAAATCTTCGTCTTATGATTATGAGGCTCTAG ATGAAGCCATGTCTATAAGTGTTGAAATGCTCAGAGAAGACATCAAAGATTATTACACAGATCTTTCCAT CCTTCAGAAGGACGTTAAGGTGCCTACAAAGGTGTTATGTATTCTCTGGGACATGGAAACTGAAGAAGTT GAAGACATACTGCAGGAGTTTGTAAATAAGTCTCTTTTATTCTGTGATCGGAATGGAAAGTCGTTTCGTT ATTATTTACATGATCTTCAAGTAGATTTTCTTACAGAGAAGAATTGCAGCCAGCTTCAGGATCTACATAA GAAGATAATCACTCAGTTTCAGAGATATCACCAGCCGCATACTCTTTCACCAGATCAGGAAGACTGTATG TATTGGTACAACTTTCTGGCCTATCACATGGCCAGTGCCAAGATGCACAAGGAACTTTGTGCTTTAATGT TTTCCCTGGATTGGATTAAAGCAAAAACAGAACTTGTAGGCCCTGCTCATCTGATTCATGAATTTGTGGA ATACAGACATATACTAGATGAAAAGGATTGTGCAGTCAGTGAGAATTTTCAGGAGTTTTTATCTTTAAAT GGACACCTTCTTGGACGACAGCCATTTCCTAATATTGTACAACTGGGTCTCTGTGAGCCGGAAACTTCAG AAGTTTATCAGCAAGCTAAGCTGCAGGCCAAGCAGGAGGTCGATAATGGAATGCTTTACCTGGAATGGAT AAACAAAAAAAACATCACGAATCTTTCCCGCTTAGTTGTCCGCCCCCACACAGATGCTGTTTACCATGCC TGCTTTTCTGAGGATGGTCAGAGAATAGCTTCTTGTGGAGCTGATAAAACCTTACAGGTGTTCAAAGCTG AAACAGGAGAGAAACTTCTAGAAATCAAGGCTCATGAGGATGAAGTGCTTTGTTGTGCATTCTCTACAGA TGACAGATTTATAGCAACCTGCTCAGTGGATAAAAAAGTGAAGATTTGGAATTCTATGACTGGGGAACTA GTACACACCTATGATGAGCACTCAGAGCAAGTCAATTGCTGCCATTTCACCAACAGTAGTCATCATCTTC TCTTAGCCACTGGGTCAAGTGACTGCTTCCTCAAACTTTGGGATTTGAATCAAAAAGAATGTCGAAATAC CATGTTTGGTCATACAAATTCAGTCAATCACTGCAGATTTTCACCAGATGATAAGCTTTTGGCTAGTTGT TCAGCTGATGGAACCTTAAAGCTTTGGGATGCGACATCAGCAAATGAGAGGAAAAGCATTAATGTGAAAC AGTTCTTCCTAAATTTGGAGGACCCTCAAGAGGATATGGAAGTGATAGTGAAGTGTTGTTCGTGGTCTGC TGATGGTGCAAGGATAATGGTGGCAGCAAAAAATAAAATCTTTTTGTGGAATACAGACTCACGTTCAAAG-32- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388 GTGGCTGATTGCAGAGGACATTTAAGTTGGGTTCATGGTGTGATGTTTTCTCCTGATGGATCATCATTTT T GACAT CTT CT GAT GACCAGACAAT CAGGCT CT GGGAGACAAAGAAAGTAT GTAAGAACT CT GCT GTAAT GT T AAAGCAAGAAGT AGAT GTTGTGTTT CAAGAAAAT GAAGT GAT GGT C CT T GCAGT T GAC CAT AT AAGA CGTCTGCAACTCATTAATGGAAGAACAGGTCAGATTGATTATCTGACTGAAGCTCAAGTTAGCTGCTGTT GCTTAAGTCCACATCTTCAGTACATTGCATTTGGAGATGAAAATGGAGCCATTGAGATTTTAGAACTTGT AAACAATAGAATCTTCCAGTCCAGGTTTCAGCACAAGAAAACTGTATGGCACATCCAGTTCACAGCCGAT GAGAAGACTCTTATTTCAAGTTCTGATGATGCTGAAATTCAGGTATGGAATTGGCAATTGGACAAATGTA TCTTTCTACGAGGCCATCAGGAAACAGTGAAAGACTTTAGACTCTTGAAAAATTCAAGACTGCTTTCTTG GTCATTTGATGGAACAGTGAAGGTATGGAATATTATTACTGGAAATAAAGAAAAAGACTTTGTCTGTCAC CAGGGTACAGTACTTTCTTGTGACATTTCTCACGATGCTACCAAGTTTTCATCTACCTCTGCTGACAAGA CTGCAAAGATCTGGAGTTTTGATCTCCTTTTGCCACTTCATGAATTGAGGGGCCACAACGGCTGTGTGCG CTGCTCTGCCTTCTCTGTGGACAGTACCCTGCTGGCAACGGGAGATGACAATGGAGAAATCAGGATATGG AATGTCTCAAACGGTGAGCTTCTTCATTTGTGTGCTCCGCTTTCAGAAGAAGGAGCTGCTACCCATGGAG GCTGGGTGACTGACCTTTGCTTTTCTCCAGATGGCAAAATGCTTATCTCTGCTGGAGGATATATTAAGTG GTGGAACGTTGTCACTGGGGAATCCTCACAGACCTTCTACACAAATGGAACCAATCTTAAGAAAATACAC GTGTCCCCTGACTTCAAAACATATGTGACTGTGGATAATCTTGGTATTTTATATATTTTACAGACTTTAG AAT AAAAT AGT T AAG CAT T AAT GT AGT T GAAC T T T T T AAAT T T T T GAAT T G GAAAAAAAT T C T AAT GAAA CCCTGATATCAACTTTTTATAAAGCTCTTAATTGTTGTGCAGTATTGCATTCATTACAAAAGTGTTTGTG GTTGGATGAATAATATTAATGTAGCTTTTTCCCAAATGAACATACCTTTAATCTTGTTTTTCATGATCAT CATTAACAGTTTGTCCTTAGGATGCAAATGAAAATGTGAATACATACCTTGTTGTACTGTTGGTAAAATT CTGTCTTGATGCATTCAAAATGGTTGACATAATTAATGAGAAGAATTTGGAAGAAATTGGTATTTTAATA CTGTCTGTATTTATTACTGTTATGCAGGCTGTGCCTCAGGGTAGCAGTGGCCTGCTTTTTGAACCACACT TACCCCAAGGGGGTTTTGTTCTCCTAAATACAATCTTAGAGGTTTTTTGCACTCTTTAAATTTGCTTTAA AAATATTGTGTCTGTGTGCATAGTCTGCAGCATTTCCTTTAATTGACTCAATAAGTGAGTCTTGGATTTA GCAGGCCCCCCCACCTTTTTTTTTTGTTTTTGGAGACAGAGTCTTGCTTTGTTGCCAGGCTGGAGTGCAG TGGCGCGATCTCGGCTCACCACAATCGCTGCCTCCTGGGTTCAAGCAATTCTCCTGCCTCAGCCTCCCGA GTAGCTGGGACTACAGGTGTGCGCACATGCCAGGCTAATTTTTGTATTTTTAGTAGAGACGGGGTTTCAC CATGTTGGCCGGGATGGTCTCGATCTCTTGACCTCATGATCTACCCGCCTTGGCCTCCCAAAGTGCTGAG ATTACAGGCGTGAGCCACCGTGCCTGGCCAGGCCCCTTCTCTTTTAATGGAGACAGGGTCTTGCACTATC ACCCAGGCTGGAGTGCAGTGGCATAATCATACCTCATTGCAGCCTCAGACTCCTGGGTTCAAGCAATCCT CCTGCCTCAGCCTCCCAAGTAGCTGAGACTACAGGCACGAGCCACCACACCCAGCTAATTTTTAAGTTTT CTTGTAGAGACAGGGTCTCACTATGTTGTCTAGGCTGGTCTTGAACTCTTGGCCTCAAGTAATCCTCCTG CCTCAGCCTCCCAAAGTGTTGGGATTGCAGATATGAGCCACTGGCCTGGCCTTCAGCAGTTCTTTTTGTG AAGTAAAACTTGTATGTTGGAAAGAGTAGATTTTATTGGTCTACCCTTTTCTCACTGTAGCTGCTGGCAG CCCTGTGCCATATCTGGACTCTAGTTGTCAGTATCTGAGTTGGACACTATTCCTGCTCCCTCTTGTTTCT TACATATCAGACTTCTTACTTGAATGAAACCTGATCTTTCCTAATCCTCACTTTTTTCTTTTTTAAAAAG CAGTTTCTCCACTGCTAAATGTTAGTCATTGAGGTGGGGCCAATTTTAATCATAAGCCTTAATAAGATTT T T C T AAGAAAT GT GAAAT AGAAC AAT T T T CAT C T AAT T C CAT T T AC T T T T AGAT GAAT G G CAT T GT GAAT GCCATTCTTTTAATGAATTTCAAGAGAATTCTCTGGTTTTCTGTGTAATTCCAGATGAGTCACTGTAACT CTAGAAGATTAACCTTCCAGCCAACCTATTTTCCTTTCCCTTGTCTCTCTCATCCTCTTTTCCTTCCTTC TTTCCTTTCTCTTCTTTTATCTCCAAGGTTAATCAGGAAAAATAGCTTTTGACAGGGGAAAAAACTCAAT AACTAGCTATTTTTGACCTCCTGATCAGGAACTTTAGTTGAAGCGTAAATCTAAAGAAACATTTTCTCTG AAATATATTATTAAGGGCAATGGAGATAAATTAATAGTAGATGTGGTTCCCAGAAAATATAATCAAAATT CAAAGATTTTTTTTGTTTCTGTAACTGGAACTAAATCAAATGATTACTAGTGTTAATAGTAGATAACTTG TTTTTATTGTTGGTGCATATTAGTATAACTGTGGGGTAGGTCGGGGAGAGGGTAAGGGAATAGATCACTC AGATGTATTTTAGATAAGCTATTTAGCCTTTGATGGAATCATAAATACAGTGAATACAATCCTTTGCATT GTTAAGGAGGTTTTTTGTTTTTAAATGGTGGGTCAAGGAGCTAGTTTACAGGCTTACTGTGATTTAAGCA AAT GT GAAAAGT GAAAC CT T AAT T T TAT CAAAAGAAAT T T CT GT AAAT GGT AT GT CT C C T T AGAAT AC C C AAATCATAATTTTATTTGTACACACTGTTAGGGGCTCATCTCATGTAGGCAGAGTATAAAGTATTACCTT TTGGAATTAAAAGCCACTGACTGTTATAAAGTATAACAACACACATCAGGTTTTAAAAAGCCTTGAATGG CCCTTGTCTTAAAAAGAAATTAGGAGCCAGGTGCGGTGGCACGTGCCTGTAATCCCAGCTCCTTGGGAGG CTAAGACAGGAGGATTCCTTGAGCCCTGGAGTTTGAGTCCAGCCTGGGTGACATAGCAAGACCCTGTCTT AAAAGAAAAAT GGGAAGAAAGACAAGGTAACAT GAAGAAAGAAGAGATACCTAGTAT GAT GGAGCT GCAA ATTTCATGGCAGTTCATGCAGTCGGTCAAGAGGAGGATTTTGTTTTGTAGTTTGCAGATGAGCATTTCTA AAGCATTTTCCCTTGCTGTATTTTTTTGTATTATAAATTACATTGGACTTCATATATATAATTTTTTTTT ACATTATATGTCTCTTGTATGTTTTGAAACTCTTGTATTTATGATATAGCTTATATGATTTTTTTGCCTT GGT AT ACAT T T T AAAAT AT GAAT T T AAAAAAT T T T T GT AAAAAT AAAAT T CACAAAAT T GT T T T GAAAAA-33- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388CA> NM 181869.2 Homo sapiens apoptotic peptidase activating factor 1 (APAF1 ), transcript variant 5, mRNA ( SEQ ID NO: 58 ) AGAGGCGGAGAAGAAGAGGTAGCGAGTGGACGTGACTGCTCTATCCCGGGCAAAAGGGATAGAACCAGAG GTGGGGAGTCTGGGCAGTCGGCGACCCGCGAAGACTTGAGGTGCCGCAGCGGCATCCGGAGTAGCGCCGG GCTCCCTCCGGGGTGCAGCCGCCGTCGGGGGAAGGGCGCCACAGGCCGGGAAGACCTCCTCCCTTTGTGT CCAGTAGTGGGGTCCACCGGAGGGCGGCCCGTGGGCCGGGCCTCACCGCGGCGCTCCGGGACTGTGGGGT CAGGCTGCGTTGGGTGGACGCCCACCTCGCCAACCTTCGGAGGTCCCTGGGGGTCTTCGTGCGCCCCGGG GCTGCAGAGATCCAGGGGAGGCGCCTGTGAGGCCCGGACCTGCCCCGGGGCGAAGGGTATGTGGCGAGAC AGAGCCCTGCACCCCTAATTCCCGGTGGAAAACTCCTGTTGCCGTTTCCCTCCACCGGCCTGGAGTCTCC CAGTCTTGTCCCGGCAGTGCCGCCCTCCCCACTAAGACCTAGGCGCAAAGGCTTGGCTCATGGTTGACAG CTCAGAGAGAGAAAGATCTGAGGGAAGATGGATGCAAAAGCTCGAAATTGTTTGCTTCAACATAGAGAAG CTCTGGAAAAGGACATCAAGACATCCTACATCATGGATCACATGATTAGTGATGGATTTTTAACAATATC AGAAGAGGAAAAAGTAAGAAATGAGCCCACTCAACAGCAAAGAGCAGCTATGCTGATTAAAATGATACTT AAAAAAGATAATGATTCCTACGTATCATTCTACAATGCTCTACTACATGAAGGATATAAAGATCTTGCTG CCCTTCTCCATGATGGCATTCCTGTTGTCTCTTCTTCCAGTGGTAAAGATTCAGTTAGTGGAATAACTTC GTATGTAAGGACAGTCCTGTGTGAAGGTGGAGTACCACAGAGGCCAGTTGTTTTTGTCACAAGGAAGAAG CT GGT GAAT GCAATT CAGCAGAAGCT CT CCAAATT GAAAGGT GAACCAGGAT GGGT GAG CATACAT GGAA TGGCAGGCTGTGGGAAGTCTGTATTAGCTGCAGAAGCTGTTAGAGATCATTCCCTTTTAGAAGGTTGTTT CCCAGGGGGAGTGCATTGGGTTTCAGTTGGGAAACAAGACAAATCTGGGCTTCTGATGAAACTGCAGAAT CTTTGCACACGGTTGGATCAGGATGAGAGTTTTTCCCAGAGGCTTCCACTTAATATTGAAGAGGCTAAAG ACCGTCTCCGCATTCTGATGCTTCGCAAACACCCAAGGTCTCTCTTGATCTTGGATGATGTTTGGGACTC TTGGGTGTTGAAAGCTTTTGACAGTCAGTGTCAGATTCTTCTTACAACCAGAGACAAGAGTGTTACAGAT TCAGTAATGGGTCCTAAATATGTAGTCCCTGTGGAGAGTTCCTTAGGAAAGGAAAAAGGACTTGAAATTT TATCCCTTTTTGTTAATATGAAGAAGGCAGATTTGCCAGAACAAGCTCATAGTATTATAAAAGAATGTAA AGTGGTGGAACGTTGTCACTGGGGAATCCTCACAGACCTTCTACACAAATGGAACCAATCTTAAGAAAAT ACACGTGTCCCCTGACTTCAAAACATATGTGACTGTGGATAATCTTGGTATTTTATATATTTTACAGACT T T AGAAT AAAAT AGT T AAG CAT T AAT GT AGT T GAAC T T T T T AAAT T T T T GAAT T G GAAAAAAAT T C T AAT GAAACCCTGATATCAACTTTTTATAAAGCTCTTAATTGTTGTGCAGTATTGCATTCATTACAAAAGTGTT TGTGGTTGGATGAATAATATTAATGTAGCTTTTTCCCAAATGAACATACCTTTAATCTTGTTTTTCATGA TCATCATTAACAGTTTGTCCTTAGGATGCAAATGAAAATGTGAATACATACCTTGTTGTACTGTTGGTAA AATTCTGTCTTGATGCATTCAAAATGGTTGACATAATTAATGAGAAGAATTTGGAAGAAATTGGTATTTT AATACTGTCTGTATTTATTACTGTTATGCAGGCTGTGCCTCAGGGTAGCAGTGGCCTGCTTTTTGAACCA CACTTACCCCAAGGGGGTTTTGTTCTCCTAAATACAATCTTAGAGGTTTTTTGCACTCTTTAAATTTGCT TTAAAAATATTGTGTCTGTGTGCATAGTCTGCAGCATTTCCTTTAATTGACTCAATAAGTGAGTCTTGGA TTTAGCAGGCCCCCCCACCTTTTTTTTTTGTTTTTGGAGACAGAGTCTTGCTTTGTTGCCAGGCTGGAGT GCAGTGGCGCGATCTCGGCTCACCACAATCGCTGCCTCCTGGGTTCAAGCAATTCTCCTGCCTCAGCCTC CCGAGTAGCTGGGACTACAGGTGTGCGCACATGCCAGGCTAATTTTTGTATTTTTAGTAGAGACGGGGTT TCACCATGTTGGCCGGGATGGTCTCGATCTCTTGACCTCATGATCTACCCGCCTTGGCCTCCCAAAGTGC TGAGATTACAGGCGTGAGCCACCGTGCCTGGCCAGGCCCCTTCTCTTTTAATGGAGACAGGGTCTTGCAC TATCACCCAGGCTGGAGTGCAGTGGCATAATCATACCTCATTGCAGCCTCAGACTCCTGGGTTCAAGCAA TCCTCCTGCCTCAGCCTCCCAAGTAGCTGAGACTACAGGCACGAGCCACCACACCCAGCTAATTTTTAAG TTTTCTTGTAGAGACAGGGTCTCACTATGTTGTCTAGGCTGGTCTTGAACTCTTGGCCTCAAGTAATCCT CCTGCCTCAGCCTCCCAAAGTGTTGGGATTGCAGATATGAGCCACTGGCCTGGCCTTCAGCAGTTCTTTT TGTGAAGTAAAACTTGTATGTTGGAAAGAGTAGATTTTATTGGTCTACCCTTTTCTCACTGTAGCTGCTG GCAGCCCTGTGCCATATCTGGACTCTAGTTGTCAGTATCTGAGTTGGACACTATTCCTGCTCCCTCTTGT TTCTTACATATCAGACTTCTTACTTGAATGAAACCTGATCTTTCCTAATCCTCACTTTTTTCTTTTTTAA AAAGCAGTTTCTCCACTGCTAAATGTTAGTCATTGAGGTGGGGCCAATTTTAATCATAAGCCTTAATAAG ATTTTTCTAAGAAATGTGAAATAGAACAATTTTCATCTAATTCCATTTACTTTTAGATGAATGGCATTGT GAATGCCATTCTTTTAATGAATTTCAAGAGAATTCTCTGGTTTTCTGTGTAATTCCAGATGAGTCACTGT AACTCTAGAAGATTAACCTTCCAGCCAACCTATTTTCCTTTCCCTTGTCTCTCTCATCCTCTTTTCCTTC CTTCTTTCCTTTCTCTTCTTTTATCTCCAAGGTTAATCAGGAAAAATAGCTTTTGACAGGGGAAAAAACT CAATAACTAGCTATTTTTGACCTCCTGATCAGGAACTTTAGTTGAAGCGTAAATCTAAAGAAACATTTTC TCTGAAATATATTATTAAGGGCAATGGAGATAAATTAATAGTAGATGTGGTTCCCAGAAAATATAATCAA AATTCAAAGATTTTTTTTGTTTCTGTAACTGGAACTAAATCAAATGATTACTAGTGTTAATAGTAGATAA CTTGTTTTTATTGTTGGTGCATATTAGTATAACTGTGGGGTAGGTCGGGGAGAGGGTAAGGGAATAGATC-34- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388 ACTCAGATGTATTTTAGATAAGCTATTTAGCCTTTGATGGAATCATAAATACAGTGAATACAATCCTTTG CATTGTTAAGGAGGTTTTTTGTTTTTAAATGGTGGGTCAAGGAGCTAGTTTACAGGCTTACTGTGATTTA AGCAAATGTGAAAAGTGAAACCTTAATTTTATCAAAAGAAATTTCTGTAAATGGTATGTCTCCTTAGAAT ACCCAAATCATAATTTTATTTGTACACACTGTTAGGGGCTCATCTCATGTAGGCAGAGTATAAAGTATTA CCTTTTGGAATTAAAAGCCACTGACTGTTATAAAGTATAACAACACACATCAGGTTTTAAAAAGCCTTGA ATGGCCCTTGTCTTAAAAAGAAATTAGGAGCCAGGTGCGGTGGCACGTGCCTGTAATCCCAGCTCCTTGG GAGGCTAAGACAGGAGGATTCCTTGAGCCCTGGAGTTTGAGTCCAGCCTGGGTGACATAGCAAGACCCTG T CTTAAAAGAAAAAT GGGAAGAAAGACAAGGTAACAT GAAGAAAGAAGAGATACCTAGT AT GAT GGAGCT GCAAATTTCATGGCAGTTCATGCAGTCGGTCAAGAGGAGGATTTTGTTTTGTAGTTTGCAGATGAGCATT TCTAAAGCATTTTCCCTTGCTGTATTTTTTTGTATTATAAATTACATTGGACTTCATATATATAATTTTT TTTTACATTATATGTCTCTTGTATGTTTTGAAACTCTTGTATTTATGATATAGCTTATATGATTTTTTTG C CT T GGT AT ACAT T T T AAAAT AT GAAT T T AAAAAAT T T T T GT AAAAAT AAAAT T CACAAAAT T GT T T T GA AAAACA

[0061] In one aspect, the present disclosure provides Apafl -specific inhibitory nucleic acids comprising a nucleic acid molecule, which is complementary to a portion of an Apafl nucleic acid sequence selected from the group consisting of SEQ ID NOs: 54-58.

[0062] The present disclosure also provides an antisense nucleic acid comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of any one of SEQ ID NOs: 54-58 (Apafl mRNA), thereby reducing or inhibiting expression of Apafl. The antisense nucleic acid may be antisense RNA, or antisense DNA. Antisense nucleic acids based on the known Apafl gene sequence can be readily designed and engineered using methods known in the art.

[0063] Antisense nucleic acids are molecules which are complementary to a sense nucleic acid strand, e.g., complementary to the coding strand of a double-stranded DNA molecule (or cDNA) or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can form hydrogen bonds with a sense nucleic acid. The antisense nucleic acid can be complementary to an entire Apafl coding strand, or to a portion thereof, e.g., all or part of the protein coding region (or open reading frame). In some embodiments, the antisense nucleic acid is an oligonucleotide which is complementary to only a portion of the mRNA coding region of Apafl. In certain embodiments, an antisense nucleic acid molecule can be complementary to a noncoding region of the Apafl coding strand. In some embodiments, the noncoding region refers to the 5' and 3' untranslated regions that flank the coding region and are not translated into amino acids. For example, the antisense oligonucleotide can be complementary to the region surrounding the translation start site of-35- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388Apafl. An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.

[0064] An antisense nucleic acid can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art. For example, an antisense nucleic acid (e.g., an antisense oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides. Examples of modified nucleotides which can be used to generate the antisense nucleic acid include 5 -fluorouracil, 5-bromouracil, 5-chlorouracil, 5-hodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thouridine, 5-carboxymethylaminometh-yluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1 -methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5-metnylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5 '-methoxy carboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopenten-yladenine, uracil-5-oxyacetic acid (v), wybutosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thlouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5-cxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).

[0065] The antisense nucleic acid molecules may be administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and / or genomic DNA encoding the protein of interest to thereby inhibit expression of the protein, e.g., by inhibiting transcription and / or translation. The hybridization can occur via Watson-Crick base pairing to form a stable duplex, or in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove of the double helix.-36- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388

[0066] In some embodiments, the antisense nucleic acid molecules are modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens. In some embodiments, the antisense nucleic acid molecule is an alpha-anomeric nucleic acid molecule. An alpha-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual P-units, the strands run parallel to each other (Gaultier et al., Nucleic Acids. Res. 15:6625-6641(1987)). The antisense nucleic acid molecule can also comprise a 2'-0 -methylribonucleotide (Inoue et al., Nucleic Acids Res. 15:6131-6148 (1987)) or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett. 215:327-330 (1987)).

[0067] The present disclosure also provides a short hairpin RNA (shRNA) or small interfering RNA (siRNA) comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of any one of SEQ ID NOs: 54-58 (mRNA of Apafl), thereby reducing or inhibiting expression of Apafl. In some embodiments, the shRNA or siRNA is about 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29 base pairs in length. Doublestranded RNA (dsRNA) can induce sequence-specific post-transcriptional gene silencing (e.g., RNA interference (RNAi)) in many organisms such as C. elegans, Drosophila, plants, mammals, oocytes and early embryos. RNAi is a process that interferes with or significantly reduces the number of protein copies made by an mRNA. For example, a double-stranded siRNA or shRNA molecule is engineered to complement and hybridize to a mRNA of a target gene. Following intracellular delivery, the siRNA or shRNA molecule associates with an RNA-induced silencing complex (RISC), which then binds and degrades a complementary target mRNA (such as mRNA of Apafl).

[0068] The present disclosure also provides a ribozyme comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of any one of SEQ ID NOs: 54-58 (Apafl mRNA), thereby reducing or inhibiting expression of Apafl. Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a complementary single-stranded nucleic acid, such as an mRNA. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach, Nature 334:585-591 (1988))) can be used to catalytically cleave Apafl transcripts, thereby inhibiting translation of Apafl.-37- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388

[0069] A ribozyme having specificity for a nucleic acid encoding Apafl can be designed based upon a nucleic acid sequence of Apafl. For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in a mRNA encoding Apafl. See, e.g., U. S. Pat. No. 4,987,071 and U. S. Pat. No. 5,116,742. Alternatively, a mRNA of Apafl can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel and Szostak (1993) Science 261:1411-1418, incorporated herein by reference.

[0070] The present disclosure also provides a synthetic guide RNA (sgRNA) comprising a nucleic acid sequence that is complementary to and specifically hybridizes with a portion of any one of SEQ ID NOs: 54-58 (mRNA of Apafl). Guide RNAs for use in CRISPR-Cas systems are typically generated as a single guide RNA comprising a crRNA segment and a tracrRNA segment. The crRNA segment and a tracrRNA segment can also be generated as separate RNA molecules. The crRNA segment comprises the targeting sequence that binds to a portion of any one of SEQ ID NOs: 54-58, and a stem portion that hybridizes to a tracrRNA. The tracrRNA segment comprises a nucleotide sequence that is partially or completely complementary to the stem sequence of the crRNA and a nucleotide sequence that binds to the CRISPR enzyme. In some embodiments, the crRNA segment and the tracrRNA segment are provided as a single guide RNA. In some embodiments, the crRNA segment and the tracrRNA segment are provided as separate RNAs. The combination of the CRISPR enzyme with the crRNA and tracrRNA make up a functional CRISPR-Cas system. Exemplary CRISPR-Cas systems for targeting nucleic acids, are described, for example, in WO2015 / 089465.

[0071] In some embodiments, a synthetic guide RNA is a single RNA represented as comprising the following elements: 5'-Xl-X2-Y-Z-3'

[0072] where XI and X2 represent the crRNA segment, where XI is the targeting sequence that binds to a portion of any one of SEQ ID NOs: 54-58, X2 is a stem sequence the hybridizes to a tracrRNA, Z represents a tracrRNA segment comprising a nucleotide sequence that is partially or completely complementary to X2, and Y represents a linker sequence. In some embodiments, the linker sequence comprises two or more nucleotides -38- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388and links the crRNA and tracrRNA segments. In some embodiments, the linker sequence comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. In some embodiments, the linker is the loop of the hairpin structure formed when the stem sequence hybridized with the tracrRNA.

[0073] In some embodiments, a synthetic guide RNA is provided as two separate RNAs where one RNA represents a crRNA segment: 5'-Xl-X2-3' where XI is the targeting sequence that binds to a portion of any one of SEQ ID NOs: 54-58, X2 is a stem sequence the hybridizes to a tracrRNA, and one RNA represents a tracrRNA segment, Z, that is a separate RNA from the crRNA segment and comprises a nucleotide sequence that is partially or completely complementary to X2 of the crRNA.

[0074] Exemplary crRNA stem sequences and tracrRNA sequences are provided, for example, in WO / 2015 / 089465, which is incorporated by reference herein. In general, a stem sequence includes any sequence that has sufficient complementarity with a complementary sequence in the tracrRNA to promote formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the stem sequence hybridized to the tracrRNA. In general, degree of complementarity is with reference to the optimal alignment of the stem and complementary sequence in the tracrRNA, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as selfcomplementarity within either the stem sequence or the complementary sequence in the tracrRNA. In some embodiments, the degree of complementarity between the stem sequence and the complementary sequence in the tracrRNA along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the stem sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the stem sequence and complementary sequence in the tracrRNA are contained within a single RNA, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin. In some embodiments, the tracrRNA has additional complementary sequences that form hairpins. In some embodiments, the tracrRNA has at least two or more hairpins. In-39- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388some embodiments, the tracrRNA has two, three, four or five hairpins. In some embodiments, the tracrRNA has at most five hairpins.

[0075] In a hairpin structure, the portion of the sequence 5' of the final “N” and upstream of the loop corresponds to the crRNA stem sequence, and the portion of the sequence 3' of the loop corresponds to the tracrRNA sequence. Further non-limiting examples of single polynucleotides comprising a guide sequence, a stem sequence, and a tracr sequence are as follows (listed 5' to 3'), where “N” represents a base of a guide sequence (e.g. a modified oligonucleotide provided herein), the first block of lower case letters represent stem sequence, and the second block of lower case letters represent the tracrRNA sequence, and the final poly-T sequence represents the transcription terminator: (a) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataa ggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 59); (b) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccg aaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT (SEQ ID NO: 60); (c) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccg aaatcaacaccctgtcattttatggcagggtgtTTTTTT (SEQ ID NO: 61); (d) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaactt gaaaaagtggcaccgagtcggtgcTTTTTT (SEQ ID NO: 62); (e) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaac ttgaaaaagtgTTTTTTT (SEQ ID NO: 63); and (f) NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTTTTTTTT(SEQ ID NQ

[0076] A variety of CRISPR enzymes are available for use in conjunction with the disclosed guide RNAs of the present disclosure. In some embodiments, the CRISPR enzyme is a Type II CRISPR enzyme. In some embodiments, the CRISPR enzyme catalyzes DNA cleavage. In some embodiments, the CRISPR enzyme catalyzes RNA cleavage. In some embodiments, the CRISPR enzyme is any Cas9 protein, for instance any naturally-occurring bacterial Cas9 as well as any chimeras, mutants, homologs or orthologs. Non-limiting examples of Cas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2,-40- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, homologues thereof, or modified variants thereof. In some embodiments, the CRISPR enzyme cleaves both strands of the target nucleic acid at the Protospacer Adjacent Motif (PAM) site. In some embodiments, the CRISPR enzyme is a nickase, which cleaves only one strand of the target nucleic acid.Formulations Including APAF1 Inhibitors of the Present Technology

[0077] The pharmaceutical compositions of the present technology can be manufactured by methods well known in the art such as conventional granulating, mixing, dissolving, encapsulating, lyophilizing, or emulsifying processes, among others. Compositions may be produced in various forms, including granules, precipitates, or particulates, powders, including freeze dried, rotary dried or spray dried powders, amorphous powders, tablets, capsules, syrup, suppositories, injections, emulsions, elixirs, suspensions or solutions. Formulations may optionally contain solvents, diluents, and other liquid vehicles, dispersion or suspension aids, surface active agents, pH modifiers, isotonic agents, thickening or emulsifying agents, stabilizers and preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. In certain embodiments, the compositions disclosed herein are formulated for administration to a mammal, such as a human.

[0078] Liquid dosage forms for oral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3 -butylene glycol, cyclodextrins, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0079] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or -41- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3 -butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U. S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid are used in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use. Compositions formulated for parenteral administration may be injected by bolus injection or by timed push, or may be administered by continuous infusion.

[0080] In order to prolong the effect of a compound of the present disclosure, it is often desirable to slow the absorption of the compound from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the compound then depends upon its rate of dissolution that, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered compound form is accomplished by dissolving or suspending the compound in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the compound in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of compound to polymer and the nature of the particular polymer employed, the rate of compound release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are also prepared by entrapping the compound in liposomes or microemulsions that are compatible with body tissues.

[0081] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and / or a) fillers or extenders such as starches, lactose, sucrose,-42- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388glucose, mannitol, and silicic acid, b) binders such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, cetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents such as phosphates or carbonates.

[0082] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0083] The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.-43- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388Methods of Treatment of the Present Technology

[0084] In one aspect, the present disclosure provides a method for treating cancer in a subject in need thereof comprising administering to the subject an effective amount of an Apafl inhibitor. In some embodiments, the method further comprises separately, sequentially or simultaneously administering at least one additional anti-cancer therapy to the subject. In another aspect, the present disclosure provides a method for enhancing efficacy of an anti-cancer therapy in a subject in need thereof comprising administering to the subject an effective amount of an Apafl inhibitor.

[0085] In any of the preceding embodiments of the methods disclosed herein, the Apafl inhibitor is a small molecule, or an inhibitory RNA that targets Apafl. Examples of Apafl -specific small molecule inhibitors include but are not limited to ZYZ-488, QM31 (SVT016426), UCN-01, SVT017686, SVT017923, SVT016448, andN-alkylglycine trimers. In some embodiments, the inhibitory RNA that targets Apafl may be a siRNA, a shRNA, an antisense oligonucleotide, a ribozyme, or a sgRNA as described herein. Additionally or alternatively, in some embodiments, the Apafl inhibitor is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, iontophoretically, transmucosally, or intramuscularly.

[0086] In any and all embodiments of the methods disclosed herein, the anti-cancer therapy comprises one or more of chemotherapy, radiotherapy, adoptive cell therapy, immune checkpoint blockade therapy or targeted therapy. The adoptive cell therapy may comprise one or more of CAR T-cell therapy, tumor-infiltrating lymphocyte (TIL) therapy, T-cell receptor (TCR) therapy, natural killer (NK) cell therapy, or dendritic cell therapy.

[0087] In any of the above embodiments of the methods disclosed herein, the chemotherapy comprises one or more of alkylating agents, topoisomerase inhibitors, endoplasmic reticulum stress inducing agents, antimetabolites, mitotic inhibitors, nitrogen mustards, nitrosoureas, alkyl sulfonates, platinum agents, taxanes, vinca agents, antiestrogen drugs, aromatase inhibitors, ovarian suppression agents, cytostatic alkaloids, cytotoxic antibiotics, endocrine / hormonal agents, or bisphosphonate therapy agents.Examples of chemotherapeutic agents include but are not limited to cyclophosphamide, fluorouracil (or 5 -fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza- -44- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserlin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, tykerb, anthracyclines (e.g., daunorubicin and doxorubicin), cladribine, midostaurin, bevacizumab, oxaliplatin, melphalan, etoposide, mechlorethamine, bleomycin, microtubule poisons, annonaceous acetogenins, chlorambucil, ifosfamide, streptozocin, carmustine, lomustine, busulfan, dacarbazine, temozolomide, altretamine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, hydroxyurea, pemetrexed, epirubicin, idarubicin, SN-38, ARC, NPC, campothecin, 9-nitrocamptothecin, 9-aminocamptothecin, rubifen, gimatecan, diflomotecan, BN80927, DX-8951f, MAG-CPT, amsacrine, etoposide phosphate, teniposide, azacitidine (Vidaza), decitabine, accatin III, 10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, cephalomannine, 10-deacetyl-7-epitaxol, 7-epitaxol, 10-deacetylbaccatin III, 10-deacetyl cephalomannine, streptozotocin, nimustine, ranimustine, bendamustine, uramustine, estramustine, mannosulfan, camptothecin, exatecan, lurtotecan, lamellarin D9-aminocamptothecin, ellipticines, aurintricarboxylic acid, HU-331, or combinations thereof.

[0088] Additionally or alternatively, in some embodiments of the methods disclosed herein, the immune checkpoint blockade therapy comprises one or more of an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-4- IBB antibody, an anti-CD73 antibody, an anti-GITR antibody, or an anti-LAG-3 antibody. Examples of immune checkpoint blockade therapy include, but are not limited to, cemiplimab, tremelimumab, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, envafolimab, atezolizumab, avelumab, durvalumab, dostarlimab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, AMP-224, MDX-1105, arelumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS (inducible T-cell costimulatory), DLBCL (diffuse large B-cell lymphoma) inhibitors, BTLA (B and T lymphocyte attenuator), PDR001, or any combination thereof.-45- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388

[0089] Additionally or alternatively, in some embodiments of the methods disclosed herein, the targeted therapy comprises one or more of VEGF / VEGFR inhibitors, EGFZEGFR inhibitors, PARP inhibitors, BCL-2 inhibitors, or CDK9 inhibitors. Examples of targeted therapy include but are not limited to bevacizumab, nimotuzumab, buparlisib, pilaralisib, sonolisib, paxalisib, dactolisib, voxtalisib, PQR309, AMG232, venetoclax, dinaciclib, ribociclib, dasatinib, imatinib, or rindopepimut.

[0090] In any and all embodiments of the methods disclosed herein, the cancer is selected from among adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, leukemias, liver cancers, lymph node cancers, lymphomas, lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, non- Hodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, and metastases thereof.

[0091] For therapeutic applications, a composition comprising an Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor disclosed herein, is administered to the subject. In some embodiments, the Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor is administered one, two, three, four, or five times per day. In some embodiments, Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor is administered more than five times per day. Additionally or alternatively, in some embodiments, the Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor is administered every day, every other day, every third day, every fourth day, every fifth day, or every sixth day. In some embodiments, the Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor is administered weekly, bi-weekly, tri -weekly, or monthly. In some embodiments, the Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor is administered for a period of one, two, three, four, or five weeks. In some embodiments, the Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor is administered for six -46- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388weeks or more. In some embodiments, the Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor is administered for twelve weeks or more. In some embodiments, the Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor is administered for a period of less than one year. In some embodiments, the Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor is administered for a period of more than one year. In some embodiments, the Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor is administered throughout the subject’s life.

[0092] In some embodiments of the methods of the present technology, the Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor is administered daily for 1 week or more. In some embodiments of the methods of the present technology, the Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor is administered daily for 2 weeks or more. In some embodiments of the methods of the present technology, the Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor is administered daily for 3 weeks or more. In some embodiments of the methods of the present technology, the Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor is administered daily for 4 weeks or more. In some embodiments of the methods of the present technology, the Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor is administered daily for 6 weeks or more. In some embodiments of the methods of the present technology, the Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor is administered daily for 12 weeks or more. In some embodiments, the Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor is administered daily throughout the subject’s life.Determination of the Biological Effect of APAF1 Inhibitors

[0093] In various embodiments, suitable in vitro or in vivo assays are performed to determine the effect of a specific Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor and whether its administration is indicated for treatment. In various embodiments, in vitro assays can be performed with representative animal models, to determine if a given Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor exerts the desired effect on reducing or eliminating signs and / or symptoms of cancer. Compounds for use in therapy can be tested in suitable animal model -47- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model system known in the art can be used prior to administration to human subjects. In some embodiments, in vitro or in vivo testing is directed to the biological function of one or more Apafl -specific inhibitory nucleic acids or Apafl -specific small molecule inhibitors.

[0094] Animal models of cancer may be generated using techniques known in the art. Such models may be used to demonstrate the biological effect of Apafl -specific inhibitory nucleic acids or Apafl -specific small molecule inhibitors in the treatment of cancer, and for determining what comprises a therapeutically effective amount of the one or more Apafl -specific inhibitory nucleic acids or Apafl -specific small molecule inhibitors disclosed herein in a given context.Modes of Administration and Effective Dosages

[0095] Any method known to those in the art for contacting a cell, organ or tissue with one or more Apafl -specific inhibitory nucleic acids or Apafl -specific small molecule inhibitors disclosed herein may be employed. Suitable methods include in vitro, ex vivo, or in vivo methods. In vivo methods typically include the administration of one or more Apafl -specific inhibitory nucleic acids or Apafl -specific small molecule inhibitors to a mammal, suitably a human. When used in vivo for therapy, the one or more Apafl -specific inhibitory nucleic acids or Apafl -specific small molecule inhibitors described herein are administered to the subject in effective amounts (i.e., amounts that have desired therapeutic effect). The dose and dosage regimen will depend upon the degree of the disease state of the subject, the characteristics of the particular Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor used, e.g., its therapeutic index, and the subject’s history.

[0096] The effective amount may be determined during pre-clinical trials and clinical trials by methods familiar to physicians and clinicians. An effective amount of one or more Apafl -specific inhibitory nucleic acids or Apafl -specific small molecule inhibitors useful in the methods may be administered to a mammal in need thereof by any of a number of well-known methods for administering pharmaceutical compounds. The Apafl -specific inhibitory nucleic acids or Apafl -specific small molecule inhibitors may be administered systemically or locally.-48- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388

[0097] The one or more Apafl -specific inhibitory nucleic acids or Apafl -specific small molecule inhibitors described herein can be incorporated into pharmaceutical compositions for administration, singly or in combination, to a subject for the treatment of cancer. Such compositions typically include the active agent and a pharmaceutically acceptable carrier. As used herein the term “pharmaceutically acceptable carrier” includes saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.Supplementary active compounds can also be incorporated into the compositions.

[0098] Pharmaceutical compositions are typically formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral (e.g., intravenous, intradermal, intraperitoneal or subcutaneous), oral, inhalation, transdermal (topical), intraocular, iontophoretic, and transmucosal administration.Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. For convenience of the patient or treating physician, the dosing formulation can be provided in a kit containing all necessary equipment (e.g., vials of drug, vials of diluent, syringes and needles) for a treatment course (e.g., 7 days of treatment).

[0099] Pharmaceutical compositions suitable for injectable use can include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL™ (BASF, Parsippany, N. J.) or phosphate buffered saline (PBS). In all cases, a composition for parenteral administration must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture -49- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

[0100] The pharmaceutical compositions having one or more Apafl -specific inhibitory nucleic acids or Apafl -specific small molecule inhibitors disclosed herein can include a carrier, which can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thiomerasol, and the like. Glutathione and other antioxidants can be included to prevent oxidation. In many cases, it will be advantageous to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin.

[0101] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, typical methods of preparation include vacuum drying and freeze drying, which can yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0102] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as -50- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0103] For administration by inhalation, the compounds can be delivered in the form of an aerosol spray from a pressurized container or dispenser, which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer. Such methods include those described in U. S. Pat. No. 6,468,798.

[0104] Systemic administration of a therapeutic compound as described herein can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. In one embodiment, transdermal administration may be performed by iontophoresis.

[0105] A therapeutic agent can be formulated in a carrier system. The carrier can be a colloidal system. The colloidal system can be a liposome, a phospholipid bilayer vehicle. In one embodiment, the therapeutic agent is encapsulated in a liposome while maintaining the agent’s structural integrity. One skilled in the art would appreciate that there are a variety of methods to prepare liposomes. (See Lichtenberg, et al., Methods Biochem. Anal., 33:337-462 (1988); Anselem, et al., Liposome Technology, CRC Press (1993)). Liposomal formulations can delay clearance and increase cellular uptake (See Reddy, Ann.Pharmacother., 34(7-8):915-923 (2000)). An active agent can also be loaded into a particle prepared from pharmaceutically acceptable ingredients including, but not limited to, soluble, insoluble, permeable, impermeable, biodegradable or gastroretentive polymers or -51- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388liposomes. Such particles include, but are not limited to, nanoparticles, biodegradable nanoparticles, microparticles, biodegradable microparticles, nanospheres, biodegradable nanospheres, microspheres, biodegradable microspheres, capsules, emulsions, liposomes, micelles and viral vector systems.

[0106] The carrier can also be a polymer, e.g., a biodegradable, biocompatible polymer matrix. In one embodiment, the therapeutic agent can be embedded in the polymer matrix, while maintaining the agent’s structural integrity. The polymer may be natural, such as polypeptides, proteins or polysaccharides, or synthetic, such as poly a-hydroxy acids.Examples include carriers made of, e.g., collagen, fibronectin, elastin, cellulose acetate, cellulose nitrate, polysaccharide, fibrin, gelatin, and combinations thereof. In one embodiment, the polymer is poly-lactic acid (PLA) or copoly lactic / glycolic acid (PGLA). The polymeric matrices can be prepared and isolated in a variety of forms and sizes, including microspheres and nanospheres. Polymer formulations can lead to prolonged duration of therapeutic effect. (See Reddy, Ann. Pharmacother., 34(7-8):915-923 (2000)). A polymer formulation for human growth hormone (hGH) has been used in clinical trials. (See Kozarich and Rich, Chemical Biology, 2:548-552 (1998)).

[0107] Examples of polymer microsphere sustained release formulations are described in PCT publication WO 99 / 15154 (Tracy, et al.), U. S. Pat. Nos. 5,674,534 and 5,716,644 (both to Zale, et al.), PCT publication WO 96 / 40073 (Zale, et al.), and PCT publication WO 00 / 38651 (Shah, et al.). U. S. Pat. Nos. 5,674,534 and 5,716,644 and PCT publication WO 96 / 40073 describe a polymeric matrix containing particles of erythropoietin that are stabilized against aggregation with a salt.

[0108] In some embodiments, the therapeutic compounds are prepared with carriers that will protect the therapeutic compounds against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such formulations can be prepared using known techniques. The materials can also be obtained commercially, e.g., from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to specific cells with monoclonal antibodies to -52- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388cell-specific antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U. S. Pat. No. 4,522,811.

[0109] The therapeutic compounds can also be formulated to enhance intracellular delivery. For example, liposomal delivery systems are known in the art, see, e.g., Chonn and Cullis, “Recent Advances in Liposome Drug Delivery Systems,” Current Opinion in Biotechnology 6:698-708 (1995); Weiner, “Liposomes for Protein Delivery: Selecting Manufacture and Development Processes,” Immunomethods, 4(3):201-9 (1994); and Gregoriadis, “Engineering Liposomes for Drug Delivery: Progress and Problems,” Trends Biotechnol., 13(12):527-37 (1995). Mizguchi, et al., Cancer Lett., 100:63-69 (1996), describes the use of fusogenic liposomes to deliver a protein to cells both in vivo and in vitro.

[0110] Dosage, toxicity and therapeutic efficacy of any therapeutic agent can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50 / ED50. Compounds that exhibit high therapeutic indices are advantageous. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0111] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of such compounds may be within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to determine useful -53- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388doses in humans accurately. Levels in plasma may be measured, for example, by high performance liquid chromatography.

[0112] Typically, an effective amount of the one or more Apafl -specific inhibitory nucleic acids or Apafl -specific small molecule inhibitors disclosed herein sufficient for achieving a therapeutic or prophylactic effect, range from about 0.000001 mg per kilogram body weight per day to about 10,000 mg per kilogram body weight per day. Suitably, the dosage ranges are from about 0.0001 mg per kilogram body weight per day to about 100 mg per kilogram body weight per day. For example dosages can be 1 mg / kg body weight or 10 mg / kg body weight every day, every two days or every three days or within the range of 1-10 mg / kg every week, every two weeks or every three weeks. In one embodiment, a single dosage of the therapeutic compound ranges from 0.001-10,000 micrograms per kg body weight. In one embodiment, one or more Apafl -specific inhibitory nucleic acid or Apafl -specific small molecule inhibitor concentrations in a carrier range from 0.2 to 2000 micrograms per delivered milliliter. An exemplary treatment regime entails administration once per day or once a week. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, or until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

[0113] In some embodiments, a therapeutically effective amount of one or more Apafl -specific inhibitory nucleic acids or Apafl -specific small molecule inhibitors may be defined as a concentration of inhibitor at the target tissue of IO’32to 10'6molar, e.g., approximately 10'7molar. This concentration may be delivered by systemic doses of 0.001 to 100 mg / kg or equivalent dose by body surface area. The schedule of doses would be optimized to maintain the therapeutic concentration at the target tissue, such as by single daily or weekly administration, but also including continuous administration (e.g., parenteral infusion or transdermal application).

[0114] The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective -54- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388amount of the therapeutic compositions described herein can include a single treatment or a series of treatments.

[0115] The mammal treated in accordance with the present methods can be any mammal, including, for example, farm animals, such as sheep, pigs, cows, and horses; pet animals, such as dogs and cats; laboratory animals, such as rats, mice and rabbits. In some embodiments, the mammal is a human.Combination Therapy

[0116] In some embodiments, one or more of the Apafl -specific inhibitory nucleic acids or Apafl -specific small molecule inhibitors disclosed herein may be combined with one or more additional therapies for the treatment of cancer.

[0117] Additionally or alternatively, in some embodiments, the one or more Apafl -specific inhibitory nucleic acids or Apafl -specific small molecule inhibitors disclosed herein may be separately, sequentially or simultaneously administered with at least one additional therapeutic agent. Examples of additional therapeutic agents include one or more of alkylating agents, topoisomerase inhibitors, endoplasmic reticulum stress inducing agents, antimetabolites, mitotic inhibitors, nitrogen mustards, nitrosoureas, alkyl sulfonates, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, VEGF / VEGFR inhibitors, EGFZEGFR inhibitors, PARP inhibitors, cytostatic alkaloids, cytotoxic antibiotics, antimetabolites, endocrine / hormonal agents, bisphosphonate therapy agents, targeted biological therapy agents, and immune checkpoint blocking agents antibodies (e.g., an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-4- IBB antibody, an anti-CD73 antibody, an anti-GITR antibody, or an anti-LAG-3 antibody).

[0118] Specific chemotherapeutic agents include, but are not limited to, cyclophosphamide, fluorouracil (or 5 -fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, proteinbound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserlin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate,-55- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388denosumab, zoledronate, trastuzumab, tykerb, anthracyclines (e.g., daunorubicin and doxorubicin), cladribine, midostaurin, bevacizumab, oxaliplatin, melphalan, etoposide, mechlorethamine, bleomycin, microtubule poisons, annonaceous acetogenins, chlorambucil, ifosfamide, streptozocin, carmustine, lomustine, busulfan, dacarbazine, temozolomide, altretamine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, hydroxyurea, pemetrexed, epirubicin, idarubicin, SN-38, ARC, NPC, campothecin, 9-nitrocamptothecin, 9-aminocamptothecin, rubifen, gimatecan, diflomotecan, BN80927, DX-8951f, MAG-CPT, amsacnne, etoposide phosphate, teniposide, azacitidine (Vidaza), decitabine, accatin III, 10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, cephalomannine, 10-deacetyl-7-epitaxol, 7-epitaxol, 10-deacetylbaccatin III, 10-deacetyl cephalomannine, streptozotocin, nimustine, ranimustine, bendamustine, uramustine, estramustine, mannosulfan, camptothecin, exatecan, lurtotecan, lamellarin D9-aminocamptothecin, amsacrine, ellipticines, aurintricarboxylic acid, HU-331, or combinations thereof.

[0119] Examples of antimetabolites include 5 -fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate, pemetrexed, and mixtures thereof.

[0120] Examples of taxanes include accatin III, 10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, cephalomannine, 10-deacetyl-7-epitaxol, 7-epitaxol, 10-deacetylbaccatin III, 10-deacetyl cephalomannine, and mixtures thereof.

[0121] Examples of DNA alkylating agents include cyclophosphamide, chlorambucil, melphalan, bendamustine, uramustine, estramustine, carmustine, lomustine, nimustine, ranimustine, streptozotocin; busulfan, mannosulfan, and mixtures thereof.

[0122] Examples of topoisomerase I inhibitor include SN-38, ARC, NPC, camptothecin, topotecan, 9-nitrocamptothecin, exatecan, lurtotecan, lamellarin D9-aminocamptothecin, rubifen, gimatecan, diflomotecan, BN80927, DX-8951f, MAG-CPT, and mixtures thereof. Examples of topoisomerase II inhibitors include amsacrine, etoposide, etoposide phosphate, teniposide, daunorubicin, mitoxantrone, amsacrine, ellipticines, aurintricarboxylic acid, doxorubicin, and HU-331 and combinations thereof.

[0123] In some embodiments, the one or more immune checkpoint blocking agents may be an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, an anti-CTLA- -56- 4920-5495-9747.1Atty. Dkt. No.: 115872-33884 antibody, an anti-TIM3 antibody, an anti -4- IBB antibody, an anti-CD73 antibody, an anti-GITR antibody, or an anti-LAG-3 antibody. In some embodiments, the one or more immune checkpoint blocking agents are selected from the group consisting of cemiplimab, tremelimumab, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, envafolimab, atezolizumab, avelumab, durvalumab, dostarlimab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, inhibitory antibodies against LAG-3 (lymphocyte activation gene 3), TIM3 (T-cell immunoglobulin and mucin-3), B7-H3, B7-H4, TIGIT (T-cell immunoreceptor with Ig and ITIM domains), AMP-224, MDX-1105, arelumab, tremelimumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS (inducible T-cell costimulatory), DLBCL (diffuse large B-cell lymphoma) inhibitors, BTLA (B and T lymphocyte attenuator), PDR001, and any combination thereof. Examples of targeted therapy include Bevacizumab, nimotuzumab, buparlisib, pilaralisib and sonolisib, paxalisib, dactolisib, voxtalisib, PQR309, AMG232, ribociclib, dasatinib, imatinib, and rindopepimut.

[0124] In any case, the multiple therapeutic agents may be administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). One of the therapeutic agents may be given in multiple doses, or both may be given as multiple doses. If not simultaneous, the timing between the multiple doses may vary from more than zero weeks to less than four weeks. In addition, the combination methods, compositions and formulations are not to be limited to the use of only two agents.Kits

[0125] The present disclosure also provides kits for the treatment of cancer, comprising one or more Apafl -specific inhibitory nucleic acids or Apafl -specific small molecule inhibitors disclosed herein. Optionally, the above described components of the kits of the present technology are packed in suitable containers and labeled for the treatment of cancer.

[0001] The above-mentioned components may be stored in unit or multi-dose containers, for example, sealed ampoules, vials, bottles, syringes, and test tubes, as an aqueous, preferably sterile, solution or as a lyophilized, preferably sterile, formulation for-57- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388reconstitution. The kit may further comprise a second container which holds a diluent suitable for diluting the pharmaceutical composition towards a higher volume. Suitable diluents include, but are not limited to, the pharmaceutically acceptable excipient of the pharmaceutical composition and a saline solution. Furthermore, the kit may comprise instructions for diluting the pharmaceutical composition and / or instructions for administering the pharmaceutical composition, whether diluted or not. The containers may be formed from a variety of materials such as glass or plastic and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper which may be pierced by a hypodermic injection needle). The kit may further comprise more containers comprising a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, culture medium for one or more of the suitable hosts. The kits may optionally include instructions customarily included in commercial packages of therapeutic or diagnostic products, that contain information about, for example, the indications, usage, dosage, manufacture, administration, contraindications and / or warnings concerning the use of such therapeutic or diagnostic products.

[0126] The kit can also comprise, e.g., a buffering agent, a preservative or a stabilizing agent. The kit can also contain a control sample or a series of control samples, which can be assayed and compared to the test sample. Each component of the kit can be enclosed within an individual container and all of the various containers can be within a single package, along with instructions for interpreting the results of the assays performed using the kit. The kits of the present technology may contain a written product on or in the kit container. The written product describes how to use the reagents contained in the kit. In certain embodiments, the use of the reagents can be according to the methods of the present technology.EXAMPLES

[0127] The present technology is further illustrated by the following Examples, which should not be construed as limiting in any way. The examples herein are provided to illustrate advantages of the present technology and to further assist a person of ordinary skill in the art with preparing or using the compositions and systems of the present technology.-58- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388The examples should in no way be construed as limiting the scope of the present technology, as defined by the appended claims. The examples can include or incorporate any of the variations, aspects, or embodiments of the present technology described above. The variations, aspects, or embodiments described above may also further each include or incorporate the variations of any or all other variations, aspects or embodiments of the present technology.Example 1: Experimental model and study participant details

[0128] Cell lines

[0129] All the mouse embryonic fibroblasts (MEFs) utilized were SV40-transformed as reported previously and maintained in Dulbecco's Modified Eagles Medium supplemented with 10% fetal bovine serum following standard culture conditions and procedures.1The tetracycline-inducible tBID-expressing ApafB '' MEFs were generated by lentiviral transduction of tet-inducible tBID followed by single cell subcloning. Cells were treated with 1 pg / mL doxycycline (Sigma) to induce the expression of tBID. The BH3s-inducible Apafl-I- MEFs were generated by retroviral transduction of MSCV-Cre-ERT2-Neo and MSCV-LoxP-GFP-LoxP-BH3-pPGK-Puro followed by single cell subcloning. The induction of BH3s following 500 nM 4-hydroxytamoxifen (4-OHT, Sigma) treatment was confirmed by immunoblot analyses. To generate pO cells, Apafl^ MEFs were cultured in the presence of 200 ng / mL ethidium bromide (Thermo Fisher Scientific) for 3 passages. Knockout cell lines were generated using CRISPR / Cas9-mediated genome editing as described.2,3Briefly, cells were transduced with lentivirus expressing Cas9 and sgRNAs targeting genes of interest. Following puromycin selection for 3 days, infected cells were subjected to single cell subcloning. Knockout clones were selected based on the lack of expression of target proteins by immunoblot analyses. CRISPR / Cas9-mediated KO of Vdacl and Vdac2 was performed inzl a / Y^’MEFs to generate Apafrl~Vdacl~ / ~Vdac2~ / ~ cells. Reconstitution of wild-type or mutant of Vdac was performed by retroviral transduction of Apafl~l~Vdac / ~Vdac2~ / ~ cells. CRISPR / Cas9-mediated KO of Bax and Bak was performed in Apafr'~ or Apafrl~Vdacl~ / ~Vdac2~ / ~ cells to generate Bax^BakdApafl'1' or Bax~ ~ Bak ^Vdacl^ Vdac2~ / ~Apafr'~ cells. The B16-F10 cell line was obtained from the American Type Culture Collection (ATCC) and cultured according to the recommendations of ATCC. MDA-MB-231 and A498 cell lines were obtained from the Developmental Therapeutics Program -59- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388(DTP) of the National Cancer Institute (NCI) and cultured in RPMI 1640 supplemented with 10% fetal bovine serum, 1% penicillin / streptomycin, 1% nonessential amino acids, 1% sodium pyruvate, 1% GlutaMAX, and 1% HEPES (Thermo Fisher Scientific). PC9 (human lung adenocarcinoma, male origin) was obtained from Dr. David Scheinberg at Memorial Sloan Kettering Cancer Center and cultured as described.4Cell lines were routinely tested for mycoplasma using the Venor™ GeM mycoplasma detection kit (Sigma).

[0130] Plasmid Construction, CRISPR / Cas9-Mediated Genome Editing, and RNA Interference

[0131] The lentiviral tetracycline-inducible hygromycin-resistance vector (TRE-gene of interest-pPGK Hygro-T2A-rtTA3) was generated from pCW-Cas9 (Addgene), LentiCRISPRv2 (Addgene), and MSCV-Hygro (Addgene) using the NEBuilderRHiFi DNA Assembly Cloning Kit (New England Biolabs). Mouse Bid with deletion of N-terminal 177 bp of coding region (tBID) was cloned into the lentiviral tetracycline-inducible hygromycin-resistance vector. sgRNAs were designed using Optimized CRISPR Design and cloned into LentiGuide-Puro (Addgene) or LentiCRISPRv2 (Addgene).5Mouse Vdac Vdac2, and Vdac3 were cloned into MSCV-Hygro (Clontech) or MSCV-IRES-GFP. The mutants of Vdac were generated by PCR-based site-directed mutagenesis.

[0132] The calcium indicator mito-G-GECO1.2 was generated by fusing the mitochondrial targeting signal derived from CMV-mito-R-GECOl (Addgene)6with G-GECO1.2 derived from CMV-GGECO1.2 (Addgene)6and subsequently cloned into pBABE-Puro (Addgene). Mouse Apafl was cloned into MSCV-IRES-GFP.1 All constructs were confirmed by DNA sequencing. Lentivirus was produced by co-transfection of 293 T cells with pCMVDR8.2 and pHCMV. VSVG using Lipofectamine 2000 (Thermo Fisher Scientific) as described.2The sequences of sgRNAs are listed in Table 2. Lentiviral shRNA targeting human PHB2 was obtained from Sigma (TRCN0000060921). Lentiviral shRNA targeting GFP has been reported previously.7

[0133] Table 2: Summary of target sequences of sgRNAs (represented by SEQ ID NOs: 23-39 in order of appearance).-60- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388Gene Target Sequencemouse Bak GCCCTGTACGTCTACCAGCGmouse Bax CAACTT CAACT GGGGC CGCGmouse Vdacl ACCAGAGCGCCCCGGATCGAmouse Vdac2 #1 GTGGAACACCGATAACACTCmouse Vdac2 #2 GTAAACCTCGCTTGGACATCmouse VdacH ACACCAACTTATTGCGACCTmouse Phb2 CAGCTCTCTTCGGATCAACAmouse Anil CGTTTGACACTGTTCGTCGTmouse Anr2 CAGCGGTAGCACCCATCGAGmouse Cues TCTTCCGCCCGAACAGACCGmouse Polg CTCCACAGCTCGACGGGTGAmouse Ppif TGAAGTTCTCGTCGGGAAAGhuman VDAC1 GCTCTGGTGCTAGGTTACGAhuman VDAC2 ACGCGCGCAAGTCTGTCCGTHuman ¥DAC3 TTTCCTAGGTCACAGTACGTCm GACG ACTAGTTAGGCGTGTALa cZ TC TCGAATACGCC CACCTCCTAT

[0134] Cell viability and caspase activity assays

[0135] Cells were plated in 12-well plates at 8 x 104or 105cells / well and treated with the indicated agents next day. Cells were treated with doxycycline (1 pg / mL) to induce tBID as well as the combination of ABT-263 (Selleck Chemicals, 2 pM) and S63845 (Chemietek, 5 or 10 pM) to induce cell death. Cells were treated with Q-VD-OPH (MedKoo Biosciences, 20 pM), necrostatin-1 (Sigma, 5 pM), GSK’872 (Sigma, 10 pM), ferrostatin-1 (Selleck Chemicals, 20 pM), deferoxamine (Selleck Chemicals, 50 pM), and cyclosporin A (Selleck Chemicals, 2 pM) to inhibit the respective cell death mechanisms. To quantify cell death, cells were harvested and stained with annexin-V (BioVision) and / or propidium iodide (2 pg / mL, Sigma) as described,? followed by flow cytometric analyses using an LSRFortessa (BD Biosciences). Data were analyzed using FACSDiva (BD Biosciences). For cell death quantification using IncuCyte assays, cells were seeded in 96-well plates at the density of 8,000 cells per well the day before experiments. Cells were treated with the indicated agents, stained with SYTOX Green (100 nM, Thermo Fisher-61- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388Scientific) to detect dead cells, and then monitored using a IncuCyteR S3 Live-Cell Analysis Instrument (Sartorius) for up to 7 days. Cell images were taken every 6-8 h. Cell death was quantified using the integrated algorithms of the IncuCyteR Basic Analysis Software to calculate the confluence of SYTOX Green-positive (green fluorescent) cells and divide that by the total cell confluence (obtained by analysis of phase-contrast images). For the quantification of caspase activities, cells were plated in 96-well plates and treated with indicated agents next day. Caspase activities were quantified using the Caspase-Gio 3 / 7 Assay System (Promega) according to the manufacturer’s protocol.

[0136] Subcellular fractionation, antibodies, and immunoblot analyses

[0137] Cells were harvested at various time points following tBID induction and incubated in digitonin permeabilization buffer (0.025% digitonin, 10 mM KC1, 5 mM MgC12, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 20 mM HEPES, pH 7.2) supplemented with complete protease inhibitor cocktail (Roche) for 5 min on ice.Organelles that contain mitochondria were removed from the soluble cytosolic fraction by pelleting at 15,000 g and then lysed with 1% Triton X-100 in PBS supplemented with complete protease inhibitor cocktail (Roche). Both cytosolic and mitochondrial fractions were subjected to immunoblot analyses. Cellular and mitochondrial lysates were separated by NuPAGE (Thermo Fisher Scientific) or SDS-PAGE (Bio-Rad) gels and transferred onto PVDF membrane (Immobilon-P; Millipore). Antibody detection was accomplished using enhanced chemiluminescence method (Western Lightning Plus-ECL, PerkinElmer) and LAS-3000 Imaging system (FUJIFILM) or Amersham Imager 680 (GE Healthcare). The primary antibodies used for immunoblots are listed as follows: anti-BID (Cell Signaling Technology (CST), #2003), anti-BIM (Covance), 2 anti-PUMA (CST, E2P7G), anti-BAX (Santa Cruz, N20; CST, #2772; Proteintech, 60267-1G), anti-BAK (Upstate, NT; CST, D4E4), anti-VDAC1 (Calbiochem, Porin 31HL; Abeam, ab186321 or ab16814), anti-VDAC2 (Covance rabbit polyclonal antibody against the peptide of amino acid 212-228;8 CST, #9412), anti-VDAC3,8 anti-ANT1 (MitoSciences, MSA02; Abeam, ab110322), anti-ANT2 (CST, E2B9D), anti-PHBl (CST, #2426), anti-PHB2 (Upstate, 07-234), anti-Cyclophilin D (Affinity BioReagents, PA1-028), anti-ATP synthase P (BD Biosciences, # 612518), anti-COX IV (CST, #4844), anti-TOM20 (CST, D8T4N), anti-PolG (Abeam, ab128899), anti-Cytochrome c (Pharmingen, #556433), anti-LDHA (CST, C28H7), anti- -62- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388APAF1 (CST, # 8723; Chemicon, # 06-957), anti -Procaspase-9 (CST, #9502), anti-Procaspase-3 (BD bioscience, #611048), anti -FIS 1 (Axxora, ALX-210-907), anti-P-Actin (Sigma, A1978), and anti-tubulin (Sigma, DM1A).

[0138] Identification of the mitochondrial BAK complex

[0139] Mitochondria isolation was performed as previously described.1,9’10Mitochondria isolated from Bax- / -Bak- / -MEFs ox Bax~~ Bak MEFs reconstituted with the N-terminal Protein C-tagged BAK were left untreated or treated with 10 pM of BIM BH3 peptide and subjected to sequential extraction using 1% CHAPS lysis buffer (1% CHAPS, 142.5 mM KC1, 2 mM CaCh, 20 mM Tris-Cl, pH 7.4) supplemented with EDTA-free protease inhibitor cocktail (Roche) and 2% CHAPS lysis buffer supplemented with EDTA-free protease inhibitors plus sonication. The protein lysates were loaded onto anti-Protein C affinity matrix (Roche) and eluted with 10 mM EDTA. The eluted immune complex was precipitated by trichloroacetic acid (TCA) and analyzed by 4-12% NuPAGE gels (Thermo Fisher Scientific) and silver staining (Silver Staining Plus kit, Bio-Rad). Excised gel slices of interest were sent for tryptic digestion and liquid chromatography -tandem mass spectrometric analysis at Taplin Mass Spectrometry Facility (Harvard Medical School, Boston, MA). BH3 domain peptide derived from murine BIM was synthesized by Tufts University Core Facility (Boston, MA) and dissolved in dimethyl sulfoxide (Sigma) at a concentration of 20 mM.

[0140] Gel filtration chromatography, immunoprecipitation, and protein crosslinking

[0141] The chromatographic step of Superdex 200 (HR 10 / 30, GE-Amersham) or Superdex 200 Increase (10 / 300 GL, Cytiva) column was performed on an automatic fast protein liquid chromatography (AKTApurifier, Cytiva) as previously described.10The column was equilibrated with 1% or 2% CHAPS gel filtration buffer (300 mM NaCl, 0.2 mM DTT, 20 mM HEPES pH 7.5) and calibrated with thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa), conalbumin (75 kDa), bovine serum albumin (66 kDa), ovalbumin (44 kDa), and cytochrome c (14 kDa) as indicated.Mitochondrial lysates were loaded onto the column, eluted at a flow rate of 0.3 ml / min. Fractions of 0.6 ml were collected, precipitated by trichloroacetic acid, and analyzed by 8- -63- 4920-5495-9747.1Atty. Dkt. No.: 115872-338816% SDS-PAGE (Bio-Rad) or 10% NuPAGE (Thermo Fisher Scientific) gels and immunoblots. For immunoprecipitation of the N-terminal Protein C-tagged BAK from gel filtration eluate, the indicated fractions were combined and incubated with anti-Protein C matrix in the presence of 2 mM Ca2+ supplemented with EDTA-free protease inhibitor cocktail (Roche).

[0142] The immunoprecipitates were separated by 4-12% NuPAGE (Thermo Fisher Scientific) gels and analyzed by silver staining and immunoblot analyses. For anti-HA immunoprecipitation, protein lysates in CHAPS lysis buffer (2% CHAPS, 142.5 mM KC1, 2 mM CaCh, 20 mM Tris-Cl, pH 7.4) were immunoprecipitated with anti-HA antibody (12CA5) and captured by protein A agarose matrix (Pierce). To detect BAX / BAK oligomerization, tBID-inducible cells were treated with doxycycline for 6 hours and then incubated with 5 mM BMH (bismaleimidohexane, ThermoFisher Scientific) at room temperature for 30 min.

[0143] In vitro cytochrome c release and protein cross-linking

[0144] The aliquots of gel filtration fractions were diluted with mitochondria isolation buffer (200 mM mannitol, 70 mM sucrose, 1 mM EGTA, 10 mM HEPES, pH 7.5) to make the final CHAPS concentration less than 0.1% and subsequently incubated with Bax- / -Bak- / - mitochondria (1 mg / ml) in a final volume of 100 pl. Quantification of cytochrome c release was performed using colorimetric ELISA assays (R& D Systems) as previously described9. The cytochrome c releasing activity was normalized against gel filtration buffer. For detecting BAK oligomers, BMH (Thermo Fisher Scientific) was directly added to the elution fractions at a final concentration of 2.5 mM, incubated at room temperature for 30 min, and quenched by NuPAGE sample buffer. The samples were analyzed by 4- 12% NuPAGE and anti -BAK immunoblots.

[0145] ADP import assay

[0146] ADP import assay was performed as previously described 11. Mitochondria were isolated as previously described and the mitochondrial pellets were resuspended in ADP import buffer (250 mM sucrose, 20 mM HEPES, 10 mM KC1, 5 mM succinate, 3 mM KH2PO4, 1.5 mM MgCh, 1 mM EGTA, 5 pM rotenone, pH 7.2) to get a final protein concentration at 0.5 mg / ml. The mitochondrial suspension was aliquoted into two tubes, -64- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388each containing 400 pl. The ANT inhibitor atractyloside (Sigma) was added to one of the two tubes at a concentration of 50 pM. Both samples were subsequently added with 0.5 pCi of 14C-ADP (PerkinElmer NEN® Radiochemicals) and incubated for 10 min on ice. 700 pl of ADP import buffer containing 50 pM of atractyloside was added to both samples to stop the reaction. The samples were washed twice with ADP import buffer, mixed with liquid scintillation cocktail, and the 14C-ADP counts were determined by liquid scintillation counter (LS6000; Beckman Coulter). The ANT-dependent uptake of ADP was calculated as the difference in counts between samples with or without a preincubation with atractyloside. To isolate mitoplasts, 0.5 mg / ml of purified mitochondria were treated with 0.15% (w / v) digitonin (Sigma) in mitoplast isolation buffer (220 mM mannitol, 70 mM sucrose, 2 mM HEPES pH 7.5) for 10 min on ice and spun down at 10,000 g for 10 min.

[0147] Quantification of mitochondrial DNA efflux

[0148] Cells (1.5 x 106) were harvested and divided into 2 portions. 1 / 5 of cells were spun down at 2,000 rpm for 4 min and then subjected to DNA extraction using the DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer’s protocol. The other 4 / 5 of cells were spun down and permeabilized with digitonin permeabilization buffer (0.025% digitonin, lOmM KC1, 5mM MgCh, ImM EDTA, ImM EGTA, 250mM sucrose, 20mM HEPES, pH 7.2) for 5 min on ice, followed by centrifugation at 15,000g for 10 min at 4°C. Supernatants were then spun at 100,000g at 4°C for 60 min in an ultracentrifuge (Beckman, Optima TM MAX-XP). Following ultracentrifugation, supernatants were taken as the cytosolic fraction from which DNA was purified using the QIAQuick Nucleotide Removal Kit (Qiagen) according to the manufacturer’s protocol. Both whole-cell DNA and cytosolic DNA were subjected to quantitative PCR with SYBR green master mix (Thermo Fisher Scientific) on a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific) or a QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific).

[0149] Quantitative PCR was performed using nuclear DNA primers (mouse Tert or human ACTB) and mitochondrial DNA (mtDNA) primers (D-loop). We confirmed that no nuclear Tert or ACTB DNA was detected in the cytosolic fractions, indicating that nuclear lysis did not occur using this digitonin method. CT values of mtDNA abundance obtained from the cytosolic fractions were normalized against those obtained from whole-cell -65- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388extracts. Fold increase of mtDNA efflux was calculated by comparing the normalized cytosolic mtDNA abundance. The primers used for realtime PCR are listed as follows: mouse D-loop, 5'- CATCTCGATGGTATCGGGTC-3' (SEQ ID NO: 1) and 5'-GGTGCGTCTAGACTGTGTGC-3' (SEQ ID NO: 2); mouse Tert, 5'-CTAGCTCATGTGTCAAGACCCTCTT-3' (SEQ ID NO: 3) and 5'-GCCAGCACGTTTCTCTCGTT-3' (SEQ ID NO: 4); human D-loop, 5'-GGATCACAGGTCTATCACCCTA-3' (SEQ ID NO: 5) and 5'-CAGCGTCTCGCAATGCTATC-3' (SEQ ID NO: 6); human ACTB 5'-GGCACCCAGCACAATGAAGATC-3' (SEQ ID NO: 7) and 5'-TGAGGACCCTGGATGTGACAG-3' (SEQ ID NO: 8)

[0150] In vitro mitochondrial DNA release from mitoplasts

[0151] Mitochondria isolated from tBID-inducible Apafl ^ MEFs without or with doxycycline treatment were sequentially extracted with 1% and 2% CHAPS to generate 1% CHAPS insoluble, 2% CHAPS soluble mitochondrial subfraction that was subsequently analyzed by Superdex 200 Increase 10 / 300 GL gel filtration chromatography. The fractions eluted at the indicated molecular weight were collected. To isolate mitoplasts, 0.5 mg / ml of mitochondria isolated from Bak / Bax / ' VdacT / ~Vdac2~ / ~ApafT / ~ MEFs were treated with 0.15% (w / v) digitonin (Sigma) in mtDNA release buffer (142.5mM KC1, 250 pM CaCh, 20 mM Tris-Cl, pH7.4) supplemented with EDTA-free protease inhibitor cocktail (Roche) for 10 min on ice and spun down at 10,000 g for 10 min. The gel filtration fractions were sequentially concentrated and diluted with mtDNA release buffer to make the final CHAPS concentration less than 0.1% and subsequently incubated with mitoplasts in a final volume of 100 pl at 30 °C for 30 min. The samples were spun down at 10,000g at 4°C for 10 min. Pellets were collected from which DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen); and supernatants were then spun at 100,000g at 4°C for 60 min in an ultracentrifuge (Beckman, Optima TM MAX-XP). DNA from supernatants following ultracentrifugation was purified using the QIAQuick Nucleotide Removal Kit (Qiagen) according to the manufacturer’s protocol. Quantitative PCR using D-loop primers was performed on a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific) or a QuantStudio™ 3 Real-Time PCR System (Thermo Fisher Scientific). CT values of mtDNA-66- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388abundance obtained from supernatants following ultracentrifugation were normalized against those obtained from pellets following centrifugation at 10,000g. The mtDNA releasing activity was calculated by comparing the normalized mtDNA release from mitoplasts incubated with buffer alone.

[0152] Quantification of mitochondrial calcium

[0153] Cells were seeded in a 4-well chamber slide (Nunc™ Lab-Tek™ II) at the density of 4.5xl04cells per well the day before experiments. Cells were untreated or treated with doxycycline (Sigma, 1 pg / mL) or the combination of ABT-263 (Selleck Chemicals, 2 pM) and S63845 (Chemi etek, 10 pM) for 4 hours. Cells were fixed with 4% paraformaldehyde and then stained with DAPI (Sigma). Cells were imaged using a Leica SP5 Inverted Confocal Microscope using the Objective of 40x 1.2 NA water (HCX PL APO; 506341). The average intensity of green fluorescence signal for each individual cell (F0) was quantified by the formula: F0=Fc (green intensity of each cell) / S (green area of each cell). The quantification was performed using ImageJ software. For each condition, 50 to 200 cells were quantified and the relative fluorescence intensity was calculated by normalization to the average of control cells in three independent experiments. Statistics were calculated using Student’s Ltest to test whether the average relative fluorescence intensity from one sample is greater than 150% of that from the other sample.

[0154] Confocal microscopy

[0155] Cells transduced with TOMM20-GFP and TIMM50-mCherry were plated in 4-well chamber slides (EMD Millipore) and incubated with the indicated agents at 37°C for the indicated durations. Following treatment, cells were fixed with 4% paraformaldehyde (Sigma) for 15 minutes, washed three times with phosphate-buffered saline (PBS), and incubated at room temperature for 1 hour in permeabilization / b locking buffer containing 0.1% Triton X-100 (Sigma), 1% BSA (EMD Millipore), 5% goat serum (Sigma) in PBS. Cells were then incubated overnight at 4°C with primary antibodies anti-TOMM20 (Abeam, ab13970) and anti-mCherry (CST, 43590S) in blocking buffer. After three washes, cells were incubated for 1 hour in secondary antibodies including goat anti-chicken AF488 (Invitrogen, A-11039) and goat anti-rabbit AF594 (Thermo Fisher Scientific, A-11012). Following two additional washes, cells were stained with DAPI (Sigma) and imaged using a -67- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388Zeiss LSM 880 confocal microscope equipped with a 63× / 1.4 NA Plan Apo objective. To analyze the colocalization of TOMM20-GFP (green) and TIMM50-mCherry (red) signals, the images were initially converted to an 8-bit format. Default autothresholding was applied, and Manders’ colocalization coefficient was calculated using the Coloc2 plugin in Fiji software.12,13

[0156] Transmission electron microscopy

[0157] Electron Microscopy was performed by the Microscopy Core Facility at Weill Cornell Medicine. Cell pellets were fixed with a modified Karmovsky's fixative solution and a secondary fixation in reduced osmium tetroxide.14Following graded ethanol dehydration, samples were embedded in an Epon analog resin, and sectioned at 65 nm using a DiATOME diamond knife on a Leica Ultracut S ultramicrotome. Ultrathin sections were contrasted with lead citrate as described15and viewed on a JEM 1400 electron microscope (JEOL) operated at 100 kV. Digital images were captured on a Veleta 2K x 2K CCD camera (EMSIS GmbH).

[0158] Quantification and statistical analysis

[0159] Statistical significance was determined using two-tailed unpaired parametric Student’s / -test (Prism 9, GraphPad Software). Data were presented as mean ± SD with P < 0.05 considered statistically significant unless otherwise stated. Statistical significance was denoted as *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Statistics in FIGs.4A, 4B, 4C, and 4D were calculated using Student’s / -test to test whether the average relative fluorescence intensity from one sample is greater than 150% of that from the other sample. In FIG. 4A, n = 87 for s Ctrl control, n = 105 for s Ctrl doxycycline, n = 131 for s Bax / s Bak control, and n = 110 for s Bax / s Bak doxycycline. In FIG. 4B, n = 117 for Apaf1- / -kA control, n = 140 for ApafP / ' #1 doxycycline, n = 117 for ApafP / ' #2 control, n =127 for ApafP / ' #2 doxycycline, n = 76 for ApafP / ~VdacP / ~Vdac2~ / ~ #1 control, n =56 for ApafP / ~VdacP / ~Vdac2~ / ~ #1 doxycycline, n = 98 for ApafP / ' VdacP^ Vdac2' / '#2 control, and n = 80 for ApafP' VdacP' Vdac2 / ~ #2 doxycycline. In Fig. 4C, n = 101 for ApafP' #1 control, n = 111 for ApafP / ' #1 ABT263 plus S63845 (A / S), n = 125 for ApafP / ' #2 control, n = 213 for ApafP' #2 A / S, n = 195 for ApafP' VdacP' Vdac2 / ~ #1 control, n = 182 for ApafP' VdacP' Vdac2 / ~ #1 A / S, n = 137 for ApafP' VdacP^ Vdac2 / ~ #2 control, n = 107-68- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388for Apafl ' Vdad ^ Vdac2 / ~ #2 A / S. In Fig. 4D, n = 135 for Apafl ' Vdad ^ Vdac2 / ~ control, n = 120 for Apafl ^ Vdacl ^ Vdac2~ / ~ doxycycline, n = 135 for Apafl ^ Vdacl ^ Vdac2~ / ~ reconstituted with VDAC1 / 2 WT control, n = 137 for Apafl ' Vdacl ^ Vdac2~ / ~ reconstituted with VDAC1 / 2 WT doxycycline, n = 117 for Apafl ^ Vdacl ^ Vdac2~ / ~ reconstituted with VDAC1 / 2 MT control, n = 121 for Apafl ^ Vdacl ^ Vdac2~ / ~ reconstituted with VDAC1 / 2 MT doxycycline. The number of independent experiments, samples, or events were indicated in the figure legends. No data were excluded from the analyses. No statistical method was used to predetermine sample size. The in vitro experiments were not performed in a blinded manner as the investigator needed to know the treatment groups. 1. Cheng, E. H., Wei, M. C., Weiler, S., Flavell, R. A., Mak, T. W., Lindsten, T., and Korsmeyer, S. J. (2001). BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol Cell 8, 705-711. 10.1016 / s1097-2765(01)00320-3.2. Tanaka, K., Yu, H. A., Yang, S., Han, S., Selcuklu, S. D., Kim, K., Ramani, S., Ganesan, Y. T., Moyer, A., Sinha, S., et al. (2021). Targeting Aurora B kinase prevents and overcomes resistance to EGFR inhibitors in lung cancer by enhancing BIM- and PUMA-mediated apoptosis. Cancer Cell 39, 1245-1261. el246. 10.1016 / j.ccell.2021.07.006.3. Xie, Y., Sahin, M., Sinha, S., Wang, Y., Nargund, A. M., Lyu, Y., Han, S., Dong, Y., Hsieh, J. J., Leslie, C. S., and Cheng, E. H. (2022). SETD2 loss perturbs the kidney cancer epigenetic landscape to promote metastasis and engenders actionable dependencies on histone chaperone complexes. Nat Cancer 3, 188-202. 10.1038 / s43018-021-00316-3.4. Bean, G. R., Ganesan, Y. T., Dong, Y., Takeda, S., Liu, H., Chan, P. M., Huang, Y., Chodosh, L. A., Zambetti, G. P., Hsieh, J. J., and Cheng, E. H. (2013). PUMA and BIM are required for oncogene inactivation-induced apoptosis. Science signaling 6,ra20.10.1126 / scisignal.2003483.5. Sanjana, N. E., Shalem, O., and Zhang, F. (2014). Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11, 783-784. 10.1038 / nmeth.3047.-69- 4920-5495-9747.1Atty. Dkt. No.: 115872-33886. Zhao, Y., Araki, S., Wu, J., Teramoto, T., Chang, Y. F., Nakano, M., Abdelfattah, A. S., Fujiwara, M., Ishihara, T., Nagai, T., and Campbell, R. E. (2011). An expanded palette of genetically encoded Ca2+indicators. Science 333, 1888-1891. 10.1126 / science.1208592.7. Wang, G. X., Tu, H. C., Dong, Y., Skanderup, A. J., Wang, Y., Takeda, S., Ganesan, Y. T., Han, S., Liu, H., Hsieh, J. J., and Cheng, E. H. (2017). ANp63 Inhibits Oxidative Stress-Induced Cell Death, Including Ferroptosis, and Cooperates with the BCL-2 Family to Promote Clonogenic Survival. Cell Rep 27, 2926-2939. 10.1016 / j.celrep.2017.11.030.8. Cheng, E. H., Sheiko, T. V., Fisher, J. K., Craigen, W. J., and Korsmeyer, S. J. (2003). VDAC2 inhibits BAK activation and mitochondrial apoptosis. Science 301, 513-517.10.1126 / science.1083995.9. Kim, H., Rafiuddin-Shah, M., Tu, H. C., Jeffers, J. R., Zambetti, G. P., Hsieh, J. J., and Cheng, E. H. (2006). Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies. Nat Cell Biol 8, 1348-1358. 10.1038 / ncb1499.10. Kim, H., Tu, H. C., Ren, D., Takeuchi, O., Jeffers, J. R., Zambetti, G. P., Hsieh, J. J., and Cheng, E. H. (2009). Stepwise activation of BAX and BAK by tBID, BIM, and PUMA initiates mitochondrial apoptosis. Mol Cell 36, 487-499. 10.1016 / j.molcel.2009.09.030.11. Vander Hei den, M. G., Chandel, N. S., Schumacker, P. T., and Thompson, C. B. (1999). BclxL prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP / ADP exchange. Mol Cell 3, 159-167. 10.1016 / sl097-2765(00)80307-x.12. Schindelin, J., Arganda-Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., and Schmid, B. (2012). Fiji: an open-source platform for biological-image analysis. Nature methods 9, 676-682.-70- 4920-5495-9747.1Atty. Dkt. No.: 115872-338813. Manders, E. M., Verbeek, F., and Aten, J. (1993). Measurement of co-localization of objects in dual-colour confocal images. Journal of microscopy 169, 375-382.14. Ito, S. (1968). Formaldehyde-glutaraldehyde fixatives containing tri nitro compounds. J. Cell Biol. 39, 168A-169A.15. Venable, J. H., and Coggeshall, R. (1965). A SIMPLIFIED LEAD CITRATE STAIN FOR USE IN ELECTRON MICROSCOPY. J Cell Biol 25, 407-408. 10.1083 / jcb.25.2.407.Example 2: Identification of a 600 kPa BAK-associated macromolecular complex

[0160] To characterize BAK-associated death machinery and address whether BAK associates with different protein complexes to regulate cytochrome c versus mtDNA efflux, Bax- / -Bak- / -mouse embryonic fibroblasts (MEFs) were reconstituted with the N-terminal Protein C-tagged BAK to facilitate the affinity purification of BAK-associated complexes. We focused on BAK because it is an integral MOM protein whereas BAX translocates from the cytosol to the mitochondria upon apoptosis.10,32To enhance the extraction of detergentresistant BAK and its associated integral membrane proteins, mitochondria were sequentially extracted with 1% and 2% CHAPS in conjunction with sonication. The 1% CHAPS insoluble, 2% CHAPS soluble mitochondrial subfraction was subjected to antiProtein C affinity chromatography followed by silver staining and tandem mass spectrometry analysis.

[0161] Table 1. Summary of the peptide sequences of BAK-associated proteins revealed by liquid chromatography-tandem mass spectrometry (represented by SEQ ID NOs: 9-22 in order of appearance).-71- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388Gene Peptide Sequence35 kDa BAK-associated proteins PHB2 K. IVQAEGEAEAAK35 kDa BAK-associated proteins PHB2 K. MLGEALSK35 kDa BAK-associated proteins PHB2 R. AQFLVEK35 kDa BAK-associated proteins PHB2 R. EYTAAVEAK35 kDa BAK-associated proteins VDAC1 K. VNGSLETK35 kDa BAK-associated proteins VDAC1 RVTQSNFAVGYK35 kDa BAK-associated proteins VDAC2 K. VSGTLETK35 kDa BAK-associated proteins VDAC2 K. YQLDPTASISAK35 kDa BAK-associated proteins VDAC3 K. ASGNLETK35 kDa BAK-associated proteins VDACS K. GYGFGMVK32 kDa BAK-associated proteins VDAC3 K. ASGNLETK32 kDa BAK-associated proteins VDAC3 K. DVFNK32 kDa BAK-associated proteins VDACS K. GYGFGMVK32 kDa BAK-associated proteins ANTI R. GNLANVIR

[0162] Interestingly, VDAC1-3 isoforms, ANTI, and prohibitin 2 (PHB2) were co-purified with BAK at nearly 1:1 stoichiometry (FIGs. 1A and 7A). In comparison, we also treated mitochondria with the peptide derived from the BH3 domain of BIM (an activator BH3) to activate BAK.7,11Notably, the composition of BAK-associated proteins was not altered by the addition of BIM BH3 peptide (FIG. 1A). The identity of these proteins was further verified by immunoblot analyses (FIG. IB). Mitochondrial matrix protein cyclophilin D, an essential component of the mitochondrial permeability transition pore complex (mPTP),33and the MIM protein ATP synthase 0 served as negative controls. These data suggest that BAK interacts with proteins that are known to associate at the contact sites between the MOM and the MIM.34Indeed, BAK was detected in both the MOM and the contact site subfractions when sub -mitochondrial vesicles were separated on a continuous sucrose gradient (FIG. 7A).

[0163] To further investigate this complex, gel filtration chromatography was performed on the 1% CHAPS insoluble, 2% CHAPS soluble mitochondrial subfraction. This approach differs from our previous method used to demonstrate BAK oligomerization, where the entire 2% mitochondrial lysates were analyzed.10,12,35BAK, VDACs, ANTI, and -72- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388PHB2 were co-eluted in high molecular weight fractions at approximately 600 kDa, suggesting that these proteins may form a 600 kDa complex. Of note, the mitochondrial matrix protein cyclophilin D and MIM proteins cytochrome c oxidase subunit IV (COX IV) and ATP synthase 0 were not co-eluted, highlighting the specificity of this 600 kDa complex (FIG. 1C). To further confirm that these proteins form a 600 kDa complex, high molecular weight fractions collected from gel filtration chromatography were subjected to anti-Protein C immunoprecipitation. Remarkably, the silver-stained gel displayed patterns similar to those shown in FIG. 1A (FIG. ID). Immunoblot analyses further confirmed the identity of these proteins (FIG. IE). Notably, this 600 kDa complex appears to exist in healthy cells, distinguishing it from oligomerized BAK that can only be detected upon activation by BH3s.10,12,35Example 3: BAK exists in three protein complexes with distinct death-regulatory activity

[0164] To further interrogate the existence of different BAK-associated complexes, gel filtration chromatography was performed on WT MEFs using either 1% CHAPS soluble or 1% CHAPS insoluble, 2% CHAPS soluble mitochondrial subfraction obtained from mitochondria before or after incubation with recombinant truncated BID (tBID, an activator BH3) protein to activate BAK in vitro. Interestingly, BAK was identified in three distinct complexes (FIG. IF). In both 1% CHAPS soluble and 1% CHAPS insoluble, 2% CHAPS soluble mitochondrial subfractions, BAK was eluted as a 60 kDa complex before tBID treatment and shifted to 160 kDa in response to tBID treatment (FIG. IF). We reasoned that the 160 kDa complex probably represents oligomerized BAK that is induced by tBID to mediate cytochrome c release whereas the 60 kDa complex probably contains inactive BAK. In addition, BAK was eluted around 600 kDa from the 1% CHAPS insoluble, 2% CHAPS soluble subfraction both before and after tBID treatment (FIG. IF), consistent with the observation that BIM BH3 peptide did not alter the composition of proteins co-purified with BAK (FIG. 1A). The 600 kDa complex was less abundant in the 1% CHAPS soluble subfraction, consistent with its lower solubility in 1% CHAPS. Notably, recombinant tBID protein was mainly co-eluted with the 600 kDa complex (FIG. IF).

[0165] To further assess the 160 kDa BAK complex induced by tBID, the 160 kDa fractions collected from gel filtration chromatography were analyzed by anti-Protein C -73- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388affinity chromatography. Surprisingly, no distinct proteins were detected associated with BAK (FIG. 1G). Hence, the 160 kDa complex probably represents a BAK hexamer composed of six 25 kDa BAK monomers. To understand the functional significance of various BAK complexes, we assessed protein crosslinking and cytochrome c releasing activity using the 60, 160, and 600 kDa fractions collected from gel-filtration chromatography. Only BAK present in the 160 kDa fraction could be cross-linked into dimers and trimers by the BMH crosslinker (FIG. 1H) and could release cytochrome c from Bax ■ ’Ba^ ’mitochondria (FIG. II). These data indicate that the 600 kDa BAK complex is functionally distinct from the 160 kDa BAK (probably a hexamer) complex that possesses cytochrome c releasing activity (FIG. II).

[0166] To determine whether BAK is present in the aforementioned complexes upon activation by endogenously expressed tBID, we engineered a doxycycline-inducible tBID system mApafl^ MEFs. This system facilitated the study of MIMP as tB ID-induced mtDNA efflux persists for an extended period in Apafl- / -cells due to their prolonged death kinetics. Furthermore, it allowed us to examine whether BAX forms distinct complexes and whether it is recruited to the 600 kDa BAK complex, given that most BAX was absent from mitochondria isolated from healthy cells as used in FIG. IF. Mitochondria were isolated from tBID-inducible Apafl ^ MEFs before or after 6 hours of tBID induction, and the 1% CHAPS insoluble, 2% CHAPS soluble mitochondrial subfraction was assessed by gel filtration chromatography. Again, we observed that BAK shifted from 60 kDa to 160 kDa in response to tBID induction while BAK was also eluted around 600 kDa together with VDAC1 / 2, ANTI, and PHB2 regardless of tBID induction (FIG. 2A). Consistent with the notion that BAX translocates from the cytosol to the mitochondria upon apoptosis, more BAX was detected after tBID induction. BAX was eluted only at 40 kDa before tBID induction and shifted to 140 kDa and 600 kDa after tBID induction (FIG. 2A). These findings indicate that BAX, once activated by tBID, is recruited to the 600 kDa complex. We reasoned that the 40 kDa BAX complex contains inactive BAX while the 140 kDa BAX complex is composed of a BAX hexamer analogous to the 160 kDa BAK hexameric complex, representing the MOMP machinery. This is consistent with the reported BAX oligomers induced by the detergent Fos-12 in vitro, composed of 6-8 BAX monomers capable of permeabilize the MOM.36Notably, tBID is also recruited to the 600 kDa -74- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388complex although the elution profile of endogenously induced tBID differed somewhat from that of exogenously added recombinant tBID (FIGs. 2A and IF).Example 4: Loss of VDAC1 or VDAC2 inhibits BAK / BAX-dependent caspaseindependent cell death

[0167] To interrogate the function of the 600 kDa BAK / BAX complex, lentiviral transduction of Cas9 and sgRNAs targeting each Vdac gene was performed in tBID-inducible Apafl^ MEFs. Targeting Vdacl or Vdac2 but not Vdac3 protected Apafl^ cells from cell death triggered by tBID or the combined inhibition of BCL-2 / BCL-XL / MCL-1 using ABT-263 (a dual inhibitor of BCL-2 and BCL-XL) and S63845 (an MCL-1 inhibitor) (FIG. 2B).22Targeting both Vdacl and Vdac2 provided even greater protection. In contrast, targeting both Vdacl and Vdac2 failed to prevent and even slightly increased apoptosis in WT MEFs (FIG. 2C). These findings were further validated in tBID-inducible Apafl ^ Vdac 1' Ardac2' ' MEFs (FIG. 2D). Consistent with our published results,4BAK / BAX activation killed Apafl ^ MEFs at a much slower rate than WT MEFs (FIG. 7B). The combination of ABT-263 and S63845 failed to activate BAK / BAX in the absence of activator BH3s (FIG. 7C), consistent with the notion that these BH3 memetics displace endogenous activator BH3s to indirectly activate BAK / BAX as reported.12,17Furthermore, retroviral transduction of tBID, BIM, or PUMA killed Apafl ^ MEFs, which could be inhibited by the loss of VDAC 1 / 2 (FIG. 7D). Reported genetic loss-of-function studies have shown that VDAC 1-3 are dispensable for MOMP.37In fact, deficiency of Vdac2 or all Vdac isoforms accelerates caspase-dependent apoptosis.35,37,38

[0168] In accordance with the viability data, KO of both Vdacl and Vdac2 did not inhibit tB ID-induced cytochrome c release to the cytosol even though it appeared to reduce baseline mitochondrial cytochrome c expression levels (FIG.2E). Furthermore, tB ID-induced oligomerization of BAK / BAX captured by protein crosslinking was still detected in Apafl~ / ~Vdacr'~Vdac2~l~ cells (FIG. 2F). It has been reported that VDAC2 not only keeps BAK as an inactive monomeric form but also stabilizes its mitochondrial targeting.35,38'40Consistent with this notion, our data reveal that BAK becomes BAX-like in the absence of VDAC2 — once activated upon death signals, it can translocate from the cytosol to the mitochondria (FIG. 2E) and form homo-oligomers even though BAK is unstable and less abundant in Apafl~ / ~Vdacl~ / ~Vdac2~ / ~ cells (FIG. 2F). On a side note, a small molecule has -75- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388been reported to interact with VDAC2, promoting the ability of VDAC2 to inhibit BAK-driven apoptosis.41

[0169] Our findings that loss of VD AC 1 / 2 inhibits tB ID-induced death in Apafl-deficient cells without inhibition of MOMP (cytochrome c release, FIG. 2E) indicate that BAK / B AX-dependent, caspase-independent cell death is not simply a consequence of MOMP. Furthermore, MEFs could survive and proliferate in the absence of cytochrome c and cytochrome c deficiency had no effect on tB ID-induced killing o Apafl ^ cells (FIG.7E). We next examined the potential engagement of other known cell death mechanisms in BAK / B AX activation-induced caspase-independent cell death (baciptosis) using specific inhibitors of various cell death mechanisms (FIG. 7F). None of these inhibitors prevented baciptosis, excluding the involvement of pyroptosis (inhibited by the pan-caspase inhibitor Q-VD-OPH), necroptosis (inhibited by the RIPK1 inhibitor necrostatin-1 and the RIPK3 inhibitor GSK’872), ferroptosis (inhibited by ferrostatin-1 and deferoxamine), and mPTP (inhibited by cyclosporin A). In contrast, depletion of mtDNA through CRISPR / Cas9-mediated KO of mtDNA polymerase gamma (Polg) protected Apafl ^ cells from tB ID-induced cell death (FIG. 7G). Similar results were observed in cells pretreated with ethidium bromide to deplete mtDNA (FIG. 7H). These data support the notion that mtDNA release into the cytosol contributes to baciptosis. Notably, the protective effect of mtDNA depletion is unlikely to result from the disruption of oxidative phosphorylation as cytochrome c depletion also halts this process but does not confer similar protection (FIG.7E). Electron microscopy revealed that tBID induction in WT MEFs exhibited characteristic apoptotic features such as chromatin condensation with margination along the nuclear membrane and plasma membrane blebbing (FIG. 71). In contrast, tBID induction in Apafl ^ MEFs displayed necrotic features, including disruption of the plasma membrane and organelles, induction of autophagy and mitophagy, and an increase in lysosomes (FIG.71). Consistent with our findings, developmental cell death in the interdigital webs of Apafl~ / _mice also exhibits necrotic rather than apoptotic morphology.42Example 5: Loss of VDAC1, VDAC2, or prohibitins impairs BAK / BAX-dependent mtDNA efflux

[0170] As mtDNA efflux contributes to baciptosis that is inhibited by the loss of VDAC1 / 2, we hypothesized that the 600 kDa BAK / B AX complexes might be involved in -76- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388mtDNA efflux. To test this, we first confirmed that tB ID-induced mtDNA efflux is dependent on BAK and BAX (FIG. 3A). Similar to their role in apoptosis, BAK and BAX were found to function redundantly in activating baciptosis (FIG. 8A). Remarkably, loss of both VDAC1 and VDAC2 greatly reduced tB ID-induced mtDNA efflux in ApafP' cells (FIG. 3B). Likewise, mtDNA efflux triggered by the combined inhibition of BCL-2 / BCL-XL / MCL-1 was also markedly suppressed by the loss of VD AC 1 / 2 (FIG. 3C). Retroviral transduction of VDAC1 or VDAC2, but not VDAC3, restored tB ID-induced mtDNA efflux and baciptosis in ApafP / ~VdacPl'Vdac2'1' cells (FIG. 3D), which is in accordance with our loss-of-function study (FIG. 2B). Together, our data indicate that VDAC1 and VDAC2 but not VDAC3 play critical roles in BAK / B AX-dependent mtDNA efflux and baciptosis.

[0171] To determine whether loss of VD AC 1 / 2 affects the formation of distinct BAK / B AX complexes, gel filtration chromatography was performed on the 1% CHAPS insoluble, 2% CHAPS soluble mitochondrial subfraction from tBID-inducible Apafl ^ Vdad'l'Vdac2'1' cells both before and after tBID induction. Consistent with the notion that BAK becomes BAX-like in the absence of VDAC2, both BAK and BAX were eluted only at low molecular weight fractions (40-60 kDa) before tBID induction, and shifted to 140-160 kDa and 600 kDa after tBID induction (FIG. 3E). These findings indicate that loss of VDAC1 / 2 does not affect the formation of the MOMP machinery, as supported by both the gel filtration chromatography (FIG. 3E) and protein crosslinking data (FIG. 2F).Furthermore, loss of VDAC1 / 2 did not alter the elution profiles of tBID, ANTI, and PHB2 (FIG. 3E). Overall, loss of VD AC 1 / 2 does not grossly affect the formation of the 600 kDa BAK / B AX complex.

[0172] As mtDNA efflux from the mitochondrial matrix to the cytosol requires passage through both the MOM and the MIM while VDAC1 / 2 are localized at the MOM, we hypothesized that PHB2 and / or ANTI, MIM proteins associated with the 600 kDa BAK / B AX complex, participate in mtDNA efflux. Of note, we were unable to generate stable cell lines deficient for Phb2 due to its essentiality.43Nonetheless, transient lentiviral transduction of Cas9 and the sgRNA targeting Phb2 markedly suppressed tB ID-induced mtDNA efflux and baciptosis (FIGs. 3F-3H). Consistent with the known interdependence of PHB1 and PHB2 that form a large heterodimeric IMM complex,43,44targeting Phb2 reduced both PHB2 and PHB1 proteins (FIG. 3F). In fact, PHB1 was also co-eluted with -77- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388the 600 kDa BAK / BAX complex (FIG. 8B) and could be co-precipitated with BAK by immunoprecipitation (FIG. 8C). As ANTI interacts with its homolog ANT2 and ANT2 is also co-eluted with the 600 kDa complex (FIG. 8B), we next assessed the roles of both ANTI and ATN2. Lentiviral transduction of Cas9 and sgRNAs targeting both Anti and Ant2 had minimal effect on tB ID-induced baciptosis and mtDNA efflux (FIGs. 3I-3J). Thus, although ANTs are associated with the 600 kDa complex, they are not key mediators of BAK / B AX-dependent mtDNA efflux and baciptosis. This is similar to the RNA Polymerase II complex, where not every protein directly influences transcription but instead serves a structural or auxiliary role.45Furthermore, while VD AC-mediated mitochondrial calcium influx could potentially activate mPTP, CRISPR / Cas9-mediated KO of cyclophilin D had little effect on tB ID-induced mtDNA efflux and baciptosis (FIG. 8D), excluding the involvement of mPTP in BAK / B AX activation-induced mtDNA efflux. Finally, the roles of VDAC1, VDAC2, and PHB2 in BAK / B AX activation-induced mtDNA efflux and baciptosis were further validated in the B16-F10 mouse melanoma cell line (FIGs. 8E-8G).Example 6: The roles of VDAC1, VDAC2, and prohibitins in BAK / BAX activation-induced mtDNA efflux and cell death in apoptosome-deficient human cancers

[0173] Given that various mechanisms inactivating the apoptosome have been reported in human cancers,29we investigated whether baciptosis can occur in these contexts and examined the roles of VDAC1, VDAC2, and prohibitins in this process. We first screened commonly used human cancer cell lines for minimal effector caspase activity following BAK / BAX activation using the combination of ABT-263 and S63845 (ABT-263 / S63845). Notably, ABT-263 / S63845 effectively triggered MOMP — cytochrome c efflux in A498, PC9, and MDA-MB-231 cell lines while little effector caspase activity was detected in A498 and PC9 cell lines likely due to their low expression of APAF1 (FIGs. 4A, 4B, 9A, and 9B). Consistent with the robust effector caspase activation observed in MDA-MB-231 cells following the treatment with ABT-263 / S63845, these cells exhibited faster death kinetics compared to A498 and PC9 cells, and only the death in MDA-MB-231 cells could be blocked by Q-VD-OPH (FIG. 9C). These findings indicate that BAK / BAX activation induces baciptosis in A498 and PC9 cells.

[0174] To determine whether human BAK and BAX proteins are present in the aforementioned complexes upon activation, mitochondria were isolated from A498 cells -78- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388before and after the treatment with ABT-263 / S63845. The 1% CHAPS insoluble, 2% CHAPS soluble mitochondrial subfraction was assessed by gel filtration chromatography (FIG. 4C). Similar to mouse BAK, human BAK shifted from 60 kDa to 160 kDa following ABT-263 / S63845 treatment, while it was also co-eluted with VDAC1 / 2 and PHB1 / 2 around 600 kDa both before and after the treatment (FIG. 4C). Consistent with mouse BAX, human BAX was eluted exclusively at 40 kDa before ABT-263 / S63845 treatment and shifted to 140 kDa and 600 kDa after treatment (FIG. 4C). Notably, BIM was predominantly eluted at ~50 kDa before treatment but shifted to 600 kDa after ABT-263 / S63845 treatment (FIG. 4C). These findings suggest that ABT-263 / S63845 displaces BIM from antiapoptotic BCL-2 members to activate BAK and BAX present in both the 40-60 kDa and 600 kDa complexes — one for the formation of the MOMP machinery and the other for the mtDNA-releasing machinery, respectively.

[0175] We further investigated the roles of VDACs and prohibitins in ABT-263 / S63845-induced mtDNA efflux and cell death in A498 and PC9 cells. Lentiviral transduction of Cas9 and the sgRNA targeting VDAC1 or VDAC2 but not VDAC3 significantly suppressed ABT-263 / S63845-induced mtDNA efflux and baciptosis in both A498 and PC9 cells (FIGs. 4D, 4E, 9D, and 9E). Due to the essentiality of prohibitins and thereby the intolerance of CRISPR / Cas9-mediated KO of PHB2 in human cells, we could only transiently knockdown (KD) PHB2 using lentiviral transduction of shRNAs. Transient KD of PHB2 reduced both PHB2 and PHB1 protein levels and significantly suppressed ABT-263 / S63845-induced mtDNA efflux (FIGs. 4F, 4G, 9D, and 9E). However, we could not assess the protective effect of PHB1 / 2 loss on baciptosis, as PHBl / 2-deficient cells died before ABT-263 / S63845-induced baciptosis occurred. In contrast, loss of VDAC1 or VDAC2 failed to inhibit ABT-263 / S63845-induced apoptosis in MDA-MB-231 cells or A498 cells reconstituted with APAF1 (FIGs. 4H, 41, and 9F-9H). Collectively, these findings demonstrate that human cancer cells replicate the observations made in mouse cells and confirm that apoptosome-deficient cancers are capable of undergoing baciptosis following BAK / BAX activation, a process regulated by VDAC1 and VDAC2.-79- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388Example 7: The ability of VDAC1 / 2 to mediate BAK / BAX-dependent mtDNA efflux is impaired by calcium-binding site mutations

[0176] Mitochondrial ATP / ADP exchange is carried out by the combined action of VDAC and ANT, which exchange matrix ATP for cytosolic ADP during oxidative phosphorylation.34As the 600 kDa BAK-associated complex includes VDACs and ANT, we hypothesized that recruitment of tBID to the 600 kDa complex may inhibit mitochondrial ATP / ADP exchange. To test this hypothesis, we employed a BH3-inducible system where GFP flanked by loxP sites was placed upstream of BH3s to prevent the expression of BH3s from the LTR promoter. Upon treatment with 4-hydroxytam oxifen (4-OHT), the floxed GFP cassette was deleted by Cre-ERT2 such that BH3 expression was induced and killed Apafl ^ MEFs (FIGs. 10A-10C). Significantly, 4-OHT treatment in tBID-inducible but not control cells greatly reduced ADP import into the mitochondria (FIG. 10D) To determine whether defects in the outer membrane VDAC or inner membrane ANT contribute to the impaired ADP import, we performed ADP import assays using mitoplasts that only retain the inner membrane protein ANT. The ADP uptake was fully restored in mitoplasts despite tBID induction (FIG. 10D), suggesting that tBID inhibits VDAC- but not ANT -mediated ADP import into the mitochondria. We next examined whether other activator BH3s, BIM and PUMA, also inhibit ADP / ATP exchange. Indeed, induction of either BIM or PUMA led to inhibition of ADP import into the mitochondria (FIG. 10E). Given that loss of VDAC 1 / 2 inhibits rather than promotes baciptosis, activator BH3-induced closure of the VDAC nucleotide channel is unlikely a direct cause of baciptosis. Instead, VDAC closure may reflect the underlying conformation changes in VDAC that are required to mediate mtDNA efflux.

[0177] It has been reported that VDAC in the closed state for metabolite transport becomes highly permeable to Ca2+, resulting in mitochondrial calcium influx46The calcium-binding sites in VDAC1 have been mapped to E73 and E203.47Notably, E73 is conserved in VDAC2 (E85) but not in VDAC3 (Q73) (FIG. 10F), which may explain why VDAC3 is not involved in BAK / BAX-dependent mtDNA efflux and baciptosis (FIGs. 2B and 3D). Accordingly, we hypothesized that BAK / BAX activation-induced closure of VDAC 1 / 2 channel could increase mitochondrial calcium influx. To interrogate this hypothesis, we employed a mitochondria-targeted fluorescent protein-based calcium-80- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388indicator (mito-G-GECO1.2) to determine whether BAK / BAX activation by tBID increases mitochondrial calcium.48Indeed, tBID induction increased mitochondrial calcium influx that was abrogated by double deficiency of Bak and Bax (FIGs. 5A and 11 A). Importantly, loss of both VDAC1 and VDAC2 greatly suppressed tB ID-induced mitochondrial calcium influx (FIGs. 5B and 11B). The combination of ABT-263 and S63845 also increased mitochondrial calcium influx that was dependent on VDAC1 / 2 (FIGs. 5C and 11C). To determine whether calcium-binding by VDAC1 / 2 contributes to tB ID-induced mitochondrial calcium influx, retroviral transduction ofWT or the calcium -binding site mutants (VDAC1 E73Q / E203Q; VDAC2 E85Q / D215N) of VDAC1 / 2 was performed in Apafk Vdacl''Vdac2'' cells. The ability of VDAC1 / 2 to restore BAK / BAX activation-induced increase of mitochondrial calcium influx was impaired by calcium-binding site mutations (FIGs. 5D and 11D) We next assessed the effect of calcium-binding site mutations on the ability of VD AC 1 / 2 to induce mtDNA efflux and baciptosis. Retroviral transduction of WT but not the calcium-binding site mutants of VD AC 1 / 2 restored tB ID-induced mtDNA efflux and baciptosis in ApafB Vdacl''Vdac2'' cells (FIG. 5E). It is noteworthy that transduction of the calcium-binding site mutants of VD AC 1 / 2 fully restored the stability and mitochondrial localization of BAK as well as the baseline cytochrome c levels as WT VDAC1 / 2 while it still failed to mediate mtDNA efflux and baciptosis (FIG.5F).

[0178] VDAC1 / 3 oligomers have been reported to mediate mtDNA release from mitochondria under oxidative stress in non-dying cells, a process independent of BAK and BAX.49The N-terminal lysine residues of VDAC1 have been shown to mediate its interaction with mtDNA, which is required for VDAC1 to form oligomers and release mtDNA49Therefore, we compared the effects of N-terminal lysine mutations and calcium-binding site mutations of VDAC1 on promoting BAK / BAX activation-induced mtDNA efflux. Retroviral transduction of both WT and the N-terminal lysine mutant (K12A / R15A / K20A) of VDAC1 restored tB ID-induced mtDNA efflux and baciptosis in Apafl~ / ~Vdacrl~Vdac2~l~ cells (FIG. 5G). In contrast, the calcium-binding site mutant (E73QZE203Q) of VDAC1 failed to rescue these phenotypes (FIG. 5G). Altogether, our data indicate that BAK / BAX activation by activator BH3s probably induces conformational-81- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388changes in VDAC1 / 2, converting them from transporting nucleotides to calcium and enabling the cytosolic release of mtDNA.Example 8: Loss of VDAC1 / 2 does not prevent BAK / BAX activation-induced mitochondrial inner membrane herniation

[0179] To determine whether the 600 kDa BAK-associated complex containing tBID can directly permeabilize the MIM, we incubated Bax^Bak^Vdac l~ / ~Vdac2~ / ~Apafl~ / ~ mitoplasts with the 60, 160 and 600 kDa fractions collected from gel -filtration chromatography. Remarkably, the 600 kDa complex containing tBID displayed the highest mtDNA releasing activity whereas the 600 kDa complex without tBID showed only minimal activity in comparison with the 60 and 160 kDa complexes (FIG. 6A). These data strongly support that the 600 kDa BAK-associated complex containing tBID is the mtDNA releasing machinery. Of note, incubation of purified mitochondria with the 600 kDa complex containing tBID resulted in minimal mtDNA efflux (data not shown), suggesting that this macromolecular complex may not assemble effectively across both the MOM and the MIM under in vitro conditions.

[0180] Given that MIM herniation through BAK / BAX macropores has been proposed to mediate mtDNA release,25,26electron microscopy (EM) was performed to determine whether loss of VD AC 1 / 2 inhibits MIM herniation. MIM herniation was observed in both Apafl ^ and Apafl~ / ~Vdacr'~Vdac2~l~ cells following tBID induction to a similar extent (FIG.6B) Since MIM herniation would lead to reduced colocalization between the MIM and the MOM, we next validated our EM findings by expressing TOMM20-GFP and TIMM50-mCherry in tBID-inducible Apafl ' and Apafl~ / ~Vdacl'l'Vdac2'1' cells. This approach allowed for fluorescence imaging of the MOM (TOMM20-GFP) and the MIM (TIMM50-mCherry), respectively. Indeed, tBID induction resulted in a significant but comparable reduction in the colocalization of the MOM and the MIM in both Apafl ^ and Apafl ^ Vdac!' 'Vdac2' ' cells (FIGs. 6C and 12A).

[0181] Collectively, these data indicate that MIM herniation is probably a consequence of the formation of BAK / BAX macropore in the MOM rather than an active mechanism driving mtDNA efflux. Consistent with this notion, it has been reported that only a minority of herniated MIM exhibits loss of membrane integrity.25It is plausible that MIM herniation still occurs in the absence of VD AC 1 / 2 given that loss of VD AC 1 / 2 does not prevent -82- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388BAK / BAX oligomerization and MOMP. The observed MIM herniation in VDAC1 / 2-deficient cells helps explain why the loss of VD AC 1 / 2 neither completely blocks mtDNA efflux nor promotes the long-term clonogenic survival of Apafl ^ cells following BAK / BAX activation (FIGs. 3B, 3C, 12B, and 12C). Altogether, our data strongly support that the 600 kDa BAK / BAX complex containing tBID, VDAC1 / 2, and prohibitins is the active machinery responsible for the majority of mtDNA release whereas MIM herniation represents a passive process contributing to mtDNA leakage from mitochondria after BAK / BAX activation.

[0182] Presented herein is biochemical evidence that BAK and BAX, once activated by activator BH3s, assemble into two distinct protein complexes at the mitochondria to regulate cytochrome c and mtDNA efflux, respectively (FIG. 6C). The MOMP machinery capable of releasing cytochrome c probably consists of a BAK or BAX hexamer, which is consistent with recently reported in vitro studies of both BAK and BAX.36,50The identification of BAK / BAX hexamers as the cytochrome c releasing machinery helps explain why BAK / BAX pores responsible for cytochrome c efflux are too small to be visualized by super-resolution microscopy.25On the other hand, the active mtDNA releasing machinery comprises BAK, BAX, activator BH3s, VDAC1 / 2 (MOM proteins), and prohibitins (MIM proteins), which span both mitochondrial outer and inner membranes. Our discovery of the mtDNA releasing machinery being different from the cytochrome c releasing MOMP machinery indicates that mtDNA efflux is not simply a consequence of MOMP but involves distinct active biochemical processes. On the contrary, MIM herniation through BAK / BAX macropores formed after cytochrome c efflux is a consequence of MOMP, which is independent of VDAC1 / 2. Our data support a model in which the 600 kDa complex containing activator BH3s such as tBID, BAK / BAX, VDAC1 / 2, and prohibitins is responsible for the majority of mtDNA release, whereas MIM herniation constitutes a passive process that accounts for a minority of mtDNA leakage from mitochondria following MOMP induction (FIG. 6C).

[0183] Although BAK / BAX-dependent mtDNA efflux can occur during caspase-mediated apoptosis, it probably does not contribute to apoptotic cell death due to its late occurrence and the fast killing kinetics mediated by caspases. Conversely, mtDNA efflux contributes to BAK / B AX-mediated cell death in the absence of caspases because prevention -83- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388of mtDNA efflux by KO of Vdctcl. Vdac2, or Phb2 as well as depletion of mtDNA in Apafl ' cells inhibit this form of cell death. These findings in mouse cells were fully validated in human cancer cell lines. It appears that when APAF1 expression levels fall below a certain threshold, effector caspase activation is inhibited. Conversely, reconstitution of APAF1 expression in APAF1 -deficient cells must surpass this threshold to restore effector caspase activation. Importantly, the present disclosure demonstrated that apoptosome-deficient human cancers can undergo baciptosis following BAK / BAX activation, a process regulated by VDAC1 and VDAC2. These findings suggest that therapy-induced activation of BAK / BAX in apoptosome-deficient cancers may have a more pronounced impact on tumor-immune interactions compared to apoptosome-proficient cancers that typically undergo immunologically silent apoptosis. Further investigation is needed to better understand how APAF1 expression levels and baciptosis activation influence therapeutic outcomes in cancer patients.

[0184] The fact that apoptosis is defined by characteristic ultrastructural features that are completely absent in cells undergoing BAK / BAX activation-induced caspaseindependent cell death justifies the introduction of a new nomenclature for this type of cell death (FIG. 71). This necrotic type of cell death is termed baciptosis. While this form of cell death has previously been described as MOMP-induced caspase-independent cell death,51these findings unequivocally demonstrate its independence from MOMP. Future work is needed to determine how mtDNA release into the cytosol initiates cell death and whether mitochondrial matrix proteins, RNAs, or metabolites are also released through the 600 kDa complex along with their respective biological significance. As cytochrome c is a crucial component of the mitochondrial electron transport chain, it is conceivable that cytochrome c depletion through MOMP may be sufficient to initiate caspase-independent cell death in cell types that are fully dependent on oxidative phosphorylation. In such a scenario, loss of VDAC1 / 2 may actually exacerbate rather than inhibit BAK / BAX activation-induced caspase-independent cell death due to the shutdown of oxidative phosphorylation. Hence, the pro-death function of VD AC 1 / 2 could be masked by their pro-survival function in a context-dependent manner. In fact, we observed that MEFs tolerate the loss of VDAC1 and VDAC2 much better than other cell types, which is consistent with the more pronounced protection against baciptosis conferred by VDAC1 / 2 loss in MEFs.-84- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388

[0185] VDAC1, VDAC3, and mPTP have been implicated in mtDNA efflux in several different settings, including mitochondrial stress caused by EndoG deletion in MEFs, NETosis of neutrophils, and pyroptosis in macrophages49,52However, neither VDAC3 nor cyclophilin D is involved in BAK / B AX-dependent mtDNA efflux (FIGs.3D and 8D). The inability of VDAC3 to mediate BAK / B AX-dependent mtDNA efflux is probably due to its lack of a critical calcium-binding amino acid residue (FIG. 10F). We demonstrated that BH3-mediated activation of BAK / B AX converts VDAC1 / 2 from transporting nucleotides to calcium. Calcium binding site mutations in VDAC1 / 2 not only reduce BAK / B AX activation-induced mitochondrial calcium influx but also impair their ability to mediate mtDNA efflux and baciptosis. Notably, loss of VD AC 1 / 2 provides greater protection against baciptosis than depletion of prohibitins or mtDNA (FIGs. 2D, 3F, 7G, and 7H). Given that prohibitins do not have known roles in regulating mitochondrial calcium but are still involved in controlling mtDNA efflux, we speculate that VDACl / 2-dependent mitochondrial calcium influx may be more closely linked to cell death than to mtDNA efflux. Interestingly, the crystal structure of calcium binding at Glu-73 across two antiparallel VDAC1 monomers has been reported,53suggesting that calcium binding may induce and stabilize VDAC1 dimerization, followed by oligomer formation. However, this would only happen when high levels of calcium are present and / or when the local lipid interface is disrupted,53events that can occur following BAK / B AX activation. The structural basis of calcium-induced VDAC1 oligomers is clearly different from the reported VDAC1 oligomers induced by the interaction between its N-terminal lysine residues and mtDNA.49Future structural characterization of the 600 kDa complex is expected to provide mechanistic insights into the regulation of BAK / B AX-dependent mtDNA efflux during cell death.Example 9: Materials and Methods

[0186] Animal Models

[0187] All animal experiments were performed in accordance with the Institutional Animal Care and Use Committee at Memorial Sloan Kettering Cancer Center (MSKCC). C57BL / 6J (strain # 000664) and B6. Cg-Rag2tm} }Csn / J (strain # 008449), C57BL / 6J-StinglgtU (strain # 017537), and B6. l29P2- / 12 / ?7"" / ' " DcrJ (strain # 002087) mice were purchased from the Jackson Laboratory. Both male and female mice were included in the -85- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388study and no sex-dependent effects were observed. All mice analyzed were sex and age matched (7-9 weeks old). To establish intradermal melanoma tumors, 5xl05tet-tBID-Apfal+ / +, te D-Apafl1-, tet-CASP3CAM, or tet-coRIPK3 B16-F10 cells in 50 pL PBS were injected intradermally on the right flank of C57BL / 6 mice. Once tumors reached 200 mm3, mice were given doxycycline water (2 mg / mL doxycycline (Sigma), 50 mg / mL sucrose (Fisher)) until tumors were no longer palpable (~2-3 weeks). For rechallenge experiments, mice with regressed tumors were maintained on doxycycline water for 2 more weeks. Two weeks after doxycycline withdrawal, mice were rechallenged with the same B16 cells (5xl05) on opposite flanks. Tumor volume was measured with calipers 2-3 times weekly and calculated as (width2* length) / 2. To assess the abscopal effect, C57BL / 6J mice were injected intradermally with tet-tBID Apafl ^ B16 cells (5xl05) or PBS on left flanks. One week later, mice were injected with parental B16 cells (IxlO5) on opposite flanks. One more week later, mice were given doxycycline and monitored for tumor growth and survival. To deplete specific immune cells during rechallenge, isotype control (BioXCell, 2A3, BE0089), anti-CD8 (BioXCell, 2.43, BE0061), or anti-CD4 (BioXCell, GK1.5, BE0003) antibodies were injected intraperitoneally in mice twice a week (200 pg / dose) beginning one day prior to rechallenge and continued until endpoint. To deplete NK cells, anti-NKl.l (PK136) was injected intraperitoneally once weekly (200 pg / dose) beginning one day prior to rechallenge and continued until endpoint as described h Antibody-mediated depletion of specific immune cells was confirmed by flow cytometric analyses of peripheral blood. For the treatment with targeted therapies and anti-PD-1, 7-8 weeks-old C57BL / 6 mice were injected subcutaneously with B16-F10 or YUMM1.7 cells in 0.2 mL 50% Matrigel (BD Biosciences). Once tumors were palpable, mice were randomized and given vehicle, anti-PD-1, combined venetoclax and dinaciclib, or combined anti-PD-1, venetoclax, and dinaciclib. Treatment continued for 4 additional weeks after tumors became impalpable, with mice bearing Apafl~'~ B 16 tumors treated for up to 7 weeks in total and mice bearing Apafl'1' YUMM1.7 tumors treated for up to 9 weeks in total. Anti-PD-1 (BioXCell, Clone RMP1-14, BE0146) or its isotype control (BioXCell, BE0089) was diluted with PBS and injected intraperitoneally (200 pg / dose) on day 9, 11, and 13, and then every other day. Venetoclax (MedKoo Biosciences, 100 mg / kg, oral gavage) and Dinaciclib (Selleck Chemicals, 25 mg / kg, intraperitoneal injection) were administrated on day 8, 10,-86- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388and 12, and then twice weekly as described2. Q-VD-OPH (MedKoo Biosciences, 20 mg / kg, intraperitoneal injection) was administrated daily starting on day 7. Emricasan (MedKoo Biosciences, 20 mg / kg, intraperitoneal injection) was administrated twice daily for 3 to 5 days per week. Mice were treated for 4 more weeks after the tumors have completely regressed. For the FACS analysis of tumor-infiltrating lymphocytes, mice bearing palpable B16 tumors were untreated or treated with venetoclax and dinaciclib every other day for 2-3 times.

[0188] Plasmid construction and CRISPR / Cas9-mediated genome editing

[0189] The lentiviral tetracycline-inducible hygromycin-resi stance vector (TRE-gene of intereset-pPGKHygro-T2A-rtTA3) was generated from pCW-Cas9 (Addgene), LentiCRISPRv2 (Addgene), and MSCV-Hygro (Addgene) using the NEBuilderRHiFi DNA Assembly Cloning Kit (New England Biolabs). The lentiviral tetracycline-inducible puromycin-resi stance vector (TRE-gene of interest-pPGK-Puro-T2A-rtTA3) was generated by replacing the hygromycin-resistance cassette in the lentiviral tetracycline-inducible hygromycin-resi stance vector with the puromycin resistance cassette. Mouse Bid with deletion of N-terminal 177 bp of coding region (tBID) was cloned into the lentiviral tetracycline-inducible hygromycin-resistance vector. A previously reported constitutively active mutant of mouse Caspase-3 (CASP3CAM) 3 as well as mouse Ripk3 fused to a constitutively oligomerizing domain (co-RIPK3) 4 were cloned into the lentiviral tetracycline-inducible puromycin-resi stance vector. sgRNAs were designed using Optimized CRISPR Design (http: / / crispr.mit.edu / ) and cloned into LentiGuide-Puro (Addgene) 5. All constructs were confirmed by DNA sequencing. Lentivirus was produced by co-transfection of 293T cells with pCMVDR8.2 and pHCMV. VSVG using Lipofectamine 2000 (Thermo Fisher Scientific) as described6. The sequences of sgRNAs are listed as follows: mouse Apafl. GCGGAGGCTCACAGTATTAT (SEQ ID NO: 40); mouse Bax, CAACTTCAACTGGGGCCGCG (SEQ ID NO: 41); mouseM, GCCCTGTACGTCTACCAGCG (SEQ ID NO: 42); and mouse Cgas, CGAGGCGCGGAAAGTCGTAA (SEQ ID NO: 43).

[0190] Cell culture and viability assay-87- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388

[0191] The B16-F10 cell line was obtained from the American Type Culture Collection (ATCC) and cultured according to the recommendations of ATCC. Cell lines engineered with hygromycin and / or puromycin-resistant markers were cultured with hygromycin B (500 pg / mL, Roche) and / or puromycin (2 pg / mL, Sigma), respectively. B16-F10 cells were transduced with lentivirus expressing tet-inducible tBID, CASP3CAM, or coRIPK3 followed by single cell subcloning. Knockout cell lines were generated using CRISPR / Cas9-mediated genome editing as described5. Briefly, B16-F10 cells were transiently transfected with LentiGuide-Puro expressing sgRNAs targeting genes of interest. Following puromycin selection for 3 days, transfected cells were subjected to single cell subcloning. Knockout clones were selected based on the lack of expression of target proteins and Cas9 protein by immunoblot analyses. Cell lines were routinely tested for mycoplasma using the Venor™ GeM mycoplasma detection kit (Sigma). Cells were treated with 1 pg / mL doxycycline (Sigma) to induce the expression of respective proteins. To quantify cell death, cells were harvested and stained with annexin-V (BioVision) and / or propidium iodide (2 pg / mL, Sigma) as described 7, followed by flow cytometric analyses using an LSRFortessa (BD Biosciences). Data were analyzed using FACSDiva (BD Biosciences).

[0192] Immunoblot analysis

[0193] Cells were lysed in RIPA buffer supplemented with complete protease inhibitors (Roche) and homogenized by FastPrep-24 homogenizer (MP Biomedicals). Protein concentration was determined by the Pierce BCA protein assay kit (Thermo Fisher Scientific). Extracted proteins were resolved by 10 % or 4-12% NuPAGE gels (Thermo Fisher Scientific) and transferred onto PVDF membranes (Immobilon-P, Millipore).Antibody detection was accomplished using enhanced chemiluminescence method (Western Lightning Plus-ECL, PerkinElmer) and Amersham Imager 680 (GE Healthcare). Antibodies used for immunoblot analyses are listed as follows: rabbit anti-APAFl (Cell Signaling Technology (CST), D7G4), rabbit anti-BID (CST, #2003), rabbit anti-cleaved caspase 3 (CST, #9661), mouse anti-caspase 3 (BD Biosciences, Clone 46), rabbit anti-PARP (CST, #9542), rabbit anti-GSDME (Abeam, EPR19859), rabbit anti-BAX (Santa Cruz, N20), rabbit anti-BAK (CST, D4E4), rabbit anti-RIPK3 (CST, D4G2A), rabbit antiphospho-MLKL (S345) (Abeam, EPR9515(2)), rabbit anti-MLKL (CST, D6W1K), rabbit antiphospho-IRF3 (S396) (CST, 4D4G), rabbit anti-IRF3 (CST, D83B9), rabbit anti- -88- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388phospho-STING (S365) (CST, D8F4W), rabbit anti-STING (CST, D2P2F), rabbit anti-phospho-TBKl (S172) (CST, D52C2), mouse anti-TBKl (CST, E9H5S), rabbit anti-phospho-p65 (S596) (CST, 93H1), rabbit anti-p65 (CST, D14E12), and mouse anti-tubulin (Sigma, DM1A).

[0194] Extracellular ATP and LDH measurement

[0195] Cells were seeded in an opaque white 96-well plate (10,000 cells / well) the day before experiments. Cells were treated with 1 pg / mL doxycycline (Sigma) and extracellular ATP was measured using the RealTime-Glo Extracellular ATP Assay kit (Promega) according to the manufacturer’s protocol. Luminescence was measured with a SpectraMax M5 plate reader at the indicated times. For the measurement of extracellular LDH, cells were seeded in a 96-well plate (8,000 cells / well) the day before experiments. Cells were treated with 1 pg / mL doxycycline (Sigma) and culture media were removed at the indicated times and analyzed for LDH using the LDH-Glo™ Cytotoxicity Assay (Promega) according to the manufacturer’s protocol. Maximum LDH release was determined by treating cells with 0.2% Triton X-100 for 15 minutes. Luminescence was measured with a SpectraMax M5 plate reader.

[0196] Reverse transcription and quantitative real-time PCR

[0197] Total RNA was extracted from cells or tissues using Trizol (Thermo Fisher Scientific). Reverse transcription was performed with oligo-dT plus random decamer primers (Thermo Fisher Scientific) using Superscript II (Thermo Fisher Scientific).Quantitative PCR was performed with SYBR green master mix (Thermo Fisher Scientific) in duplicates using the gene specific primers on a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). Data were normalized against fl-Actin. The primers used for real-time PCR are listed as follows: mouse Ijhbl, 5'-CCTTTGCCATCCAAGAGATGCTC-3' (SEQ ID NO: 44) and 5'-AGTTGAGGACATCTCCCACGTC-3' (SEQ ID NO: 45); mouse Actb, 5'-GCCATGTACGTAGCCATCCAGGC-3' (SEQ ID NO: 46) and 5'-CTCCAGGGAGGAAGAGGATGCGGC-3' (SEQ ID NO: 47).

[0198] Quantification of mitochondrial DNA efflux-89- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388

[0199] Cells (1.5 x 106) were harvested and divided into 2 portions. 1 / 5 of cells were spun down at 2000 rpm for 4 minutes and then subjected to DNA extraction using the DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer’s protocol. The other 4 / 5 of cells were spun down and permeabilized with digitonin permeabilization buffer (0.025% digitonin, 10mM KCl, 5mM MgCl2, 1mM EDTA, 1mM EGTA, 250mM sucrose, 20mM HEPES, pH 7.2) for 5 minutes on ice, followed by centrifugation at 15,000g for 10 minutes at 4°C. Supernatants were then spun at 100,000g at 4°C for 60 minutes in an ultracentrifuge (Beckman, Optima TM MAX-XP). Following ultracentrifugation, supernatants were taken as the cytosolic fraction from which DNA was purified using the QIAquick Nucleotide Removal Kit (Qiagen) according to the manufacturer’s protocol. Both whole-cell DNA and cytosolic DNA were subjected to quantitative PCR with SYBR green master mix (Thermo Fisher Scientific) on a ViiA 7 Real-Time PCR System (Thermo Fisher Scientific). Quantitative PCR was performed using nuclear DNA primers (Terf) and mitochondrial DNA (mtDNA) primers (D-loop). We confirmed that no nuclear Tert DNA was detected in the cytosolic fractions, indicating that nuclear lysis did not occur using this digitonin method. CT values of mtDNA abundance obtained from the cytosolic fractions were normalized against those obtained from whole-cell extracts. Fold increase of mtDNA efflux was calculated by comparing the normalized cytosolic mtDNA abundance. The primers used for real-time PCR are listed as follows: mouse D-loop, 5'-CATCTCGATGGTATCGGGTC-3' (SEQ ID NO: 48) and 5'-GGTGCGTCTAGACTGTGTGC-3' (SEQ ID NO: 49); mouse Tert, 5'-CTAGCTCATGTGTCAAGACCCTCTT-3' (SEQ ID NO: 50) and 5'-GCCAGCACGTTTCTCTCGTT-3' (SEQ ID NO: 51).

[0200] Dissociation of tumors and lymph nodes

[0201] B16 tumors were dissected and minced in RPMI-1640 (Thermo Fisher Scientific). Tumors were then incubated with Liberase-TL (1.67 Wunsch units / mL, Roche) and DNase-I (0.2 mg / mL, Roche) in RPMI at 37°C for 30 minutes with gentle agitation every 10 minutes before being passed through a 70 pm strainer (Thermo Fisher Scientific) to generate homogeneous single cell suspensions. Live cells were purified using Ficoll-Paque PREMIUM 1.084 (Cytiva) and subsequently used for flow cytometric analyses. The tumor-draining inguinal lymph nodes were harvested and teased with a 27G needle in RPMI -90- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388+ 10% FBS (Thermo Fisher Scientific) to release immune cells, followed by incubation with Collagenase III (Worthington) for 35 minutes at 37°C (agitated once at 15 minutes). Collagenase was then neutralized with EDTA and passed through a 70 pm strainer to generate single cell suspensions for subsequent flow cytometric analyses.

[0202] Flow cytometry analysis

[0203] Cells isolated from tumors or lymph nodes were incubated with surface antibodies cocktail and Fixable Live / Dead dye (eBioscience) in the presence of FcR blocking reagent (Miltenyi Biotec) for 30 minutes on ice. Cells were washed (3X) and fixed in Fix / Perm buffer (Tonbo Biosciences) for at least 30 minutes at 4°C. Next, cells were washed (3X) and stained with the intracellular antibody cocktail for 30 minutes on ice. Finally, samples were washed and acquired using an LSRFortessa flow cytometer (BD Biosciences). Acquired data was analyzed using FlowJo (Tree Star). Antibodies for CD45 (30-F11, 1:200), CD8a (53-6.7, 1:200), CD4 (RMA4-5, 1:200), CDllc (N418, 1:500), I-A / I-E (M5 / 114.15.2, 1:600), F4 / 80 (T45-2342, 1:200), Ly-6C (AL-21, 1:500), and CD44 (IM7, 1:200) were purchased from BD Biosciences, and antibodies for TCR-b (H57-597, 1:200), CDllb (MI / 70, 1:400), and H-2Kb (AF6-88.5, 1:100) were purchased from BioLegend. Antibodies for FOXP3 (FJK-16s, 1:200), Ki-67 (SolA15, 1:300), and APC-conjugated OVA257-264 (SIINFEKL (SEQ ID NO: 53)) peptide bound to H2-Kb monoclonal antibody (eBio25-D1.16, 1:200) were purchased from eBioscience and the antibody for granzyme B (GB 11, 1: 100) was purchased from Thermo Fisher Scientific. The APC-conjugated H2-Kb-OVA tetramer (1: 100) and Alexa647-conjugated H-2Db-EGSRNQDWL (SEQ ID NO: 52) (GP100) tetramer (1: 100) were obtained from the NIH Tetramer Core Facility. Data was analyzed using FlowJo (Tree Star).

[0204] MHC-I quantification

[0205] Cells were incubated with PE-conjugated pan-H2 antibody (BD Biosciences, Clone Ml / 42) in the presence of FcR blocking reagent (Miltenyi Biotec) on ice in the dark for 30 minutes. Cells were then washed (3X) and resuspended in FACS buffer with DAPI (4',6-diamidino-2-phenylindole, Thermo Fisher Scientific). Samples were acquired on an LSRFortessa flow cytometer (BD Biosciences). To quantify the number of surface MHC-I molecules on each cell, the Quantibrite PE Fluorescence Quantification Kit (BD-91- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388Biosciences) was utilized according to manufacturer’s protocol. Quantibrite beads were run on the flow cytometer immediately prior to cell sample acquisition and were run for each independent experiment. Data was analyzed using FlowJo (Tree Star).

[0206] Immunofluorescence analysis

[0207] Cryopreserved OCT blocks were sectioned at 5-10 pm, placed on slides, and maintained at -80°C. The slides were fixed with cold methanol, blocked with 5% serum in PBS for 30 minutes, and incubated overnight with primary antibodies: CDllc (N418, 1:100), F4. / 80 (T45-2342, 1:200), and H-2Kb (AF6-88.5, 1:100). The slides were then incubated with secondary antibodies (1:500, Jackson ImmunoResearch) for 1 hour: Alexa Fluor 647-labelled anti-rat, Alexa Fluor 594-labelled anti-mouse, or Alexa Fluor 488- labelled anti-Armenian hamster After counterstaining with DAPI, the slides were mounted using VECTASHIELD mounting medium ( Vector Laboratories) and imaged on a Zeiss Axio Imager Z2 (20x / 0.8 NA, 40x / 0.9 NA). For each condition, tumor tissue was imaged at ten different fields of view using the same settings. Quantification was performed using Fiji Image! software v2.0.0-rc-65 / 1.54i, calculating the percentage area for each image by the formula: O (area of overlap or yellow signal) / S (area of green fluorescence signal) * 100. The data was plotted using Prism, and statistical significance was calculated using an unpaired Student's Ltest.

[0208] Assessment of calreticulin exposure

[0209] Cells were incubated with rabbit anti-calreticulin antibody (MBL International, JM-3077-100, 1: 100) for 30 minutes on ice. Next, cells were washed and incubated with goat anti-rabbit-AF488 (Thermo Fisher Scientific, 1:200) for 30 minutes on ice. Cells were then washed and stained with DAPI prior to acquisition on an LSRFortessa flow cytometer (BD Biosciences). The geometric mean fluorescence intensity of surface calreticulin on live cells was analyzed using FlowJo (Tree Star).

[0210] Cytokine assessment by ELISA and Luminex

[0211] Tumors were homogenized in Lysing Matrix D tubes (MP Bio) with a Bead Mill 24 (Fisher) in Mammalian Protein Extraction Reagent (M-PER, Thermo Fisher Scientific) supplemented with 150 mM NaCl and complete protease inhibitors (Roche). Cellular debris-92- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388was removed by centrifugation at 12,000g for 10 minutes at 4°C. Supernatant was transferred to a clean tube and spun at 12,000g for 5 minutes at 4°C to remove remaining debris. Protein concentrations in tumor lysates were measured with the Pierce BCA protein assay kit (Thermo Fisher Scientific). Tumor lysates were normalized to 4 mg / mL prior to use in cytokine assays. The IFN-P ELISA (PBL) and 2’, 3’-cGAMP ELISA (Arbor Assays) were performed according to the manufacturers’ protocols.

[0212] Luminex assays were performed by the Immunomonitoring Laboratory at Washington University in St. Louis. Briefly, 50 pL of sample was added to each well containing premixed, washed beads in a 96-well black microtiter plate. The plate was sealed and incubated on an orbital shaker at 650 rpm at room temperature for 30 minutes, then incubated at 4°C overnight on an orbital shaker. After incubation, the plate was washed (3X), followed by incubation with 25 pL of detection antibody mix for 1 hour on a shaker at room temperature. Next, samples were washed (3X) and incubated with 50 pL of SA-PE reagent for 30 minutes on a shaker. Finally, Read buffer was added and the data was collected using the LuminexR FLEXMAP 3DR detection instrument operated with xPONENT Software V4.2 (Luminex Corp). Fifty beads per region for each bead were analyzed for mean fluorescence intensity (MFI) and compared to a 7-point standard curve. A 5-parameter curve fit algorithm was applied to the data using Belysa version 1.1.0 software (Merck EMD Millipore) to calculate the pg / mL of each analyte.

[0213] Single-cell RNA sequencing

[0214] B16 tumors were dissected and dissociated as described above. Cells were stained with DAPI and live cells were sorted using a FACSAria (BD Biosciences) by the Flow Cytometry Core Facility at MSKCC. The single-cell RNA sequencing (scRNA-seq) libraries were prepared by the Single Cell Research Initiative (SRCI) at MSKCC following the user guide manual (CG00052 Rev E) provided by the 10X Genomics and Chromium Single Cell 5' Reagent Kit (v2). Between 5,000 and 10,000 cells were targeted for each sample. Encapsulated cells were subjected to reverse transcription (RT) reaction at. 53°C for 60 minutes. After the R T step, the emulsion droplets were broken and barcoded-cDNA was purified with Dynabeads, followed by 14-cycles of PCR amplification (98°C for 45 s; [98°C for 20 s, 63°C for 30 s, 72°C for 60 s] x 12-cycles; 72°C for 60 s). 50 ng of PCR-amplified -93- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388barcoded cDNA was used for 5' gene expression library construction. PCR-amplified cDNA was fragmented with the reagents provided in the kit and purified with SPRI beads to obtain an average fragment size of 600 bp. Next, the DNA library was ligated to the sequencing adaptor followed by indexing PCR (98°C for 45 s; 14-16 cycles of 98°C for 20 s, 54 °C for 30 s, 72°C for 20 s; 72°C for 1 minute). The resulting DNA library was double-size purified with SPRI beads and sequenced on Illumina NovaSeq S4 platform by the Integrated Genomics Operation (IGO) at MSKCC (R1 - 26 cycles, i7 - 10 cycles, i5 - 10 cycles, R2 -90 cycles).

[0215] Processing of single-cell RNA sequencing libraries and clustering

[0216] The single-cell RNA sequencing files were preprocessed and aligned to the mmlO reference genome (v3.0.0) using Cell Ranger v3.1.0 to obtain a filtered raw count matrix, which was further filtered and processed using Scanpy8. The count matrix was library size normalized and log transformed. Briefly, cells devoid of expression or with >20% mitochondrial gene transcripts were eliminated. Next, to maintain quality and remove doublets, cells with the library size of 2000-4400 transcripts were selected. Lastly, the count matrix was further filtered to remove genes expressed in <10 cells as well as cells with <1000 genes expressed. After filtering, this dataset had 39,360 cells and 18,376 genes. Note, tumor cells were harvested, sequenced, and processed in the same batch. The preprocessed and filtered dataset was then clustered using the Leiden algorithm in PhenoGraph, using 30 principal components (PCs) and 15 nearest neighbors for graph construction9. For annotation purposes, marker genes from a mouse cell atlas 10 in addition to the standard B16 markers DCT, MLANA, and TYRP1 were used, and cells were assigned into broad classes: initial cancer, immunogenic cancer, T cells, myeloid cells, and endothelial cells. A total of 2679 T cells (CD3 -expressing) were re-clustered and annotated using neural topic model (as described below) in addition to the expression profile of standard T cell marker genes. A small cluster of cells that displayed significantly low expression of T cell markers (CD3E, CD3G, CD3D) but high expression of NK cell markers (NCR1, KLRB1C) was labeled as NK cells. Similarly, myeloid cells were separated from the main dataset, re-clustered, and annotated using expression pattern of standard myeloid marker genes.-94- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388

[0217] Topic modeling for single-cell RNA sequencing data

[0218] To understand complicated pathways and study the relationship between the clusters with topic modeling, Latent Dirichlet Allocation (LDA) models, also known as a Grade of Membership (GoM) models, were trained on scRNA-seq data using CountClust vl.18.0 11,12. First, genes that were detected in fewer than 10 cells or more than 95% of cells were removed. Topic models were fitted to the raw counts with the number of topics, K, selected based on the Bayes Factor (BF) value. The weight of topics across single cells, co, was used to evaluate the importance of topics for each cluster of cells. The top n genes for each topic were identified by selecting genes that maximize the minimum KL-divergence of that topic’s relative gene expression compared to other topics, assuming a Poisson distribution. Using the R function gost from gprofiler2 v0.2.1 13, pathway analysis was performed on top n=30 and n=100 genes of each topic in order to interpret their biological significance.

[0219] Differential expression tests

[0220] We performed multiple differential gene expression analyses using the rank_genes_groups() function in scanpy with method=’wilcoxon’. For the T cell clusters enriched in CD8 (0, 1, 2, 3, 4, 8, 10, and 15), differential gene expression was performed by comparing tet-tBID Apafl ^ cells treated with doxycycline for 48 hours to tet-tBID Apafl+ / +B16 cells treated with doxycycline for 48 hours. For myeloid cells, differential gene expression analysis was performed by comparing tet-tBID Apafl ^ cells treated with doxycycline for 48 hours to tet-tBID Apafl+ / +B 16 cells treated with doxycycline for 48 hours in each cluster. Prior to performing differential gene expression analysis in cancer cells, the initial cancer cells and immunogenic cancer cells were reclustered separately. Tumor-immune doublet clusters were identified and removed based on the expression of T cell and myeloid marker genes. Differential gene expression analysis was then performed by comparing non-doublet immunogenic cancer cells from the tet-tBID Apafl ^ sample treated with doxycycline for 48 hours (A5_48hrs) with non-doublet initial cancer cells from the tet-tBID Apafl ^ sample (A5_0hrs).

[0221] Survival and transcriptome analysis of human cutaneous melanoma patients-95- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388

[0222] Normalized RNA-seq counts by trimmed mean of M values (TMM) from the TCGA cutaneous melanoma cohort was downloaded by TCGAbiolinks (v2.22.4). The singscore R package14was used to calculate the single sample baciptosis signature enrichment scores for the TCGA cutaneous melanoma cohort. Patients were divided into two groups: the top 50% with the highest signature enrichment scores (highest 50%) and the bottom 50% with the lowest signature enrichment scores (lowest 50%). Kaplan-Meier survival analysis for these two groups was conducted using the TCGAbiolinks R package15. The relationship between signature scores an APAFl mRNA expression was visualized using a scatter plot. Pearson correlation coefficients and associated p-values were calculated using a two-sided test by ggpubr (v0.6.0). Baciptosis-upregulated genes (FDR < 0.05 and log2(FC) > 0) specifically identified in tumor-infiltrating CD8 T cells, Clq-high monocytes, or CCR7+ dendritic cells via scRNA-seq were further filtered by excluding genes commonly used to annotate these populations 16. Baciptosis-upregulated genes (FDR < 0.05 and log2(FC) > 1) in cancer cells detected by both scRNA-seq and bulk RNA-seq were defined as the baciptosis signature of cancer cells.

[0223] Quantification and statistical analysis

[0224] Statistical significance was determined using two-tailed unpaired parametric Student’s / -test when comparing two groups and two-way ANOVA when comparing three or more groups (Prism 8.0, GraphPad Software). Data were presented as mean ± SD with C < 0.05 considered statistically significant unless otherwise stated. Statistical significance was denoted as *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. When comparing the survival data in Kaplan-Meier analysis, log-rank (Mantel-Cox) test was used (Prism 8.0, GraphPad Software). The number of independent experiments, samples, or events were indicated in the figure legends. No statistical method was used to predetermine sample size. For the in vivo experiments, animals were randomly assigned to experimental groups. The starting tumor burden in the treatment and control groups was similar before treatment. For the in vitro experiments, all samples were analyzed equally with no subsampling; therefore, there was no requirement for randomization. For the in vivo experiments, tumor measurements by digital caliper were performed in a blinded manner.-96- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388The in vitro experiments were not performed in a blinded manner as the investigator needed to know the treatment groups in order to complete the study.1. Weizman, O. E., Adams, N. M., Schuster, I. S., Krishna, C., Pritykin, Y., Lau, C., Degli-Esposti, M. A., Leslie, C. S., Sun, J. C., and O'Sullivan, T. E. (2017). ILC1 Confer Early Host Protection at Initial Sites of Viral Infection. Cell 177, 795-808.e712.10.1016 / j.cell.2017.09.052.2. Inoue- Yamauchi, A., Jeng, P. S., Kim, K., Chen, H.-C., Han, S., Ganesan, Y. T., Ishizawa, K., Jebiwott, S., Dong, Y., and Pietanza, M. C. (2017). Targeting the differential addiction to anti-apoptotic BCL-2 family for cancer therapy. Nature Communications S, ncommsl6078.3. Srinivasula, S. M., Ahmad, M., MacFarlane, M., Luo, Z., Huang, Z., Fernandes-Alnemri, T., and Alnemri, E. S. (1998). Generation of constitutively active recombinant caspases-3 and-6 by rearrangement of their subunits. Journal of Biological Chemistry 273, 10107-10111.4. Snyder, A. G., Hubbard, N. W., Messmer, M. N., Kofman, S. B., Hagan, C. E., Orozco, S. L., Chiang, K., Daniels, B. P., Baker, D., and Oberst, A. (2019). Intratumoral activation of the necroptotic pathway components RIPK1 and RIPK3 potentiates antitumor immunity. Science immunology 4.5. Sanjana, N. E., Shalem, O., and Zhang, F. (2014). Improved vectors and genomewide libraries for CRISPR screening. Nature Methods 77, 783-784. 10.1038 / nmeth.3047.6. Tanaka, K., Yu, H. A., Yang, S., Han, S., Selcuklu, S. D., Kim, K., Ramani, S., Ganesan, Y. T., Moyer, A., Sinha, S., et al. (2021). Targeting Aurora B kinase prevents and overcomes resistance to EGFR inhibitors in lung cancer by enhancing BIM- and PUMA-mediated apoptosis. Cancer Cell 39, 1245-1261. el246. 10.1016 / j.ccell.2021.07.006.7. Wang, G. X., Tu, H.-C., Dong, Y., Skanderup, A. J., Wang, Y., Takeda, S., Ganesan, Y. T., Han, S., Liu, H., and Hsieh, J. J. (2017). ANp63 inhibits oxidative stress-induced cell death, including ferroptosis, and cooperates with the BCL-2 family to promote clonogenic survival. Cell reports 27, 2926-2939.-97- 4920-5495-9747.1Atty. Dkt. No.: 115872-33888. Wolf, F. A., Angerer, P., and Theis, F. J. (2018). SCANPY: large-scale single-cell gene expression data analysis. Genome Biol 19, 15. 10.1186 / sl3059-017-1382-0.9. Levine, J. H., Simonds, E. F., Bendall, S. C., Davis, K. L., Amir el, A. D., Tadmor, M. D., Litvin, O., Fienberg, H. G., Jager, A., Zunder, E. R., et al. (2015). Data-Driven Phenotypic Dissection of AML Reveals Progenitor-like Cells that Correlate with Prognosis. Cell 162, 184-197. 10.1016 / j.cell.2015.05.047.10. Han, X., Wang, R., Zhou, Y., Fei, L., Sun, H., Lai, S., Saadatpour, A., Zhou, Z., Chen, H., Ye, F., et al. (2018). Mapping the Mouse Cell Atlas by Microwell-Seq. Cell 173, 1307.10.1016 / j.cell.2018.05.012.11. Quadrini, K. J., Patti-Diaz, L., Maghsoudlou, J., Cuomo, J., Hedrick, M. N., and McCloskey, T. W. (2021). A flow cytometric assay for HLA-DR expression on monocytes validated as a biomarker for enrollment in sepsis clinical trials. Cytometry B Clin Cytom 100, 103-114.10.1002 / cyto.b.21987.12. M, T. (2012). On Estimation and Selection for Topic Models. AISTATS JMLR 13. Raudvere, U., Kolberg, L., Kuzmin, I., Arak, T., Adler, P., Peterson, H., and Vilo, J. (2019). g: Profiler: a web server for functional enrichment analysis and conversions of gene lists (2019 update). Nucleic Acids Res 47, W191-W198. 10.1093 / nar / gkz369.14. Foroutan, M., Bhuva, D. D., Lyu, R., Horan, K., Cursons, J., and Davis, M. J. (2018). Single sample scoring of molecular phenotypes. BMC bioinformatics 19, 1-10.15. Colaprico, A., Silva, T. C., Olsen, C., Garofano, L., Cava, C., Garolini, D., Sabedot, T. S., Malta, T. M., Pagnotta, S. M., and Castiglioni, I. (2016). TCGAbiolinks: an R / B ioconductor package for integrative analysis of TCGA data. Nucleic acids research 44, e71-e71.16. Chen, B., Khodadoust, M. S., Liu, C. L., Newman, A. M., and Alizadeh, A. A. (2018). Profiling Tumor Infiltrating Immune Cells with CIBERSORT. Methods Mol Biol 1711, 243-259. 10.1007 / 978-l-4939-7493-l_12.Example 10: Engineering programmed cancer cell death models

[0225] To compare the effects of different types of cancer cell death on tumor regression and antitumor immunity, we employed the well-established B16-F10 mouse -98- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388melanoma cell line.33,34To model apoptosis, we engineered a tetracycline (tet)-inducible truncated BID (tBID, an active form of BID14) system in B16 melanoma cells (FIGs. 13A-13C). To model baciptosis, CRISPR / Cas9-mediated KO of ApafL the central component of the apoptosome, was performed in tBID-inducible B16 cells (FIGs. 13A-13C), which mimics frequent silencing of APAF1 in human melanoma.27Notably, tBID induction in parental B16 cells displayed characteristic features of apoptosis such as chromatin condensation with margination along the nuclear membrane and plasma membrane blebbing (FIG. 19A). In contrast, tBID induction mApafM B16 cells displayed necrotic features, including disruption of the plasma membrane and organelles, induction of autophagy and mitophagy, and an increase in lysosomes (FIG. 19B). Consistent with our findings, developmental cell death of interdigital webs mApafM mice also shows morphological features of necrosis rather than apoptosis.35The absence of the characteristic ultrastructural features that define apoptosis in cells undergoing baciptosis along with the novel mtDNA releasing machinery identified (Cheng unpublished data) warrants the introduction of a new nomenclature for this type of cell death. To model necroptosis, we engineered B16 cells expressing tet-inducible RIPK3 fused to a constitutively oligomerizing domain (tet-coRIPK3).6In addition, we generated B16 cells expressing a tet-inducible constitutively active mutant of caspase-336(tet-CASP3CAM) that cleaves GSDME to model pyroptosis (FIGs. 13B and 13C). Consistent with our published data,37baciptosis killed cells at slower kinetics than other types of cell death (FIG. 13B). Induction of tBID and CASP3CAMdifferentially activated caspases to cleave GSDME (FIG. 13C) whereas only CASP3CAMinduction resulted in the engagement of GSDME-dependent release of cytosolic lactate dehydrogenase (LDH) into culture media before cellular demise (FIGs. 20A and 20B). These findings suggest that mitochondrial apoptosis dominates over pyroptosis in tB ID-induced Apafl+ / +cells such that dying apoptotic cells might be phagocytosed before undergoing cell membrane lysis. As reported,4,5induction of coRIPK3 resulted in the phosphorylation of MLKL and NF-KB p65 and this cell death was abrogated by Mikl KO (FIGs. 13C and 20C)

[0226] tBID induction in Apafl~'~ but not Apafl+ / +B 16 cells resulted in the accumulation of 2’, 3’-cGAMP and the release of IFN-P into culture media while mtDNA efflux into the cytosol was detected in both cell lines (FIG. 13D), which is consistent with -99- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388the reported caspase-mediated inactivation of cGAS and IRF3.22Of note, tB ID-induced mtDNA efflux in Apafl+ / +cells occurs almost simultaneously with the onset of annexin- V positivity (FIGs. 13B and 13D), suggesting that this efflux, which happens at the verge of cell death,20may not occur until the cells are phagocytosed in vivo. Neither pyroptosis nor necroptosis induced mtDNA efflux, 2’, 3’-cGAMP accumulation, and IFN-P release into culture media (FIG. 13D). Notably, tB ID-induced mtDNA efflux sustained for an extended period of time in Apafl- / -cells that remain viable for at least 24 hours after tBID induction, leading to robust IFN-P (Ifnb1) upregulation (FIGs. 13B and 13E). As expected, tB ID-induced baciptosis was abrogated by KO of both Bax and Bak (FIGs. 13F and 20D). KO of Cgas impaired tB ID-induced accumulation of 2’, 3’-cGAMP, phosphorylation of STING-TBK1-IRF3, and IFN-P (Ifnb1) upregulation, but did not affect cell death or mtDNA efflux (FIGs. 13F and 13G). We subsequently assessed two common features associated with ICD — extracellular release of ATP and calreticulin exposure on the cell surface, which are known to serve as 'find-me' and 'eat-me' signals, respectively.2,3All types of cell death except baciptosis induced extracellular release of ATP while all types induced calreticulin exposure (FIGs. 20E and 20F).Example 11: Characterizing immunogenic features of programmed cancer cell death in vivo

[0227] We next conducted in vivo characterization of the aforementioned inducible cancer cell death models. Intradermal implantation of tet-tBID Apafl+ / +(apoptosis), tet-tBID Apafl" (baciptosis), tet-CASP3CAM(pyroptosis), or tet-coRIPK3 (necroptosis) B16 cells was performed in syngeneic C57BL / 6J mice. Induction of all these distinct types of cell death led to complete tumor regression (FIG. 13H). To assess antitumor memory, mice bearing regressed tumors following induction of each specific type of cancer cell death were rechallenged with the same cell line on the opposite flanks (FIG. 131). Strikingly, baciptosis protected all mice from tumor rechallenge while apoptosis, pyroptosis, and necroptosis conferred 0%, 25%, and 37.5% protection against tumor rechallenge, respectively (FIGs.131 and 20G). Remarkably, baciptosis-induced immunological memory was long-lasting in that 5 / 8 (62.5%) of mice protected from the first tumor rechallenge were still protected from the second rechallenge 6 months later (FIGs. 13J and 20H). Baciptosis-induced immunological memory was further confirmed in two additional independently generated -100- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388tet-tBID Apafl⁻ / ⁻ B16 cell lines (FIG.201). Although necroptosis-induced immunological memory has been shown in the presence of the model antigen OVA or an immunodominant viral gp70 antigen,5,12,13our data demonstrated that intratumoral induction of necroptosis is less protective than baciptosis against tumor rechallenge in the absence of OVA or gp70.

[0228] Assessment of intratumoral cytokines revealed that baciptosis induced more pro-inflammatory cytokines than apoptosis and necroptosis (FIG. 14A). Caspase 3-mediated pyroptosis also stimulated robust cytokine production (FIG. 14A), which is consistent with GSDME-dependent release of cytosolic contents into the tumor microenvironment (FIG. 20A). Notably, baciptosis not only uniquely induced IFN-P but also upregulated more TNF-a, CCL4, and CCL5 than other cell death modalities (FIGs. 14A and 14B). Flow cytometric analyses of tumor-infiltrating leukocytes revealed that baciptosis induced more effector differentiation of CD8 cytotoxic T lymphocytes marked by granzyme B (GZMB) expression than other cell death modalities (FIGs. 14C and 21A). Necroptosis induced more proliferation but less effector differentiation of CD8 T cells than baciptosis (FIGs. 14C and 23A) All four cell death models led to the activation of conventional natural killer (cNK) cells and an increase of tumor-infiltrating monocytes while apoptosis uniquely increased tumor-infiltrating neutrophils (FIG. 14D).

[0229] To assess the impact of baciptosis versus apoptosis on the generation of tumorspecific CD8 T cells, tet-tBID Apafl+ / +and Apafl- / -B16 cells were transduced with retrovirus expressing the PresentER-mCherry-SIINFEKL (SEQ ID NO: 53) (FIGs. 21B, and 21C).38SIINFEKL (SEQ ID NO: 53) is the H2-Kb-restricted peptide presented from the model antigen OVA that can be used to track the response of tumor-specific CD8 T cells to cell death using an H2-Kb- SIINFEKL (SEQ ID NO: 53) tetramer. Intratumoral induction of baciptosis but not apoptosis led to the expansion and activation of SIINFEKL (SEQ ID NO: 53) -specific CD8 T cells in tumor-draining lymph nodes (FIGs. 14E and 21D). We next investigated whether baciptosis is capable of inducing the expansion of CD8 T cells against GP100, an unmutated melanocyte differentiation antigen expressed by melanoma cells. Indeed, baciptosis but not apoptosis significantly increased GP100-specific CD8 T cells in tumor-draining lymph nodes (FIG. 14F). Together, these findings indicate that baciptosis is capable of inducing robust cross-priming of CD8 T cells in vivo. This is consistent with a report showing that irradiation of caspase9-deficient MC38 colon -101- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388adenocarcinoma cell line can enhance cross-priming of tumor-specific T cell activity in an in vitro bone marrow-derived dendritic cell and T cell co-culture system.25Example 12: Single-cell transcriptome analyses

[0230] To further interrogate the differential effects of cancer cell baciptosis versus apoptosis on tumor-immune interactions, droplet-based 5’ single-cell RNA-seq (scRNA-seq) was performed on tet-tBID Apafl+ / +and Apafl- / -B16 tumors at 0 and 48 hours following tBID induction. In total, we obtained scRNA profiles from 39,360 cells, including cancer, T, myeloid, NK, and endothelial cells (FIG. 22A). Notably, cancer cells in tumors undergoing baciptosis displayed immunogenic signatures, including highly enriched signatures of IFN responses, TNF-a / NF-KB signaling, and IL6 JAK STAT3 signaling (data not shown). In general, baciptosis increased T cell infiltration more than apoptosis whereas apoptosis recruited more myeloid cells (FIG. 22A). Phenograph clustering of T cells revealed 16 clusters, many of which were preferentially induced by either apoptosis or baciptosis (FIGs. 15A and 22B-22D). Among CD8 T cell clusters, CO, C2, C8, and CIO were mainly induced by baciptosis, Cl and C3 by apoptosis, C4 by both baciptosis and apoptosis, whereas C15 was present in all tumors regardless of cell death induction (FIGs.15A, 15B, 22B, and 22C). A subset of T cells had high scores for both T cell and myeloid signatures, which probably represent T cell-myeloid doublets and can be further divided into 4 distinct clusters, C5-7 and C9 (FIG. 15A). Interestingly, all these doublets were induced by cell death; and CD8 T cell-dendritic cell (DC) doublets (C5 and C7) were mainly induced by baciptosis whereas CD8 T cell-macrophage doublets (C6 and C9) were induced by both forms of cell death. (FIG. 15A).

[0231] To quantitatively analyze and compare single-cell gene expression programs across different T cell clusters, we performed topic modeling using Latent Dirichlet allocation (LDA) with 10 topics (FIG. 15C).39,40In the context of scRNA-seq analysis, a topic model generates the gene counts from single-cell transcriptomes from multiple learned topics, where each topic represents a gene expression program corresponding to a multinomial probability over genes.41Overall, baciptosis led to higher expression of T-cell receptor (TCR) components (topic 1) and proliferation (topic 4) in T cells, while apoptosis was associated with oxidative phosphorylation (topic 3) (FIG. 15C). This is especially obvious when comparing CO versus Cl, and C2 versus C3. It is noteworthy that C8, a -102- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388cluster unique to baciptosis (FIG. 15A), was highly enriched for the effector / cytolytic program (topic 7) (FIG. 15C). To further interrogate the differential impact of baciptosis versus apoptosis on tumor-infiltrating CD8 T cells, gene set enrichment analysis (GSEA) was performed on genes that were differentially expressed (false discovery rate (FDR) < 0.05) in CD8 T cells (CO-4, 8, 10, and C15) from baciptotic tumors compared to those from apoptotic tumors. Enrichment of IFN response signatures was identified in CD8 T cells from baciptotic tumors (data not shown), consistent with paracrine signaling due to tumor-intrinsic activation of IFN-P upon baciptosis. Furthermore, CD8 T cells in baciptotic tumors showed enriched signatures of the proteasome, antigen processing and cross-presentation, DNA synthesis, and cell killing (data not shown), all of which could potentiate antitumor immunity. To evaluate the prognostic value of baciptosis-upregulated genes (FDR < 0.05 and log2(fold change; FC) > 0) specifically in tumor-infiltrating CD8 T cells but not in other cell types (data not shown), we assessed the RNA-seq data of cutaneous melanoma from The Cancer Genome Atlas (TCGA). Significantly, high expression of the baciptosis-specific signature of CD8 T cells correlated with improved survival of melanoma patients from the TCGA cohort (FIG. 15D). Notably, we have removed genes commonly used to annotate CD8 T cells from the CD8-specific baciptosis signature in this analysis,42suggesting that the prognostic value is not simply caused by the presence of CD8 T cells.

[0232] Phenograph clustering of myeloid cells revealed 7 clusters, all of which were preferentially increased upon apoptosis (FIGs. 15E, 15F, 22E and 22F). As CO (Clq-high monocyte) and C2 (CCR7+DC1) were two major myeloid populations induced by baciptosis (FIGs. 15F and 22E), we next investigated whether baciptosis induced different gene signatures from apoptosis in these two myeloid populations. In fact, GSEA of differentially expressed genes (FDR < 0.05) in these clusters revealed enrichment of IFN response signatures in baciptotic tumors compared to apoptotic ones (data not shown). Importantly, CCR7+DC1 in baciptotic tumors showed enriched signatures of antigen processing and cross-presentation, DNA replication, and PIK3-AKT-mTOR signaling (data not shown), suggesting that baciptosis may enhance the activity, proliferation, and survival of DCs. In addition, the ER stress signature was enriched in Clq-high monocytes form baciptotic tumors (data not shown). We also investigated whether baciptosis-upregulated genes specifically in these two myeloid populations have potential prognostic value (data -103- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388not shown). To ensure the prognostic value was not simply due to the presence of these myeloid cells, we removed genes commonly used to annotate these populations from the signatures.42Notably, high expression of the baciptosis signature in Clq-high monocytes but not in CCR7+DC1 correlated with improved survival of melanoma patients from the TCGA cohort (FIGs. 15G and 22G). Interestingly, baciptosis upregulated a shared gene set of 56 genes in CD8 T cells, Clq-high monocytes, and CCR7+DC1, and their expression negatively correlated with APAF1 mRNA expression in the TCGA cohort (FIG. 22H), consistent with their upregulation in APAF1 -deficient B16 cells. Lastly, baciptosis-upregulated genes in cancer cells also possess prognostic value (FIG. 15H). In summary, tumor-intrinsic activation of baciptosis appears to trigger a type I IFN autocrine and paracrine circuit involving tumor cells, CD8 T cells, monocytes, and DCs. Moreover, individual immune cell subpopulations appear to differentially activate additional unique programs beyond the type I IFN response in baciptotic tumors.Example 13: Elucidating mechanisms underlying baciptosis-induced immunogenicity

[0233] To determine whether local induction of baciptosis is sufficient to eradicate distant tumors through the so-called abscopal effect, we established tBID-inducible Apafl ^ B16 tumors on left flanks and parental B16 tumors on right flanks of mice (FIG. 16A). Impressively, doxycycline-induced activation of baciptosis on left flank tumors (tet-tBID Apafl ^ Bl 6) almost completely suppressed contralateral tumor growth (FIGs. 16A and 23A), indicating that baciptosis is capable of generating an abscopal effect. To identify the lymphocyte subsets essential for maintaining baciptosis-induced immunological memory, mice with regressed tB ID-induced Apafl ^ tumors were treated with depleting antibodies targeting CD8, CD4, or NK1.1 prior to tumor rechallenge. 70% of the mice depleted of CD8 T cells and 40% of the mice depleted of CD4 T cells were unable to reject tumors upon rechallenge (FIGs. 16B, 16C, and 23B). On the other hand, all the mice depleted of NK1.1+cells successfully rejected rechallenged tumors similar to the isotype controls (FIGs. 16B, 16C, and 23B). These findings indicate that CD8 T cells are the main effectors and CD4 T cells play a lesser but significant role in the maintenance of immunological memory.Consistent with the requirement of both CD8 and CD4 T cells, none of the Rag2~ / ~ mice with regressed baciptotic tumors were protected from rechallenge (FIGs. 16D, 23C, and 23D). Of note, intratumoral induction of baciptosis led to complete tumor regression in -104- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388Rag2~ / ~ mice (FIG. 23C). We subsequently investigated the role of the cGAS-STING-IFN-0 axis in baciptosis-induced immunological memory. Importantly, knockout of Cgas significantly inhibited but did not entirely eliminate the immunological memory phenotype even though it completely abrogated the intratumoral accumulation of 2’, 3’-cGAMP and IFN-P (FIGs. 16E, 16F, and 23E). Similar results were obtained when baciptosis was induced in B16 tumors in Stinggt / gt-deficient (Stinggt / gt) mice (FIGs. 16E and 23E). These data indicate that activation of cGAS-STING-IFN-P signaling contributes significantly yet incompletely to baciptosis-induced immunological memory.

[0234] Our findings that necroptosis is less effective than baciptosis in activating immunological memory in the absence of model antigens suggest that one of the major advantages of baciptosis may be related to IFN-P-induced upregulation of MHC-I for antigen presentation in tumor cells.43Hence, we assessed whether baciptosis increases MHC-I surface expression on tumor cells and to what extent it depends on the induction of IFN-p. Indeed, tBID induction mApafl / _B16 cells increased MHC-I surface expression (FIG. 16G). In contrast, activation of apoptosis, pyroptosis, or necroptosis failed to induce MHC-I expression in B16 cells (FIG. 16H). Interestingly, despite the complete inhibition of IFN-P upon Cgas deletion in baciptotic cells (FIGs. 13F and 16F), loss of cGAS only partially compromised the MHC-I upregulation (FIG. 16G), which mirrored the in vivo phenotype (FIG. 16E). It has been reported that inhibiting NF-KB impairs antitumor effects of caspase-independent cell death.24Given that cGAS-STING signaling has been shown to activate NF-kB that cooperates with IRF3 to induce type I IFNs,44it is conceivable that inhibiting NF-kB could affect the cGAS-STING-IFN-P signaling axis. Notably, necroptosis induced more robust NF-KB activation than baciptosis yet failed to upregulate MHC-I in our system (FIGs. 13C and 16H), suggesting that NF-KB activation per se is insufficient to upregulate MHC-I. Overall, our data indicate the involvement of cGAS-STING-IFN-P-independent signaling events in baciptosis-induced enhancement of MHC-I expression and antitumor immunological memory.

[0235] In addition to cross-presenting antigens derived from dead cells to CD8 T cells, it has been occasionally reported that DCs can acquire and present intact peptide-MHC class I complexes derived from tumor cells to CD8 T cells via a process called “MHC-I cross- -105- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388dressing45,46It is noteworthy that baciptosis not only increased cell surface MHC-I but also upregulated the expression of proteasomal genes, lysosomal proteases, and genes involved in the presentation of peptides by MHC-I in tumor cells (data not shown), which could potentially enhance antigen processing and presentation. Additionally, tumor cells die much slower upon baciptosis than apoptosis, necroptosis, and caspase-3 -mediated pyroptosis (FIG. 13B). Accordingly, we hypothesize that cancer cells undergoing baciptosis are capable of effectively processing and presenting antigens on MHC-I molecules, leading to the ensuing efficient intercellular transfer of intact MHC-I complexes from tumor cells to antigen-presenting cells (APCs). To prove this hypothesis, we implanted tet-tBID Apafl+ / +and Apafl^ B16 cells in B2m ~ mice in which immune cells lack the MHC-I expression. Consistent with our in vitro study (FIGs. 16G and 16H), FACS analyses showed that baciptosis but not apoptosis increased surface H2-Kbexpression in tumor cells (FIGs. 17A and 17B). Importantly, baciptosis but not apoptosis also increased surface H2-Kbin cDCl, cDC2, macrophages, and monocytes (FIGs. 17A and 17B), indicating the transfer of H2-Kbfrom baciptotic tumor cells to APCs. These findings were further confirmed by immunofluorescence analyses showing that CD1 lc+or F4 / 80+APCs from baciptotic tumors in B2m / _mice displayed H2-Kbat the plasma membrane (FIGs. 17C and 17D).

[0236] As the MHC-I of tumor cells is required for cross-dressing but not for crosspresentation mediated priming of CD8 T,45,46we implanted tet-tBID B2m~ / ~Apafl~ / ~ B16 cells in C57BL / 6J mice to assess the functional contribution of MHC-I cross-dressing to baciptosis-induced immunological memory. Following baciptosis-induced tumor regression, mice were rechallenged with B2m+'+B16 cells. Baciptosis-induced immunological memory was significantly impaired by the deletion of B2m in tumor cells during the initial challenge with 62.5% (5 / 8) of mice losing baciptosis-mediated protection (FIGs. 17E and 23F). These findings indicate that intratumoral activation of baciptosis can promote cross-dressing of APCs with tumor-derived peptide-MHC class I complexes, thereby contributing to antitumor memory. Nonetheless, 37.5% (3 / 8) of mice bearing regressed B2m~ / ~Apafl~ / ~ B16 tumors were still protected from tumor rechallenge, indicating that baciptosis is also capable of inducing CD8 T cells priming through classical cross-presentation of tumor-specific antigens by DCs. This is consistent with the observed enrichment of antigen processing and cross-presentation signatures in CCR7+DC1 from baciptotic tumors (data not shown).-106- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388Notably, necroptosis could only enhance cross-presentation,3and conferred 37.5% protection against tumor rechallenge (FIG. 131). In summary, baciptosis effectively activates and expands tumor-specific T cells through a two-pronged strategy that involves both cross-dressing and cross-presentation.Example 14: Pharmacological activation of baciptosis in apoptosome-deficient cancer

[0237] We next sought to assess the feasibility of pharmacological activation of BAK / BAX in tumors. Using high-throughput screening, we have previously identified the combination of venetoclax (a BCL-2 inhibitor) and dinaciclib (a CDK9 inhibitor that downregulates MCL-1 and BCL-XL) as a broadly effective therapeutic strategy for cancer.47Consistently, the combination of venetoclax and dinaciclib killed Apafl+ / +B16 cells faster than Apafl / _B16 cells but only induced IFN-P (Ifnb1) in the latter (FIGs. 18A and 18B).Intriguingly, dinaciclib, an inhibitor of CDK9-mediated transcriptional elongation,47induced the transcription of Ifnb1 when combined with venetoclax in Apafl- / -cells. Flow cytometric analyses of tumor-infiltrating lymphocytes revealed that the combination of venetoclax and dinaciclib induced more GZMB expression and proliferation of CD8 T cellsin Apafl- / -and Apafl+ / +B16 tumors (FIG. 18C), which is analogous to tBID induction (FIG. 14C). Consistent with previous reports,33,34most tumor-infiltrating CD8 T cells expressed PD-1 (FIG. 18C). Of note, anti-PD-1 antibodies including nivolumab and pembrolizumab constitute the core ICB strategy in modern antitumor immunotherapy.1

[0238] We then compared the in vivo antitumor efficacy of this combination therapy against Apafl+ / +versus Apafl ^ B16 melanoma as well as assessed its potential to enhance the response to anti-PD-1. While neither anti-PD-1 nor the combination of venetoclax and dinaciclib provided benefits to mice bearing Apafl+ / +B16 tumors, the combination of anti- PD-1, venetoclax, and dinaciclib slightly prolonged the survival of these mice (FIGs. 18D and 24A). All these therapies had more effects on mice bearing Apafl ^ B16 tumors than those with Apafl+ / +B16 tumors (FIGs. 18D, 18E, 24A, and 24B). Strikingly, 70% (7 / 10) of mice bearing Apafl ^ B16 tumors were cured by the combination of anti-PD-1, venetoclax, and dinaciclib, whereas the combination of venetoclax and dinaciclib cured 25% (2 / 8) of mice, and anti-PD-1 alone showed no survival benefits (FIGs. 18E and 24B).Similar results were obtained in another murine melanoma model YUMM1.7 that harbors the BrafV600Emutation (FIGs. 18F, 18G, 24C, and 24D). 72.7% (8 / 11) of mice bearing -107- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388Apafl ^ YUMM1.7 tumors were cured by the combination of anti-PD-1, venetoclax, and dinaciclib, whereas the combination of venetoclax and dinaciclib cured 40% (4 / 10) of mice (FIGs. 18G and 24D)

[0239] Based on our reported findings that tBID is more potent than the inhibition of BCL-2 / BCL-XL / MCL-1 in activating BAK / BAX,48it is conceivable that tBID is more effective than the combination of venetoclax and dinaciclib in causing tumor regression (FIGs. 13H and 18E). Notably, inhibitors of anti-apoptotic BCL-2 members displace BID / BIM / PUMA from BCL-2 / BCL-XL / MCL-1 to activate BAK / BAX indirectly.15 16Consistent with the potent activation of BAK / BAX by tBID, tBID induction is sufficient to induce regression of both Apafl+ / +and Apafl- / -tumors (FIG. 13H). Although the combination of venetoclax and dinaciclib has minimum antitumor activity against Apafl+ / +tumors, it can activate immunogenic baciptosis in Apafl- / -tumors, which recruits activated PD1+CD8 T cells to tumors and thereby sensitizes tumors to anti-PD-1 therapy (FIG. 18).Consequently, 25-40% of mice bearing APAF1 -deficient melanomas were cured by venetoclax / dinaciclib (FIGs. 18E and 18G), whereas none of the mice bearing APAF1-proficient melanomas were cured (FIGs. 18D and 18F). The addition of anti-PD-1 further increased the cure rate to 70-72% in mice bearing APAF1 -deficient melanomas whereas anti-PD-1 alone had no anti -tumor effect (FIGs. 18E and 18G). Overall, these data strongly support that pharmacological activation of baciptosis by targeting the BCL-2 family can sensitize apoptosome-defective cancers to ICB, thereby leading to tumor eradication.EQUIVALENTS

[0240] The present technology is not to be limited in terms of the particular embodiments described in this application, which are intended as single illustrations of individual aspects of the present technology. Many modifications and variations of this present technology can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and apparatuses within the scope of the present technology, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to fall within the scope of the present technology. It is to be understood that this present technology is not limited to particular methods, reagents,-108- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0241] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0242] As will be understood by one skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a nonlimiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can be subsequently broken down into subranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 cells refers to groups having 1, 2, or 3 cells. Similarly, a group having 1-5 cells refers to groups having 1, 2, 3, 4, or 5 cells, and so forth.

[0243] All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.REFERENCES1. Wang, X. (2001). The expanding role of mitochondria in apoptosis. Genes Dev 15, 2922-2933.2. Green, D. R., and Kroemer, G. (2004). The pathophysiology of mitochondrial cell death. Science 305, 626-629. 10.1126 / science.1099320.3. Wei, M. C., Zong, W. X., Cheng, E. H., Lindsten, T., Panoutsakopoulou, V., Ross, A. J., Roth, K. A., MacGregor, G. R., Thompson, C. B., and Korsmeyer, S. J. (2001).-109- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292, 727-730. 10.1126 / science.1059108.4. Cheng, E. H., Wei, M. C., Weiler, S., Flavell, R. A., Mak, T. W., Lindsten, T., and Korsmeyer, S. J. (2001). BCL-2, BCL-X(L) sequester BH3 domain-only molecules preventing BAX- and BAK-mediated mitochondrial apoptosis. Mol Cell 8, 705-711.10.1016 / s1097-2765(01)00320-3.5. Youle, R. J., and Strasser, A. (2008). The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9, 47-59. 10.1038 / nrm2308.6. Jeng, P. S., Inoue- Yamauchi, A., Hsieh, J. J., and Cheng, E. H. (2018). BH3-Dependent and Independent Activation of BAX and BAK in Mitochondrial Apoptosis. Curr Opin Physiol 3, 71-81. 10.1016 / j.cophys.2018.03.005.7. Letai, A., Bassik, M. C., Walensky, L. D., Sorcinelli, M. D., Weiler, S., and Korsmeyer, S. J. (2002). Distinct BH3 domains either sensitize or activate mitochondrial apoptosis, serving as prototype cancer therapeutics. Cancer Cell 2, 183-192. 10.1016 / s1535-6108(02)00127-7.8. Chen, L., Willis, S. N., Wei, A., Smith, B. J., Fletcher, J. I., Hinds, M. G., Colman, P. M., Day, C. L., Adams, J. M., and Huang, D. C. (2005). Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function. Mol Cell 17, 393-403. 10.1016 / j.molcel.2004.12.030.9. Kim, H., Rafiuddin-Shah, M., Tu, H. C., Jeffers, J. R., Zambetti, G. P., Hsieh, J. J., and Cheng, E. H. (2006). Hierarchical regulation of mitochondrion-dependent apoptosis by BCL-2 subfamilies. Nat Cell Biol 8, 1348-1358. 10.1038 / ncb1499.10. Kim, H., Tu, H. C., Ren, D., Takeuchi, O., Jeffers, J. R., Zambetti, G. P., Hsieh, J. J., and Cheng, E. H. (2009). Stepwise activation of BAX and BAK by tBID, BIM, and PUMA initiates mitochondrial apoptosis. Mol Cell 36, 487-499.10.1016 / j.molcel.2009.09.030.11. Gavathiotis, E., Suzuki, M., Davis, M. L., Pitter, K., Bird, G. H., Katz, S. G., Tu, H. C., Kim, H., Cheng, E. H., Tjandra, N., and Walensky, L. D. (2008). BAX activation is initiated at a novel interaction site. Nature 455, 1076-1081. 10.1038 / nature07396. 12. Ren, D., Tu, H. C., Kim, H., Wang, G. X., Bean, G. R., Takeuchi, O., Jeffers, J. R., Zambetti, G. P., Hsieh, J. J., and Cheng, E. H. (2010). BID, BIM, and PUMA are essential for activation of the BAX- and BAK-dependent cell death program. Science 330, 1390-1393. 10.1126 / science.1190217.13. Edwards, A. L., Gavathiotis, E., LaBelle, J. L., Braun, C. R., Opoku-Nsiah, K. A., Bird, G. H., and Walensky, L. D. (2013). Multimodal interaction with BCL-2 family proteins underlies the proapoptotic activity of PUMA BH3. Chem Biol 20, 888-902.10.1016 / j.chembiol.2013.06.007.14. Leshchiner, E. S., Braun, C. R., Bird, G. H., and Walensky, L. D. (2013). Direct activation of full-length proapoptotic BAK. Proc Natl Acad Sci U S A 110, E986-995.10.1073 / pnas.1214313110.15. Chen, H. C., Kanai, M., Inoue- Yamauchi, A., Tu, H. C., Huang, Y., Ren, D., Kim, H., Takeda, S., Reyna, D. E., Chan, P. M., et al. (2015). An interconnected hierarchical -110- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388model of cell death regulation by the BCL-2 family. Nat Cell Biol 77, 1270-1281.10.1038 / ncb3236.16. Czabotar, P. E., Westphal, D., Dewson, G., Ma, S., Hockings, C., Fairlie, W. D., Lee, E. F., Yao, S., Robin, A. Y., Smith, B. J., et al. (2013). Bax crystal structures reveal how BH3 domains activate Bax and nucleate its oligomerization to induce apoptosis. Cell 752, 519-531. 10.1016 / j.cell.2012.12.031.17. Inoue- Yamauchi, A., Jeng, P. S., Kim, K., Chen, H. C., Han, S., Ganesan, Y. T., Ishizawa, K., Jebiwott, S., Dong, Y., Pietanza, M. C., et al. (2017). Targeting the differential addiction to anti-apoptotic BCL-2 family for cancer therapy. Nat Commun 8, 16078. 10.1038 / ncommsl6078.18. Adams, J. M., and Cory, S. (2018). The BCL-2 arbiters of apoptosis and their growing role as cancer targets. Cell Death Differ 25, 27-36. 10.1038 / cdd.2017.161.19. Diepstraten, S. T., Anderson, M. A., Czabotar, P. E., Lessene, G., Strasser, A., and Kelly, G. L. (2022). The manipulation of apoptosis for cancer therapy using BH3- mimetic drugs. Nat Rev Cancer 22, 45-64. 10.1038 / s41568-021-00407-4.20. Roberts, A. W., Davids, M. S., Pagel, J. M., Kahl, B. S., Puvvada, S. D., Gerecitano, J. F., Kipps, T. J., Anderson, M. A., Brown, J. R., Gressick, L., etal. (2016). Targeting BCL2 with Venetoclax in Relapsed Chronic Lymphocytic Leukemia. N Engl J Med 374, 311-322. 10.1056 / NEJMoal513257.21. Roberts, A. W., Wei, A. H., and Huang, D. C. S. (2021). BCL2 and MCL1 inhibitors for hematologic malignancies. Blood 138, 1120-1136. 10.1182 / blood.2020006785. 22. Kotschy, A., Szlavik, Z., Murray, J., Davidson, J., Maragno, A. L., Le Toumelin- Braizat, G., Chanrion, M., Kelly, G. L., Gong, J. N., Moujalled, D. M., et al. (2016). The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models. Nature 538, 477-482. 10.1038 / naturel9830.23. White, M. J., McArthur, K., Metcalf, D., Lane, R. M., Cambier, J. C., Herold, M. J., van Delft, M. F., Bedoui, S., Lessene, G., Ritchie, M. E., et al. (2014). Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN production. Cell 159, 1549- 1562. 10.1016 / j.cell.2014.11.036.24. Rongvaux, A., Jackson, R., Harman, C. C., Li, T., West, A. P., de Zoete, M. R., Wu, Y., Yordy, B., Lakhani, S. A., Kuan, C. Y., et al. (2014). Apoptotic caspases prevent the induction of type I interferons by mitochondrial DNA. Cell 159, 1563-1577.10.1016 / j. cell.2014.11.037.25. McArthur, K., Whitehead, L. W., Heddleston, J. M., Li, L., Padman, B. S., Oorschot, V., Geoghegan, N. D., Chappaz, S., Davidson, S., San Chin, H., et al. (2018). BAK / BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis. Science 359. 10.1126 / science.aao6047.26. Riley, J. S., Quarato, G., Cloix, C., Lopez, J., O'Prey, J., Pearson, M., Chapman, J., Sesaki, H., Carlin, L. M., Passos, J. F., et al. (2018). Mitochondrial inner membrane permeabilisation enables mtDNA release during apoptosis. Embo j 37.10.15252 / embj.201899238.4920-5495-9747.1Atty. Dkt. No.: 115872-338827. Ning, X., Wang, Y., Jing, M., Sha, M., Lv, M., Gao, P., Zhang, R., Huang, X., Feng, J. M., and Jiang, Z. (2019). Apoptotic Caspases Suppress Type I Interferon Production via the Cleavage of cGAS, MAVS, and IRF3. Mol Cell 77, 19-31. el7.10.1016 / j.molcel.2019.02.013.28. Marchi, S., Guilbaud, E., Tait, S. W., Yamazaki, T., and Galluzzi, L. (2023).Mitochondrial control of inflammation. Nature Reviews Immunology 23, 159-173.29. Schafer, Z. T., and Kombluth, S. (2006). The apoptosome: physiological, developmental, and pathological modes of regulation. Dev Cell 10, 549-561.10.1016 / j.devcel.2006.04.008.30. Sanchis, D., Mayorga, M., Ballester, M., and Cornelia, J. X. (2003). Lack of Apaf-1 expression confers resistance to cytochrome c-driven apoptosis in cardiomyocytes. Cell Death Differ 10, 977-986. 10.1038 / sj.cdd.4401267.31. Johnson, C. E., Huang, Y. Y., Parrish, A. B., Smith, M. I., Vaughn, A. E., Zhang, Q., Wright, K. M., Van Dyke, T., Wechsler-Reya, R. J., Kornbluth, S., and Deshmukh, M. (2007). Differential Apaf-1 levels allow cytochrome c to induce apoptosis in brain tumors but not in normal neural tissues. Proc Natl Acad Sci U S A 104, 20820-20825.10.1073 / pnas.0709101105.32. Wolter, K. G., Hsu, Y. T., Smith, C. L., Nechushtan, A., Xi, X. G., and Youle, R. J.(1997). Movement of Bax from the cytosol to mitochondria during apoptosis. J Cell Biol 139, 1281-1292. 10.1083 / jcb.139.5.1281.33. Baines, C. P., Kaiser, R. A., Purcell, N. H., Blair, N. S., Osinska, H., Hambleton, M. A., Brunskill, E. W., Sayen, M. R., Gottlieb, R. A., Dorn, G. W., et al. (2005). Loss of cyclophilin D reveals a critical role for mitochondrial permeability transition in cell death. Nature 434, 658-662. 10.1038 / nature03434.34. Brdiczka, D. G., Zorov, D. B., and Sheu, S. S. (2006). Mitochondrial contact sites: their role in energy metabolism and apoptosis. Biochim Biophys Acta 1762, 148-163.10.1016 / j.bbadis.2005.09.007.35. Cheng, E. H., Sheiko, T. V., Fisher, J. K., Craigen, W. J., and Korsmeyer, S. J. (2003).VDAC2 inhibits BAK activation and mitochondrial apoptosis. Science 301, 513-517.10.1126 / science.1083995.36. Hauseman, Z. J., Harvey, E. P., Newman, C. E., Wales, T. E., Bucci, J. C., Mintseris, J., Schweppe, D. K., David, L., Fan, L., Cohen, D. T., et al. (2020). Homogeneous Oligomers of Pro-apoptotic BAX Reveal Structural Determinants of Mitochondrial Membrane Permeabilization. Mol Cell 79, 68-83. e67. 10.1016 / j.molcel.2020.05.029.37. Baines, C. P., Kaiser, R. A., Sheiko, T., Craigen, W. J., and Molkentin, J. D. (2007).Voltage-dependent anion channels are dispensable for mitochondrial -dependent cell death. Nat Cell Biol 9, 550-555. 10.1038 / ncbl575.38. Ren, D., Kim, H., Tu, H. C., Westergard, T. D., Fisher, J. K., Rubens, J. A., Korsmeyer, S. J., Hsieh, J. J., and Cheng, E. H. (2009). The VDAC2-BAK rheostat controls thymocyte survival. Sci Signal 2, ra48. 10.1126 / sci signal.2000274.-112- 4920-5495-9747.1Atty. Dkt. No.: 115872-338839. Setoguchi, K., Otera, H., and Mihara, K. (2006). Cytosolic factor- and TOM- independent import of C-tail-anchored mitochondrial outer membrane proteins. Embo j 25, 5635-5647. 10.1038 / sj.emboj.7601438.40. Hosoi, K. I., Miyata, N., Mukai, S., Furuki, S., Okumoto, K., Cheng, E. H., and Fujiki, Y. (2017). The VDAC2-BAK axis regulates peroxisomal membrane permeability. J Cell Biol 276, 709-722. 10.1083 / jcb.201605002.41. van Delft, M. F., Chappaz, S., Khakham, Y., Bui, C. T., Debrincat, M. A., Lowes, K. N., Brouwer, J. M., Grohmann, C., Sharp, P. P., Dagley, L. F., et al. (2019). A small molecule interacts with VDAC2 to block mouse BAK-driven apoptosis. Nat Chem Biol 75, 1057-1066. 10.1038 / s41589-019-0365-8.42. Chautan, M., Chazal, G., Cecconi, F., Gruss, P., and Golstein, P. (1999). Interdigital cell death can occur through a necrotic and caspase-independent pathway. Curr Biol 9, 967-970. 10.1016 / s0960-9822(99)80425-4.43. Merkwirth, C., and Langer, T. (2009). Prohibitin function within mitochondria:essential roles for cell proliferation and cristae morphogenesis. Biochim Biophys Acta 7793, 27-32. 10.1016 / j.bbamcr.2008.05.013.44. Ko, K. S., Tomasi, M. L., Iglesias- Ara, A., French, B. A., French, S. W., Ramani, K., Lozano, J. J., Oh, P., He, L., Stiles, B. L., et al. (2010). Liver-specific deletion of prohibitin 1 results in spontaneous liver injury, fibrosis, and hepatocellular carcinoma in mice. Hepatology 52, 2096-2108. 10.1002 / hep.23919.45. Schier, A. C., and Taatjes, D. J. (2020). Structure and mechanism of the RNA polymerase II transcription machinery. Genes & development 34, 465-488.46. Tan, W., and Colombini, M. (2007). VDAC closure increases calcium ion flux.Biochim Biophys Acta 1768, 2510-2515. 10.1016 / j.bbamem.2007.06.002.47. Israelson, A., Abu-Hamad, S., Zaid, H., Nahon, E., and Shoshan-Barmatz, V. (2007).Localization of the voltage-dependent anion channel- 1 Ca2+-binding sites. Cell Calcium 41, 235-244. 10.1016 / j.ceca.2006.06.005.48. Zhao, Y., Araki, S., Wu, J., Teramoto, T., Chang, Y. F., Nakano, M., Abdelfattah, A. S., Fujiwara, M., Ishihara, T., Nagai, T., and Campbell, R. E. (2011). An expanded palette of genetically encoded Ca2+indicators. Science 333, 1888-1891.10.1126 / science.1208592.49. Kim, J., Gupta, R., Blanco, L. P., Yang, S., Shteinfer-Kuzmine, A., Wang, K., Zhu, J., Yoon, H. E., Wang, X., Kerkhofs, M., et al. (2019). VDAC oligomers form mitochondrial pores to release mtDNA fragments and promote lupus-like disease. Science 366, 1531-1536. 10.1126 / science. aav4011.50. Cowan, A. D., Smith, N. A., Sandow, J. J., Kapp, E. A., Rustam, Y. H., Murphy, J. M., Brouwer, J. M., Bernardini, J. P., Roy, M. J., Wardak, A. Z., et al. (2020). BAK core dimers bind lipids and can be bridged by them. Nat Struct Mol Biol 27, 1024-1031.10.1038 / s41594-020-0494-5.51. Lartigue, L., Kushnareva, Y., Seong, Y., Lin, H., Faustin, B., and Newmeyer, D. D.(2009). Caspase-independent mitochondrial cell death results from loss of respiration,-113- 4920-5495-9747.1Atty. Dkt. No.: 115872-3388not cytotoxic protein release. Mol Biol Cell 20, 4871-4884. 10.1091 / mbc.e09-07- 0649.52. Xian, H., Watari, K., Sanchez-Lopez, E., Offenberger, J., Onyuru, J., Sampath, H., Ying, W., Hoffman, H. M., Shadel, G. S., and Karin, M. (2022). Oxidized DNA fragments exit mitochondria via mPTP- and VDAC-dependent channels to activate NLRP3 inflammasome and interferon signaling. Immunity 55, 1370-1385. el378.10.1016 / j.immuni.2022.06.007.53. Ujwal, R., Cascio, D., Colletier, J. P., Faham, S., Zhang, J., Toro, L., Ping, P., and Abramson, J. (2008). The crystal structure of mouse VDAC1 at 2.3 A resolution reveals mechanistic insights into metabolite gating. Proc Natl Acad Sci U S A 105, 17742- 17747. 10.1073 / pnas.0809634105-114- 4920-5495-9747.1

Claims

Atty. Dkt. No.: 115872-3388WHAT IS CLAIMED IS1. A method for treating cancer in a subject in need thereof comprising administering to the subject an effective amount of an Apafl inhibitor.

2. The method of claim 1, further comprising separately, sequentially or simultaneously administering at least one additional anti-cancer therapy to the subject.

3. A method for enhancing efficacy of an anti-cancer therapy in a subject in need thereof comprising administering to the subject an effective amount of an Apafl inhibitor.

4. The method of claim 2 or 3, wherein the anti-cancer therapy comprises one or more of chemotherapy, radiotherapy, adoptive cell therapy, immune checkpoint blockade therapy or targeted therapy.

5. The method of any one of claims 1-4, wherein the Apafl inhibitor is a small molecule, or an inhibitory RNA that targets Apafl.

6. The method of claim 5, wherein the small molecule is ZYZ-488, QM31 (SVT016426), UCN-01, SVT017686, SVT017923, SVT016448, or N-alkylglycine trimers.

7. The method of claim 5, wherein the inhibitory RNA that targets Apafl is a siRNA, a shRNA, an antisense oligonucleotide, or a sgRNA.

8. The method of any one of claims 1-7, wherein the cancer is selected from among adrenal cancers, bladder cancers, blood cancers, bone cancers, brain cancers, breast cancers, carcinoma, cervical cancers, colon cancers, colorectal cancers, corpus uterine cancers, ear, nose and throat (ENT) cancers, endometrial cancers, esophageal cancers, gastrointestinal cancers, head and neck cancers, Hodgkin's disease, intestinal cancers, kidney cancers, larynx cancers, leukemias, liver cancers, lymph node cancers, lymphomas, lung cancers, melanomas, mesothelioma, myelomas, nasopharynx cancers, neuroblastomas, nonHodgkin's lymphoma, oral cancers, ovarian cancers, pancreatic cancers, penile cancers, pharynx cancers, prostate cancers, rectal cancers, sarcoma, seminomas, skin cancers, stomach cancers, teratomas, testicular cancers, thyroid cancers, uterine cancers, vaginal cancers, vascular tumors, and metastases thereof.-115- 4920-5495-9747.1Atty. Dkt. No.: 115872-33889. The method of any one of claims 1-8, wherein the Apafl inhibitor is administered orally, topically, intranasally, systemically, intravenously, subcutaneously, intraperitoneally, intradermally, intraocularly, iontophoretically, transmucosally, or intramuscularly.

10. The method of any one of claims 4-9, wherein the chemotherapy comprises one or more of alkylating agents, topoisomerase inhibitors, endoplasmic reticulum stress inducing agents, antimetabolites, mitotic inhibitors, nitrogen mustards, nitrosoureas, alkylsulfonates, platinum agents, taxanes, vinca agents, anti-estrogen drugs, aromatase inhibitors, ovarian suppression agents, cytostatic alkaloids, cytotoxic antibiotics, endocrine / hormonal agents, or bisphosphonate therapy agents.

11. The method of any one of claims 4-10, wherein the chemotherapy comprises one or more of cyclophosphamide, fluorouracil (or 5 -fluorouracil or 5-FU), methotrexate, edatrexate (10-ethyl-10-deaza-aminopterin), thiotepa, carboplatin, cisplatin, taxanes, paclitaxel, protein-bound paclitaxel, docetaxel, vinorelbine, tamoxifen, raloxifene, toremifene, fulvestrant, gemcitabine, irinotecan, ixabepilone, temozolmide, topotecan, vincristine, vinblastine, eribulin, mutamycin, capecitabine, anastrozole, exemestane, letrozole, leuprolide, abarelix, buserlin, goserelin, megestrol acetate, risedronate, pamidronate, ibandronate, alendronate, denosumab, zoledronate, trastuzumab, tykerb, anthracyclines (e.g., daunorubicin and doxorubicin), cladribine, midostaurin, bevacizumab, oxaliplatin, melphalan, etoposide, mechlorethamine, bleomycin, microtubule poisons, annonaceous acetogenins, chlorambucil, ifosfamide, streptozocin, carmustine, lomustine, busulfan, dacarbazine, temozolomide, altretamine, 6-mercaptopurine (6-MP), cytarabine, floxuridine, fludarabine, hydroxyurea, pemetrexed, epirubicin, idarubicin, SN-38, ARC, NPC, campothecin, 9-nitrocamptothecin, 9-aminocamptothecin, rubifen, gimatecan, diflomotecan, BN80927, DX-8951f, MAG-CPT, amsacrine, etoposide phosphate, teniposide, azacitidine (Vidaza), decitabine, accatin III, 10-deacetyltaxol, 7-xylosyl-10-deacetyltaxol, cephalomannine, 10-deacetyl-7-epitaxol, 7-epitaxol, 10-deacetylbaccatin III, 10-deacetyl cephalomannine, streptozotocin, nimustine, ranimustine, bendamustine, uramustine, estramustine, mannosulfan, camptothecin, exatecan, lurtotecan, lamellarin D9-aminocamptothecin, ellipticines, aurintricarboxylic acid, HU-331, or combinations thereof.-116- 4920-5495-9747.1Atty. Dkt. No.: 115872-338812. The method of any one of claims 4-11, wherein the immune checkpoint blockade therapy comprises one or more of an anti-PD-1 antibody, an anti-PD-Ll antibody, an anti-PD-L2 antibody, an anti-CTLA-4 antibody, an anti-TIM3 antibody, an anti-4- IBB antibody, an anti-CD73 antibody, an anti-GITR antibody, or an anti-LAG-3 antibody.

13. The method of any one of claims 4-12, wherein the immune checkpoint blockade therapy comprises one or more of cemiplimab, tremelimumab, ipilimumab, nivolumab, pidilizumab, lambrolizumab, pembrolizumab, envafolimab, atezolizumab, avelumab, durvalumab, dostarlimab, MPDL3280A, BMS-936559, MEDI-4736, MSB 00107180, AMP-224, MDX-1105, arelumab, IMP321, MGA271, BMS-986016, lirilumab, urelumab, PF-05082566, IPH2101, MEDI-6469, CP-870,893, Mogamulizumab, Varlilumab, Galiximab, AMP-514, AUNP 12, Indoximod, NLG-919, INCB024360, CD80, CD86, ICOS (inducible T-cell costimulatory), DLBCL (diffuse large B-cell lymphoma) inhibitors, BTLA (B and T lymphocyte attenuator), PDR001, or any combination thereof.

14. The method of any one of claims 4-13, wherein the targeted therapy comprises one or more of VEGF / VEGFR inhibitors, EGF / EGFR inhibitors, PARP inhibitors, BCL-2 inhibitors, or CDK9 inhibitors.

15. The method of any one of claims 4-14, wherein the targeted therapy comprises one or more of Bevacizumab, nimotuzumab, buparlisib, pilaralisib, sonolisib, paxalisib, dactolisib, voxtalisib, PQR309, AMG232, venetoclax, dinaciclib, ribociclib, dasatinib, imatinib, or rindopepimut.

16. The method of any one of claims 4-15, wherein the adoptive cell therapy comprises one or more of CAR T-cell therapy, tumor-infiltrating lymphocyte (TIL) therapy, T-cell receptor (TCR) therapy, natural killer (NK) cell therapy, or dendritic cell therapy.-117- 4920-5495-9747.1

Images