Effect of RBM5 knockout or overexpression on transcription in MOLM13 AML cells
Background: The oncogenic protein HOXA9 plays a critical role in leukemia transformation and maintenance, and its aberrant expression is a hallmark of most aggressive acute leukemia. Although inhibiting the upstream regulators of HOXA9 has been proven as a significant therapeutic intervention, the comprehensive regulation network controlling HOXA9 expression in leukemia has not been systematically investigated. Results: Here, we perform genome-wide CRISPR/Cas9 screening in the HOXA9-driven reporter acute leukemia cells. We identify a poorly characterized RNA-binding protein, RBM5, as the top candidate gene required to maintain leukemia cell fitness. RBM5 is highly overexpressed in acute myeloid leukemia (AML) patients compared to healthy individuals. RBM5 loss triggered by CRISPR knockout and shRNA knockdown significantly impairs leukemia maintenance in vitro and in vivo. Through domain CRISPR screening, we reveal that RBM5 functions through a noncanonical transcriptional regulation circuitry rather than RNA splicing, such an effect depending on DNA-binding domains. By integrative analysis and functional assays, we identify HOXA9 as the downstream target of RBM5. Ectopic expression of HOXA9 rescues impaired leukemia cell proliferation upon RBM5 loss. Importantly, acute protein degradation of RBM5 through auxin-inducible degron system immediately reduces HOXA9 transcription. Conclusions: We identify RBM5 as a new upstream regulator of HOXA9 and reveal its essential role in controlling the survival of AML. These functional and molecular mechanisms further support RBM5 as a promising therapeutic target for myeloid leukemia treatment. Keywords: Acute myeloid leukemia; CRISPR screen; Genome editing; HOXA9; RBM5. Overall design: To investigate the RNA binding protein, RBM5, in the regulation of MLL-r leukemia maintenance, we established CRISPR KO MOLM13 cell lines in which RBM5 has been targeted by Cas9/sgRNA against coding exons. Lentiviral cDNA was delivered in to MOLM13 for overexpression. We then performed gene expression profiling analysis using data obtained from RNA-seq of 3 biological replicates in sgNT, sgRBM5, OE RBM5 groups. Differential gene expression was compared between sgNT vs sgRBM5,  NT. Vs RBM5 OE.