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

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.