Integrated multiomics identifies inhibitors of apoptosis (IAPs) and death-inducing signaling complex (DISC) components as novel vulnerabilities to overcome immune evasion in TP53-inactivated Acute Myeloid Leukemia

Abstract

Abstract TP53-inactivated (TP53-mutant or deficient) acute myeloid leukemia (AML) is a biologically and clinically aggressive subtype associated with profound treatment resistance, immune evasion, and dismal outcomes. Notably, AML patients with TP53 mutations have limited benefit from standard induction chemotherapy or from recently approved venetoclax-based combination regimens. The consistent failure of these therapies to induce durable remissions underscores the urgent need for alternative treatment strategies. Given the central role of T cells and natural killer (NK) cells in mediating anti-leukemic immunity, developing immunotherapeutic approaches that can overcome immune resistance in TP53-inactivated AML represents a promising avenue to improve outcomes for this high-risk patient population. To test the hypothesis that TP53 inactivation confers resistance to T/NK cell cytotoxicity in AML, we engineered TP53-wildtype (WT), knockout (KO), or mutant isogenic AML cells engineered to express a model antigen (LMP2A) with primary human LMP2A-targeted TCR-T/CAR-T cells, or NK-92 and primary human NK cells as a model for NK cytotoxicity. TP53-inactivated AML cells displayed marked resistance to both LMP2A-targeted TCR-T/CAR-T, as well as to NK-92 and primary NK cell-mediated cytotoxicity compared to their TP53 WT counterparts. Further comparison among isogenic MOLM-13 cells carrying one of six different TP53-hotspot mutants (R248Q, M237I, Y220C, R175H, R273H, R282W) identified the R248Q mutant as the most resistant to immune killing. In addition to impaired cytotoxicity, TP53-inactivated AML cells suppressed T cell proliferation, activation, and cytokine secretion, while promoting an exhausted T cell phenotype. To uncover the mechanisms driving immune resistance in TP53-inactivated AML, we performed functional genomic screening by performing unbiased genome-wide CRISPR KO screens in TP53-inactivated and WT isogenic MOLM-13 cells under LMP2A TCR-T cell or NK-92-mediated immune pressure. Subsequent integration of CRISPR screen data with bulk transcriptomics and total proteomic profiling conducted on MOLM-13 isogenic cells with TP53-WT, R248Q mutation, or KO identified key regulators of immune susceptibility. Notably, inhibitors of apoptosis (IAPs: BIRC2/cIAP1, BIRC3/cIAP2, XIAP) and components of the death-inducing signaling complex (DISC)—comprising FADD, caspase-8, and cFLIP—emerged as top hits, revealing extrinsic apoptosis as a key dependency of TP53-inactivated AML. Targeting IAPs enhances this apoptotic pathway, while inhibition of caspase activity with emricasan, alone or in combination with IAP antagonists, shifts cell death toward immunogenic necroptosis, thereby potentiating immune killing.Functional validation using targeted individual CRISPR KO and pharmacological inhibitors confirmed that antagonizing IAPs restored T and NK cell–mediated killing of TP53-inactivated AML. We found that IAP inhibitors birinapant and tolinapant, as well as IAP PROTAC-degrader CST-626, potently sensitized TP53-inactivated AML to CAR-T/TCR-T and NK-92-mediated cytotoxicity. Strikingly, emricasan, a clinically advanced caspase inhibitor, was also able to sensitize TP53-inactivated AML cells to TCR-T, CAR-T, and NK-mediated cytotoxicity. Moreover, combining emricasan with IAP antagonists synergistically enhanced immune-mediated killing in TP53-inactivated AML. Mechanistically, emricasan inhibits the formation of the caspase-8/cFLIP heterodimer and subsequent DISC complex, thereby promoting non-apoptotic, immunogenic cell death in conjunction with IAP antagonists. This approach restored immune killing and amplified effector T/NK cell responses against TP53-inactivated AML. Collectively, TP53 inactivation drives immune evasion in AML by rewiring death receptor signaling and inducing T cell dysfunction. We employ integrated multiomics and functional assays to identify the IAPs, as well as the DISC—comprising FADD, caspase-8, and cFLIP—as critical, targetable checkpoints to modulate immune effector–mediated killing in TP53-inactivated AML. Pharmacological inhibition of the caspase 8-cFLIP heterodimer with emricasan alone, or combined with IAP antagonists, effectively overcomes this checkpoint and sensitizes AML cells to immune cytotoxicity. These findings establish a strong preclinical rationale for combining cell death–promoting agents with T or NK cell–based immunotherapies to overcome immune resistance in TP53-mutated high-risk AML subset.