INHIBITORS OF IRE1A ENHANCE VENETOCLAX ACTIVITY IN DE NOVO AND SECONDARY ACUTE MYELOID LEUKEMIA

Venetoclax (VEN) has emerged as a promising option for AML patients who are ineligible for or have relapsed after standard-of-care therapies. Although ~75% initially respond, most eventually relapse due to acquired resistance, underscoring the urgent need to identify mechanisms of resistance and strategies to overcome them. The central goal of this study is to identify chemical strategies for improving VEN efficacy ind e novo AML (pAML) and secondary AML (sAML). We developed a patient-derived AML (PD-AML) ex vivo platform to identify compounds that enhance VEN efficacy. Briefly, we collect fresh bone marrow samples/peripheral blood from AML patients at both diagnosis and relapse. Samples are then treated with 1.25 μM VEN alone or in combination focused 45-compound library enriched for pathways implicated in VEN resistance, such as metabolic reprogramming, inflammation, and stress adaptation. Pre- and post- drug treatment analyses include AML cell viability/abundance, immunophenotype, and genetic subtype. Compound interaction with VEN is calculated using SynergyFinder. To date, we have analyzed 29 PD-AML samples and observed VEN response patterns that are consistent with clinical outcomes: 38% of PD-AML are strongly responsive to VEN alone (>65% killing), 34% show intermediate (INT) sensitivity (~65-30% killing), and 28% are weakly (WEAK) responsive to VEN (<30% killing). Importantly, intermediate and refractory samples are enriched for KRAS, NRAS, FLT3-ITD , or TP53 mutations, aligning with patient-derived data. From our library, two of the top-scoring compounds are KIRA6 and 4μ8C, both of which inhibit the enzyme IRE1α, a key component of the unfolded protein response. VEN combined with KIRA6 or 4μ8C killed PD-AML cells significantly more than VEN alone, with the greatest increase in cytotoxicity observed in INT and WEAK VEN responders. In patients, VEN is used in combination with 5-azacytidine (AZA). Although both AZA and KIRA6 enhance VEN cytotoxicity, VEN+KIRA6 is significantly more effective than VEN + AZA in eliminating WEAK PDAML cells. We also tested VEN+IRE1α inhibitors in sAML (n=4), which also showed heterogeneous VEN sensitivity. One MPN-derived sAML (sAML_1: TP53WT; IDH2R140Q/JAK2V617F/SRSF2P95H) showed near-complete loss of viable cells with low-dose VEN, whereas therapy-related sAML with complex karyotype and TP53 deletion (sAML_2: del(7q); TP53 loss ~60% nuclei) remained >70% viable after VEN alone. However, sAML_2 was highly responsive to the combination of VEN and KIRA6 (Figure A). Two additional MDS/MPN-derived sAMLs showed intermediate VEN sensitivity that improved with IRE1α inhibitors. At the protein level, we observed that VEN reduced IRE1α protein in VEN-sensitive sAML_1, while IRE1α persisted after VEN exposure in VEN-resistant sAML_2. Using, two AML cell lines that represent VEN-sensitivity (MOLM‑13) and VEN-resistance (HNT-34), we found that VEN alone depleted IRE1a in MOLM-13 cells, not HNT-34. Notably, VEN+KIRA6 reduced IRE1α and MCL-1, a known VEN-resistance mechanism, and enhanced apoptosis induction in HNT-34 (Figure B). Our data indicate that IRE1α inhibition enhances VEN activity and can overcome VEN resistance, including high-risk TP53-altered sAML. Persistence of IRE1α after VEN exposure may serve as a functional biomarker of resistance in sAML. Cohort expansion is ongoing, focusing on TP53-altered sAML.