Targeting metabolic vulnerabilities in Acute Myeloid Leukemia: Therapeutic potential of l-asparaginase and synergy with venetoclax

Abstract Recent advances in supportive care, measurable residual disease (MRD), and genomic based risk stratification have significantly improved outcomes in acute myeloid leukemia (AML). Nonetheless, prognosis remains suboptimal, with 5-year overall survival rates of approximately 70% in children, 40% in young adults, and less than 10% in elderly patients. These findings underscore an urgent need for new treatment strategies, such as drug repositioning, to improve patient outcomes. L-asparaginase (L-asp) is a key therapeutic agent for the treatment of acute lymphoblastic leukemia (ALL); however, its efficacy in AML remains unclear. To evaluate its therapeutic potential in AML, we systematically investigated L-asp sensitivity and explored the underlying molecular mechanisms that confer enhanced susceptibility in AML cell lines.To assess L-asp sensitivity in AML, we evaluated IC50 values and growth curves in 22 diverse AML cell lines. Based on their responses, AML cell lines were categorized as highly sensitive (n=7), moderately sensitive (n=9), and resistant (n=6) groups. These classifications were recapitulated in cell line-derived xenograft models, which demonstrated consistent differences in survival outcomes. To elucidate the molecular mechanisms underlying high sensitivity to L-asp, we initially examined its asparaginase activity. All highly sensitive cell lines exhibited low expression of asparagine synthetase (ASNS), mirroring findings in ALL. ASNS expression levels positively correlated with L-asp IC50 values (Spearman r = 0.81, p < 0.001). Bisulfite sequencing and SNP array analyses revealed that ASNS downregulation was attributable to either promoter hypermethylation or genomic loss—monosomy 7 or focal deletion at 7q21.3—in all highly sensitive lines. Moreover, ASNS knockdown in resistant cell lines restored L-asp sensitivity, confirming its role as a key determinant of susceptibility. These findings demonstrate that AML cells with low ASNS expression exhibit high sensitivity to L-asp. Next, to investigate the mechanisms underlying enhanced sensitivity in moderately sensitive cell lines, we examined the glutaminase activity of L-asp. In AML, glutamine metabolism plays a critical role in cellular homeostasis by (1) fueling the TCA cycle via glutaminolysis and (2) maintaining redox balance through the synthesis of reduced glutathione (GSH), a major antioxidant that scavenges reactive oxygen species (ROS). To determine whether impaired TCA cycle activity contributes to L-asp–induced apoptosis, we assessed mitochondrial function using the Seahorse assay. All moderately sensitive AML cell lines demonstrated reduced mitochondrial respiration following L-asp treatment. Consistently, metabolomic analysis revealed decreased levels of TCA cycle intermediates. Moderate sensitive cell lines failed to upregulate compensatory pathways such as glycolysis or β-oxidation, indicating a high dependence on glutamine metabolism for energy production (glutamine-dependent AML cells).We next examined whether L-asp induces ferroptosis via disruption of redox homeostasis. Compared to resistant cells, moderately sensitive cells showed significant reductions in intracellular GSH levels and increased mitochondrial ROS following L-asp exposure. However, L-asp–induced lipid peroxidation was minimal, and the annexin V positive cells induced by L-asp were only modestly suppressed by the ferroptosis inhibitors liproxstatin-1 and deferoxamine. These findings suggest that glutamine-dependent AML cells undergo apoptosis primarily due to L-asp–induced energy deprivation, while ferroptosis induced by redox imbalance contribute minimally to cytotoxicity.To identify potential combinatorial therapeutic agents with L-asp, we performed RNA sequencing on three moderately sensitive and three resistant AML cell lines. Notably, BMF (BCL-2 modifying factor), a pro-apoptotic gene, was consistently upregulated exclusively in moderately sensitive cell lines. Aligned with these findings, the combination of L-asp and venetoclax demonstrated marked synergistic cytotoxicity in these cell lines.In summary, AML with low ASNS expression shows high sensitivity to L-asp, suggesting that treatment with L-asp is an absolute indication. Furthermore, even in glutamine-dependent AML with high ASNS expression, combination therapy with L-asp and venetoclax represents a promising therapeutic strategy.