Integrated Perturb-seq and Computational Modeling Identifies KAT6A-Menin-DOT1L Synergy and Associated Gene Program as Therapeutic Targets in Acute Myeloid Leukemia [RNA-Seq]

Background: Acute myeloid leukemia (AML) is a heterogeneous malignancy characterized by genetic and epigenetic dysregulation. Despite advances in targeted therapies against individual epigenetic regulators, clinical responses are often transient due to complex compensatory mechanisms. Identifying functional interactions between chromatin regulators and their associated transcriptional programs represents a promising strategy to overcome resistance and enhance therapeutic efficacy. Results: Using Perturb-seq combined with computational modeling and machine learning approaches, we systematically investigated 16 key epigenetic regulators across the Menin-MLL, Polycomb, and histone acetyltransferase complexes in MOLM-13 cells. Single-cell transcriptomic analysis of 31,015 cells revealed 17 distinct gene programs with differential impacts on AML pathogenesis. We identified strong synergy between KAT6A and the Menin-DOT1L axis in regulating oncogenic pathways, particularly gene program P-15 (Pro-Differentiation Program), which significantly correlates with favorable clinical outcomes in both TCGA and Beat-AML cohorts. Notably, P-15 showed high expression in NPM1-mutated AML and remission-stage patients but was suppressed in TP53-mutated cases with poor prognosis. Validating our computational predictions, dual inhibition of Menin (VTP-50469) and KAT6A (WM119, a preclinical compound) demonstrated significant therapeutic synergy in MOLM-13 cells, reducing the IC50 from 35nM to 12nM. Conversely, PCGF1 disruption conferred resistance to DOT1L inhibition, revealing a novel mechanism of therapeutic resistance. Bulk RNA-seq analysis of combination treatments confirmed that dual KAT6A-Menin inhibition uniquely modulates specific gene modules associated with myeloid differentiation. Conclusions: Our integrated experimental-computational approach, distinct from prior studies examining individual epigenetic regulators in isolation, identifies the KAT6A-Menin-DOT1L axis as a critical regulator of AML transcriptional programs in MLL-rearranged leukemia and reveals gene program P-15 as both a favorable prognostic biomarker and potential therapeutic target. These findings provide mechanistic insights into epigenetic regulation in AML and establish a framework for developing rational combination therapies targeting cooperative epigenetic dependencies while preventing compensatory resistance mechanisms.