Abstract
Aberrant HOXA9 expression is a hallmark of most aggressive acute leukemias, notably those with KMT2A (MLL) gene rearrangements. 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. Here, we generated the HOXA9-mCherry knock-in reporter cell lines to dissect HOXA9 regulation. By utilizing the reporter and CRISPR/Cas9 screens, we identified transcription factors controlling HOXA9 expression, including a novel regulator, USF2, whose depletion significantly down-regulated HOXA9 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 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.
Highlights
Dysregulation of the homeobox (HOX)-containing transcription factor HOXA9 is a prominent feature in most aggressive acute leukemias (Collins and Hess, 2016a; Alharbi et al, 2013)
When the homology arms (HAs)/knock-in cassette was co-electroporated with an all-in-one vector expressing wild-type Cas9 and the same HOXA9 single guide RNA (sgRNA), the HA/ knock-in cassette was released from the donor vector with two nuclease cleavages and delivered to the target genomic region where double-strand breaks occurred
RNA-seq data collected from SEM cells in our previous studies suggested that HOXA7, HOXA9 and HOXA10 were the only highly expressed HOXA genes in MLL gene rearrangements (MLLr) leukemia SEM cells (Hyle et al, 2019; Figure 1E), and that these patterns were indistinguishable between WT and KI populations, indicating the P2AmCherry knock-in did not alter the gene expression landscape at the HOXA cluster (Figure 1F)
Summary
Dysregulation of the homeobox (HOX)-containing transcription factor HOXA9 is a prominent feature in most aggressive acute leukemias (Collins and Hess, 2016a; Alharbi et al, 2013). Ghasemi et al reported that HOXA gene expression was maintained in the CTCF-binding site deletion mutants derived from AML OCI-AML3 cells, suggesting that transcriptional activity at the HOXA locus in NPM1-mutant AML cells does not require long-range CTCF-mediated chromatin interactions (Ghasemi et al, 2020). These data suggest that CTCF may play a cell-typedependent role on HOXA9 regulation. Our HOXA9P2A-mCherry reporter lines are robust tools for discovery of novel HOXA9 regulators
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