Rna Binding Protein Rbmx Is Required In Acute Myeloid Leukemia By Regulating The Transcriptional Activity Of The Heterochromatin Protein Hp1 Alpha

Abstract Acute myeloid leukemia (AML) is characterized by a block in the development of myeloid cells, often due to dysregulation of genes involved in key processes including self-renewal, proliferation, and differentiation. Somatic mutations and aberrant expression of RNA binding proteins (RBPs) have recently been found to be important in hematological malignancies. For example, our group and others have recently determined that increased expression of MUSASHI-2 and SYNCRIP drives aggressive leukemia. To discover novel RBP regulators of leukemia, we performed an in vivo pooled shRNA screen of 127 MSI2 direct protein interactors and associated genes (Vu et al. Nat Gen. 2017). In this screen, shRNAs specific to the RBP RBMX (RNA binding motif protein, X-linked) were selectively depleted in murine MLL-AF9 driven leukemia. RBMX has been implicated in regulating alternative splicing, chromatin cohesion, and DNA-damage response, but its function in hematopoiesis and leukemia is not known. We confirmed that depletion of RBMX with shRNAs in murine MLL-AF9 leukemia cells resulted in reduced myeloid colony formation, increased apoptosis, and increased differentiation as determined by flow analysis of myeloid cell surface markers Gr-1 and Mac-1 (mean of 61-65% shRNA versus mean of 12.95% control). Furthermore, RBMX is highly expressed among human myeloid leukemia cell lines (n=10/11) and primary AML patient samples (n=2/4). Depletion of RBMX with shRNAs led to a dramatic decrease in cell proliferation and 3-fold induction of apoptosis in several human myeloid leukemia cell lines (MOLM-13, THP-1, K562, and KCL-22). Additionally, RBMX depletion in AML cells induced myeloid differentiation and significantly delayed leukemogenesis cells in vivo (median survival of 51.5 days in control versus median 'not reached' in shRNA1 and shRNA2). To determine if there is a differential requirement of RBMX in survival of leukemia cells compared to normal hematopoietic stem and progenitor cells (HSPCs), we depleted RBMX with shRNAs in normal murine bone marrow c-Kit+ cells and found no significant changes in colony formation. Depleting RBMX with shRNAs in human cord blood derived CD34+ HSPCs resulted in reduced colony formation but no increase in apoptosis. Thus, these data suggest that there is a differential requirement for RBMX in myeloid leukemia cells compared to normal cells. To uncover the mechanism of RBMX function, we performed RNA-sequencing of human AML cells (MOLM-13) depleted for RBMX. Gene set enrichment analysis demonstrated a loss of cell cycle and DNA repair associated programs in RBMX depleted cells. Complex chromosomal karyotyping analysis of these cells revealed increased metaphases with breaks and gaps (mean of 30.67% shRNA versus mean of 13.33% control) and irregular chromatin compaction (mean of 47.67%shRNA versus mean of 20% control), while cell cycle analysis showed significantly increased S-phase arrest as determined by flow analysis of Hoechst stained cells (mean of 37-40% shRNA versus of 24.18% control). Reanalysis of RBMX transcriptome-wide binding sites in 293T cells revealed that RBMX directly binds to heterochromatin protein HP1α transcripts (Liu et al. Nucleic Acids Res. 2017). HP1α, also called CBX5, is a key heterochromatin protein that binds to histone H3-K9 tri-methylation marks to promote heterochromatin formation, which is critical in chromatin condensation and chromosome segregation. HP1α has also been determined to be required for MLL leukemia stem cell maintenance. We demonstrated that RBMX depletion resulted in a significant decrease of HP1α mRNA expression without affecting its mRNA stability in AML cells. We confirmed that RBMX depletion reduced the protein abundance of HP1α. Moreover, overexpression of HP1α rescued the effect of RBMX depletion on cell growth and apoptosis. Our study finds that RBMX binds to HP1α mRNA and regulates the transcriptional activity of the HP1α locus, which then maintains proper chromatin compaction in leukemia cells. Overall, we determine that RBMX function is critical for myeloid leukemia survival and has potential as a novel therapeutic target in AML. Disclosures No relevant conflicts of interest to declare.