Transcriptional Control of HP1a By the RNA Binding Proteins Rbmx/L1 Maintain Chromatin State in Myeloid Leukemia
Authors: Nguyen Diu T.T. , Prieto Camila , Liu Zhaoqi , Wheat Justin , Perez Alexendar , Gourkanti Saroj , Chou Timothy , Barin Ersilia , Chow Arthur , Taggart James , Hoskova Katerina , Dhodapkar Meera , Schurer Alexandra , Barlowe Trevor Stephen , Vu Ly , Leslie Christina , Steidl Ulrich G. , Rabadan Raul , Kharas Michael G. 
Published: February 2021

Mutations and aberrant expression of RNA binding proteins (RBPs) have recently been found to contribute to leukemia development (Prieto and Kharas, CSH, 2020). Previously we have identified the RBP RBMX (RNA binding motif protein, X-linked) in our shRNA in vivo screen using murine MLL-AF9 driven leukemia (Prieto et al, ASH abstract 2018). Here we uncover the role of RBMX and its retrogene RBMXL1 in normal hematopoiesis and leukemogenesis in mouse and human systems.
To investigate RBMX function in normal hematopoiesis, we created an Rbmx Mx1-Cre conditional knockout model to specifically delete Rbmx in the hematopoietic system. We gender stratified our studies since Rbmx is sex-linked. Deletion of Rbmx in both female and male primary mice as well as in non-competitive transplant animals did not affect hematopoiesis. Additionally, Rbmx knockout (KO) leads to a mild reduction in multipotent progenitors (MPP2 and MPP4) in female competitive transplanted mice although no defects in long-term hematopoiesis was observed in male competitive transplanted mice. These data suggest that Rbmx is dispensable for normal hematopoiesis.
To identify the role of RBMX in leukemogenesis we knockout Rbmx in MLL-AF9 murine leukemia cells and found it significantly reduced colony formation in vitro and delayed leukemogenesis in vivo, indicating that Rbmx is required for leukemia maintenance. We observed, however, that MLL-AF9 transformed cells from Rbmx KO donor mice showed no delay in leukemia initiation versus cells from wildtype. We then determined that while Rbmx deletion is effective with complete depletion of mRNA, the retrogene RBMXL1 expression is maintained in Rbmx deficient cells, which may compensate for Rbmx deletion in leukemia initiation. Indeed, depletion of RBMXL1 by shRNAs (KD) in Rbmx deficient leukemia cells results in a drastic reduction in colony formation, increased colony myeloid differentiation, and induced apoptosis in cells deficient for both RBMXL1 and RBMX compared to those only depleted of RBMX. Correspondingly, RBMXL1 KD in Rbmx deficient leukemia cells resulted in further delayed leukemogenesis in vivo, indicating that RBMXL1 is functionally redundant to RBMX and both genes are required for leukemia development and maintenance.
We next investigated the role of RBMX/L1 in normal human hematopoietic and leukemia cells. We found that RBMX/L1 expression were higher in AML cell lines (n=10/11) and primary AML patient samples (n=2/4) compared to healthy individuals. RBMX/L1 KD by shRNAs led to a dramatic decrease in cell proliferation, induction of apoptosis and myeloid differentiation in several human myeloid leukemia cell lines (MOLM-13, THP-1, K562, and KCL-22). Additionally, RBMX/L1 depletion significantly delayed leukemogenesis in vivo of AML cell lines (median survival of 51.5 days in control vs. median 'not reached' in shRNA1 and shRNA2), and of primary leukemia cells derived from an AML patient (median 50 days in control vs. "not reached" in shRNA2). We next depleted RBMX/L1 in human CD34+ stem and progenitor cells and observed reduced colony formation but no increase in apoptosis. Taken together, our data suggest that RBMX/L1 are differentially required in leukemia cells versus normal cells in both human and mouse systems.
To uncover the mechanism of RBMX/L1 function, we performed complex chromosomal karyotyping analysis of RBMX/L1 depleted MOLM13 cells and revealed increased metaphases with breaks and gaps. In addition, ATAC-seq analysis showed profound genome-wide changes in chromatin accessibility and compaction upon RBMX/L1 depletion. Analyses of RNA-seq and transcriptome-wide RBMX/L1 binding targets (PAR-CLIP; Liu et al. 2017) revealed that RBMX/L1 directly binds to mRNAs and affects transcription of multiple loci including the heterochromatin protein 1 alpha (HP1a) mRNA. Using single molecule RNA FISH, we uncovered that RBMX/L1 controls the nascent transcription of the HP1a locus. Forced HP1a expression rescued the RBMX/L1 depletion effects on cell growth and apoptosis. Overall, we determine that RBMX/L1 control leukemia cell survival by regulating chromatin state through its downstream target HP1a. This study describes a novel genetic compensation phenomenon in leukemia and illuminate a mechanism for RBPs directly promoting transcription. Our results suggest RBMX/L1 as well as HP1a as potential novel therapeutic targets in myeloid malignancies.

Disclosures
Steidl: Aileron Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Stelexis Therapeutics: Consultancy, Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Bayer Healthcare: Research Funding; Pieris Pharmaceuticals: Consultancy. Kharas:Accent Therapeutics: Consultancy; 28-7: Research Funding.