Overcoming acquired venetoclax resistance in acute myeloid leukemia through cell metabolism targeting.

6537 Background: Venetoclax is a selective inhibitor of the anti-apoptotic protein BCL2, often overexpressed in acute myeloid leukemia (AML), contributing to cell survival and resistance to standard therapies. It has expanded treatment options, especially for elderly or chemotherapy-ineligible patients. However, acquired resistance to venetoclax is a significant challenge, limiting long-term effectiveness. Understanding the mechanisms of resistance is crucial for improving therapeutic strategies and identifying biomarkers for personalized treatment. This study investigates these mechanisms by analyzing cellular phenotype, metabolic changes, BCL2 family gene/protein expression, and signaling pathways in venetoclax-resistant AML cell lines. Methods: Venetoclax-sensitive AML cell lines, MOLM-13 and MV4-11, were exposed intermittently to increasing concentrations of the drug to induce resistance. Cell viability was assessed using MTT assays, clonogenicity by colony formation, and apoptosis, mitochondrial damage, and DNA content by flow cytometry. Metabolic profiles were analyzed with Seahorse XF96, and signaling pathways were studied by Western blotting, qPCR, and global proteomics. Synergy assays were conducted with metformin (mitochondrial complex I inhibitor) and KPT-9274 (NAMPT inhibitor). Results: Intermittent exposure to venetoclax selected for resistant clones, MV4-11VR (IC 50 > 1000 nM; parental cells IC 50 = 2.5 nM) and MOLM-13VR (IC 50 = 723 nM; parental cells IC 50 = 3.3 nM). Venetoclax-induced apoptosis, mitochondrial damage, and DNA fragmentation were absent in resistant cells. Metabolic analysis showed increased mitochondrial metabolism in MOLM-13VR cells and enhanced glycolysis in MV4-11VR cells. Molecularly, MV4-11VR cells exhibited downregulation of BCL2L10, BAX, BCL2L11, BBC3, BIK, and BNIP3, while MOLM-13VR cells showed reduced BID, PMAIP1, BAD, BMF, and BECN1, along with increased MCL1. MOLM-13VR cells displayed enhanced MAPK signaling, and both resistant models had activation of the PI3K/AKT/mTOR pathway and upregulation of BCL-XL. Proteomic analysis revealed enhanced metabolic activity, with MV4-11VR cells enriched in fatty acid biosynthesis and carbohydrate metabolism pathways, while MOLM-13VR cells showed upregulation of aerobic respiration and ATP metabolism. Both parental and resistant cells exhibited comparable sensitivity to metformin and KPT-9274. The combination of these inhibitors with venetoclax resulted in synergistic effects, with KPT-9472 and venetoclax eliminating over 95% of resistant cells. Conclusions: This study provides key insights into the mechanisms of venetoclax resistance in AML. Targeting cellular metabolism with metformin or KPT-9274, combined with venetoclax, offers promising synergistic effects and potential strategies to overcome resistance, improving treatment outcomes in resistant AML cases.