Functional Interrogation of HOXA9 Regulome in MLLr Leukemia via Reporter-based CRISPR/Cas9 screen

Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, including human acute myeloid leukemia (AML) and subtypes of acute lymphoblastic leukemia (ALL). HOXA9 overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation and maintenance. However, our current understanding of HOXA9 regulation in leukemia is limited, hindering development of therapeutic strategies to treat HOXA9-driven leukemia. To mitigate these challenges, we generated the first HOXA9-mCherry knock-in reporter in an MLL-rearranged (MLLr) B-ALL cell line to dissect HOXA9 regulation. By utilizing the reporter and CRISPR/Cas9 mediated screens, we identified transcription factors controlling HOXA9 expression, including a novel regulator, USF2 and its homolog USF1. USF1/USF2 depletion significantly down-regulated HOXA9 expression and impaired MLLr leukemia cell proliferation. Ectopic expression of HOXA9-MEIS1 fusion protein rescued the impaired leukemia cell proliferation upon USF2 loss. Cut&Run analysis revealed the direct occupancy of USF2 onto HOXA9 promoter in MLLr leukemia cells. Collectively, the HOXA9 reporter facilitated the functional interrogation of the HOXA9 regulome and has advanced our understanding of the molecular regulation network in HOXA9-driven leukemia.