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

Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, including a vast majority of human acute myeloid leukemia (AML) and subtypes of acute lymphoblastic leukemia (ALL). HOXA9 overexpression not only predicts poor patient diagnosis and outcome, but also plays critical roles in leukemia transformation and maintenance. Besides in few clinical cases as a translocation partner, HOXA9 gene generally shows lower frequencies of somatic mutations and is rarely amplified, indicating that transcriptional and epigenetic regulatory mechanisms might be predominantly utilized. However, our current understanding on the upstream regulation of HOXA9 in acute leukemia is still very limited, hindering development of effective methods to treat leukemia driven by aberrant HOXA9 expression. To mitigate these challenges, we have established the first HOXA9 endogenous reporter in an MLL-rearranged leukemia cell line to faithfully dictate HOXA9 expression by mCherry. By utilizing the reporter cell line and CRISPR/Cas9 genome editing tools, we have exploited comprehensive loss-of-function screenings to systematically discover novel transcription factors controlling HOXA9 expression. USF2 was discovered as a novel regulator of HOXA9 that directly binds to HOXA9's promoter. USF2 depletion significantly down-regulated HOXA9 expression. Notably, in patients with MLL-rearranged B-ALL, transcriptional expression of USF2 and that of HOXA9 were positively correlated. In contrast, chromatin looping factor CTCF was dispensable for controlling HOXA9 expression in MLL-rearranged SEM cells. Collectively, these studies have functionally interrogated the regulome of HOXA9 and advance our understanding of the molecular regulation network in HOXA9-driven leukemia. Overall design: CUT&RUN ChIP-seq was performed on auxin-inducible degron system in a human B-cell lymphoblastic leukemia (B-ALL) cell line, SEM, using USF2 (NBP1-92649, Novus) antibodies against USF2 for a pulldown assay