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

Abstract

Aberrant <i>HOXA9</i> expression is a hallmark of most aggressive acute leukemias, notably those with KMT2A (MLL) gene rearrangements. <i>HOXA9</i> overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation and maintenance. However, our current understanding of <i>HOXA9</i> regulation in leukemia is limited, hindering development of therapeutic strategies. Here, we generated the <i>HOXA9-mCherry</i> knock-in reporter cell lines to dissect <i>HOXA9</i> regulation. By utilizing the reporter and CRISPR/Cas9 screens, we identified transcription factors controlling <i>HOXA9</i> expression, including a novel regulator, USF2, whose depletion significantly down-regulated <i>HOXA9</i> 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 <i>HOXA9</i> promoter in MLLr leukemia cells. Collectively, the <i>HOXA9</i> reporter facilitated the functional interrogation of the <i>HOXA9</i> regulome and has advanced our understanding of the molecular regulation network in <i>HOXA9</i>-driven leukemia.