ACSL4-associated lipid metabolism is a distinct therapeutic vulnerability in KMT2A-rearranged acute myeloid leukemia

• ACSL4 is selectively essential in KMT2A r AML cells • ACSL4 depletion impairs growth, colony formation, and leukemia expansion ex vivo and in vivo • Polyunsaturated fatty acid supplementation rescues growth defects caused by ACSL4 knockdown • KRADS12 signature reflects ACSL4 dependency in KMT2A r AML and predicts poor patient survival ACSL4 is selectively essential in KMT2A r AML cells ACSL4 depletion impairs growth, colony formation, and leukemia expansion ex vivo and in vivo Polyunsaturated fatty acid supplementation rescues growth defects caused by ACSL4 knockdown KRADS12 signature reflects ACSL4 dependency in KMT2A r AML and predicts poor patient survival Deregulated lipid metabolism contributes to leukemogenesis and the progression of acute myeloid leukemia (AML). By analyzing large-scale CRISPR-Cas9 screening data, we identified acyl-CoA synthetase long-chain family member 4 ( ACSL4 ) as a selective vulnerability in lysine methyltransferase 2A-rearranged ( KMT2A r) AML. Functional validation using CRISPR interference and short hairpin RNA knockdown confirmed that ACSL4 loss impairs the growth of KMT2A r but not non- KMT2A r AML cells. ACSL4 knockdown reduced colony formation in cells derived from patients with KMT2A r AML and murine MLL-AF9 cells and delayed leukemia onset in vivo in MLL-AF9 mice. A multi-omics approach, including transcriptomics, proteomics, and lipidomics, revealed depletion of polyunsaturated lipid species and compensatory activation of lipid metabolic pathways upon ACSL4 loss. Supplementation with exogenous polyunsaturated fatty acids (PUFAs) rescued the growth defect, linking ACSL4 dependency to defective PUFA utilization. Finally, we generated a KMT2Ar - ACSL4 dependency signature (KRADS12) that correlates with KMT2A r status and predicts poor survival in patients with AML.