Functional Interrogation ofHOXA9Regulome in MLLr Leukemia via Reporter-based CRISPR/Cas9 screen

AberrantHOXA9expression is a hallmark of most aggressive acute leukemias, including human acute myeloid leukemia (AML) and subtypes of acute lymphoblastic leukemia (ALL).HOXA9overexpression not only predicts poor diagnosis and outcome but also plays a critical role in leukemia transformation and maintenance. However, our current understanding ofHOXA9regulation in leukemia is limited, hindering development of therapeutic strategies to treatHOXA9-driven leukemia. To mitigate these challenges, we generated the firstHOXA9-mCherryknock-in reporter in an MLL-rearranged (MLLr) B-ALL cell line to dissectHOXA9regulation. By utilizing the reporter and CRISPR/Cas9 mediated screens, we identified transcription factors controllingHOXA9expression, including a novel regulator, USF2 and its homolog USF1. USF1/USF2 depletion significantly down-regulatedHOXA9expression 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 ontoHOXA9promoter in MLLr leukemia cells. Collectively, theHOXA9reporter facilitated the functional interrogation of theHOXA9regulome and has advanced our understanding of the molecular regulation network inHOXA9-driven leukemia.