A Perturb-seq map of a differentiation hub reveals synergistic vulnerabilities in KMT2A-rearranged acute myeloid leukemia.

KMT2A-rearranged acute myeloid leukemia is driven by epigenetic dependencies yet remains clinically resistant to therapies targeting individual regulators, indicating that resistance reflects compensatory regulation across an epigenetic network. A systematic understanding of this compensatory network has been lacking. To address this gap, we utilized Perturb-seq screening to systematically map the functional architecture of this network. We uncovered a compensatory epigenetic circuit, where a synergistic hub including KAT6A, Menin, and DOT1L converges to silence a core differentiation program (the 'Myeloid Program'), thereby maintaining leukemic identity. The activity of this program strongly correlated with favorable survival in large patient cohorts. While individual perturbations of hub components only partially derepress this program, their simultaneous pharmacological inhibition collapses the circuit's buffering capacity, leading to robust reactivation of the Myeloid Program and potent synergistic anti-leukemic activity. Our model also shows that disruption of antagonistic regulators of the Myeloid Program, such as the PRC1.1 component PCGF1, confers strong resistance to DOT1L inhibition. Finally, the Myeloid Program is a predictive biomarker, where high baseline activity defined a vulnerable state that could be selectively targeted by MEK, AKT, and mTOR inhibitors. Together, these findings establish a framework for identifying circuit-level epigenetic compensation and for rationally designing precision combination therapies that restore differentiation or target state-dependent vulnerabilities in acute myeloid leukemia.