Targeting OXPHOS de novo purine synthesis as the nexus of inhibitor-mediated synergistic antileukemic actions.

Using a genome-wide CRISPR screen, we identified , , and as synthetic lethal partners with gilteritinib treatment in fms-like tyrosine kinase 3 ()-internal tandem duplication (ITD) acute myeloid leukemia (AML) and genetically and pharmacologically validated their roles in gilteritinib sensitivity. The presence of -ITD is associated with an increase in anaerobic glycolysis, rendering leukemia cells highly sensitive to inhibition of glycolysis. Supportive of this, our data show the enrichment of single guide RNAs targeting 28 glycolysis-related genes upon gilteritinib treatment, suggesting that switching from glycolysis to oxidative phosphorylation (OXPHOS) may represent a metabolic adaption of AML in gilteritinib resistance. CDK9i/FLT3i, DHODHi/FLT3i, and PRMT5i/FLT3i pairs mechanistically converge on OXPHOS and purine biosynthesis blockade, implying that targeting the metabolic functions of these three genes and/or proteins may represent attractive strategies to sensitize AML to gilteritinib treatment. Our findings provide the basis for maximizing therapeutic impact of -ITD inhibitors and a rationale for a clinical trial of these novel combinations.