RNA-Binding Protein RBM5 Plays an Essential Role in Acute Myeloid Leukemia By Activating the Oncogenic Protein HOXA9

Acute myeloid leukemia (AML) represents a type of malignant hematological disease that indicates poor outcomes in children and adults. HOXA9 overexpression is observed in 50-70% of human AML and positively correlates with poor patient outcomes. Leukemia subtypes with hallmark overexpression of HOXA9 include but are not limited to those carrying KMT2A gene rearrangements (KMT2A-r), NPM1c mutations, NUP98-fusions and EZH2 mutation. Accumulating genetic evidence also indicates that HOXA9 dysregulation is sufficient and necessary for leukemic transformation, and loss of function of HOXA9 and downstream pathways consequently impaired leukemia maintenance. Although HOXA9 is not an ideal direct candidate for therapeutic interventions, uncovering the novel regulators of HOXA9 might elucidate the HOXA9 regulation mechanism that could be exploited to identify novel therapeutic targets in HOXA9-driven leukemias.

In this study, we performed genome-wide CRISPR/Cas9 screens in the HOXA9-endogenous reporter acute leukemia cells and unbiasedly identified that candidate genes are significantly enriched in RNA binding and RNA splicing regulation. An RBP protein, RBM5, was among the top essential genes. Initially, RBM5 was reported as a tumor suppressor gene in lung cancer. Thus far, the function of the encoding protein RBM5 in blood cancer remained largely unknown. To further test how RBM5 expression might correlate with AML development, we analyzed the public RNA-seq data from the TCGA clinical cancer sample datasets, RBM5 is highly expressed in AML, indicating a plausible role in AML oncogenesis. Functional validation by two individual sgRNAs against different exons of RBM5 confirmed the selectively survival essential of RBM5 in AML cell lines. Ectopic expression of sgRNA-resistant RBM5 cDNA ultimately rescued the cellular phenotype upon RBM5 loss. We utilized an alternative loss-of-function approach, RNA interference (RNAi), to further examine the functional requirement of RBM5, Two independent shRNAs targeted RBM5 mRNA in three AML cells lines. RBM5 loss significantly impaired cell growth, reduced colony numbers and triggered notable myeloid differentiation. We further transplant MOLM13 cells targeted with shRBM5 into NSG mice, and observed that RBM5 shRNAs-treated animals lagged progressively more behind with much less splenomegaly and reduced leukemia cell infiltration in bone marrow, spleen, and peripheral blood compared to the wild-type control, suggesting that RBM5 is required for AML progression in vivo. More importantly, Suppression of RBM5 in primary AML cells significantly reduced colony numbers and induced myeloid differentiation, as demonstrated by the maturation markers CD11b and CD14. In addition, the domain dropout CRISPR screen revealed that the DNA-binding domain C2H2 is required for the gene function. Integrative analysis further suggested that HOXA9 promises the direct transcriptional target of RBM5 as demonstrated by functional rescue assay and HOXA9 partially restore the gene transcriptional defect in RBM5 null leukemia cells.

In summary, our innovative research reported that a new RNA-binding protein, RBM5, is required for the survival of AML through non-canonical transcriptional regulation via a complex regulatory mechanism. These data support RBM5 as a promising AML therapeutic target in future studies.