Functional interrogation of HOXA9 regulome in MLLr leukemia via reporter-based CRISPR/Cas9 screen.

Aberrant expression is a hallmark of most aggressive acute leukemias, notably those with KMT2A (MLL) gene rearrangements. overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation and maintenance. However, our current understanding of regulation in leukemia is limited, hindering development of therapeutic strategies. Here, we generated the knock-in reporter cell lines to dissect regulation. By utilizing the reporter and CRISPR/Cas9 screens, we identified transcription factors controlling expression, including a novel regulator, USF2, whose depletion significantly down-regulated expression and impaired MLLr leukemia cell proliferation. Ectopic expression of Hoxa9 rescued impaired leukemia cell proliferation upon USF2 loss. Cut and Run analysis revealed the direct occupancy of USF2 at promoter in MLLr leukemia cells. Collectively, the reporter facilitated the functional interrogation of the regulome and has advanced our understanding of the molecular regulation network in -driven leukemia.