Metabolic reprogramming drives acquired venetoclax resistance in Acute Myeloid Leukemia and reveals targetable vulnerabilities

Abstract

Abstract Despite the clinical success of the BCL2 inhibitor venetoclax in acute myeloid leukemia (AML), treatment resistance frequently emerges, leading to relapse. Mounting evidence suggests that metabolic reprogramming contributes to venetoclax resistance, yet the underlying mechanisms remain poorly defined. This study aimed to develop and characterize venetoclax-resistant AML models and explore metabolic vulnerabilities that could be exploited therapeutically. For this purpose, we established two venetoclax-resistant AML cell lines (MV4-11VR and MOLM-13VR) through repeated intermittent exposure to increasing doses of venetoclax. Resistance was assessed via cell viability assays, annexin V-based apoptosis analysis, mitochondrial membrane potential, cell cycle profiling, and colony formation assays. Metabolic reprogramming was evaluated using measurements of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Molecular and genomic alterations were explored through transcriptomic profiling, global proteomics, and targeted sequencing. In addition, we tested the therapeutic potential of combining venetoclax with metabolic inhibitors, metformin, a mitochondrial complex I inhibitor, and KPT-9274, a NAMPT inhibitor, by calculating synergy scores and quantifying apoptotic responses. Venetoclax-resistant MV4-11VR and MOLM-13VR cells exhibited markedly elevated IC50 values (more than 200-fold compared to parental cells), impaired apoptosis induction, preserved proliferative capacity, and enhanced colony formation under venetoclax exposure. Notably, resistant cells showed distinct metabolic rewiring: MV4-11VR cells relied on enhanced glycolysis, whereas MOLM-13VR cells displayed increased oxidative phosphorylation (all p < 0.05). These findings were supported by functional assays and proteomic signatures, which showed enrichment of carbohydrate metabolism pathways in MV4-11VR and aerobic energy production in MOLM-13VR (all FDR < 0.05). At the molecular level, venetoclax resistance was accompanied by cell line-specific modulation of BCL2-family gene expression. MV4-11VR cells exhibited downregulation of multiple pro-apoptotic genes, including BAX, BCL2L11, BBC3, BIK, and BNIP3, while MOLM-13VR cells showed increased expression of MCL1 and BAD, along with reduced expression of BID, PMAIP1, and BMF. At the protein level, both models demonstrated reduced PARP1 cleavage and lower γH2AX levels upon venetoclax exposure, indicating suppression of apoptosis and DNA damage signaling. Furthermore, total BCL2 levels remained unchanged, while BCL-XL expression was stable in resistant cells but downregulated in parental cells upon drug exposure. A shared hallmark in both models was the hyperactivation of the PI3K/AKT/mTOR pathway, as evidenced by increased phosphorylation of the downstream effector RPS6. Additionally, MV4-11VR cells showed reduced ERK1/2 phosphorylation, whereas MOLM-13VR cells exhibited increased ERK1/2 activation. Genomic profiling revealed additional mechanisms contributing to resistance. MV4-11VR cells acquired a novel TP53 mutation (R158H) and showed clonal expansion of a pre-existing TP53 R248W mutation, alongside a mosaic deletion of the TP53 locus. In MOLM-13VR cells, sequencing revealed the emergence of a truncating mutation in the SPEN gene (I1326*) and acquisition of trisomy 5, further supporting distinct genetic trajectories of resistance. Therapeutically, venetoclax-resistant cells showed partial sensitivity to the metabolic inhibitors metformin and KPT-9274. Importantly, combination therapy with venetoclax significantly increased apoptosis in resistant cells. The venetoclax + KPT-9274 combination yielded strong synergy (ZIP scores up to 13.86) and eliminated over 95% of resistant cells, demonstrating the feasibility of targeting metabolic dependencies to restore drug sensitivity. Acquired venetoclax resistance in AML arises through heterogeneous yet targetable metabolic reprogramming. Distinct metabolic and molecular adaptations in resistant MV4-11 and MOLM-13 cells point to the complexity of resistance mechanisms. Nonetheless, the shared activation of survival pathways and metabolic plasticity creates vulnerabilities that can be exploited using mitochondrial or NAD+ biosynthesis inhibitors. These findings support a combinatorial therapeutic strategy targeting both apoptotic and metabolic pathways to overcome acquired venetoclax resistance in AML. Supported by FAPESP, CAPES, and CNPq.