Abstract IA13: RNA regulators and the control of self-renewal
Authors: Vu Ly , Prieto Camila , Amin Eliana M. , Minuesa Gerard , Chhangawala Sagar , Vidal Maria C. , Krivtsov Andrei , Chou Timothy , Chow Arthur , Barlowe Trevor , Taggart James , Tivnan Patrick , Deering Raquel P. , Chu Lisa P. , Gonen Mithat , Figueroa Maria E. , Paietta Elisabeth , Tallman Martin S. , Melnick Ari , Levine Ross , Al-Shahrour Fatima , Jaras Marcus , Hacohen Nir , Hwang Alexia , Garippa Ralph , Lengner Christopher , Armstrong Scott , Cowley Glenn S. , Root David , Doench John , Cerchietti Leandro , Leslie Christina , Ebert Benjamin L. , Kharas Michael G. 
Published: August 2018

Abstract
Hematopoietic malignancies result from dysregulated self-renewal pathways and an altered differentiation program. Acute myeloid leukemia (AML) is characterized by the abnormal development of blood cells in the myeloid lineage. Although many studies have focused on transcriptional regulators, activated kinases, and epigenetic regulators, it is unknown how RNA binding proteins (RBPs) maintain the normal developmental program. Somatic mutations and aberrant expression of RBPs have recently emerged to be critically important in hematologic malignancies. Our laboratory and others have demonstrated that MSI2 RBP expression predicts a poor prognosis and drives the aggressiveness of myeloid leukemia. We found that MSI2 enhances translation of Myc, Hoxa9, and Ikzf2 and is required for the self-renewal of MLL-AF9 transformed leukemia stem cells (LSCs). These data suggest that the RBP maintains a positive feedback look that controls the epigenetic landscape in leukemia. To determine if Ikzf2 contributes to the LSC program, we utilized mice that were depleted in Ikzf2 and found a delay in leukemogenesis that increased during serial transplantation. Ikzf2-deficient leukemic cells lost the self-renewal gene expression program and demonstrated increased differentiation.
To further probe the altered MSI2 interactome, our laboratory has performed proteomics analysis of MSI2 interacting proteins followed by functional shRNA screening. We curated a list of 127 MSI2 direct protein interactors and associated genes to perform an in vivo shRNA screen using MLL-AF9 leukemia cells. We identified shRNAs corresponding to 24 genes that were significantly depleted in vivo after sequencing and comparing their representation from day 16 to day 0. We confirmed knockdown and demonstrated marked reduction in myeloid colony formation in vitro after depleting 7 hits identified in our screen. Additionally, we tested these genes in normal bone marrow c-Kit positive cells and found that the most differentially required gene in leukemia cells compared to normal cells was SYNCRIP (Synaptotagmin-binding, cytoplasmic RNA-interacting protein). SYNCRIP is an RNA binding protein that has been implicated in various RNA regulatory processes, but its role in the hematopoietic system is virtually unknown. Depletion of SYNCRIP with shRNAs in murine MLL-AF9 leukemia cells resulted in an increase in myeloid differentiation, apoptosis, and delayed leukemogenesis in vivo (median survival of 35 days; control versus 61 days shRNA#1 knockdown was selected against, and “not reached” shRNA#2). To further assess SYNCRIP function in vivo, we developed a germline Syncrip knockout (KO) by injecting Cas9-DNA and Syncrip-guides RNAs into embryos and harvested E13 fetal liver cells. After Syncrip deletion was verified by immunoblotting, we observed normal numbers of HSCs and equivalent engraftment in lethally irradiated animals in both primary and secondary transplants. In contrast, we observed a delay in leukemeogenesis (median survival of 87.5 days; WT versus 118 days KO) in recipient mice after transplantation of MLL-AF9 transformed LSKs. Notably, nondeleted leukemia cells outcompeted the SYNCRIP deleted cells based on a reemergence of SYNCRIP expression. These data suggest that SYNCRIP is differentially required in myeloid leukemia cells compared to normal cells. Furthermore, we found that SYNCRIP was highly expressed in a wide variety of human AML cell lines and in primary AML patients (n=4/5). SYNCRIP depletion with shRNAs resulted in reduced cell proliferation and the induction of apoptosis in human AML cell lines (MOLM13, NOMO-1, KASUMI-1 and NB4) and a marked decrease in engraftment of primary AML patient cells.
To gain insights into SYNCRIP function, we performed RNA-sequencing of leukemia cells after shRNA-mediated depletion. Gene set enrichment analysis (GSEA) negatively enriched for the MLL-AF9, HOXA9, and stem cell programs in SYNCRIP-KD cells and positively enriched for MSI2's direct mRNA binding targets and an MSI2-deficient LSC signature. Reciprocal immunoblotting in the presence or absence of RNAse demonstrated that SYNCRIP and MSI2 interaction is RNA dependent. We validated their shared targets by performing SYNCRIP RNA-immunoprecipitation (RIP) for previously identified MSI2's direct mRNAs targets (HOXA9 and c-MYC). SYNCRIP depletion resulted in reduced protein abundance of HOXA9 and c-MYC. Forced MSI2 expression partially rescued the colony formation and HOXA9 expression in SYNCRIP-KD cells. To assess the functional downstream targets of SYNCRIP in leukemia, we overexpressed HOXA9 and c-MYC in SYNCRIP-KD cells and observed that HOXA9 expression, but not c-MYC, partially rescues the effect of SYNCRIP depletion on myeloid colony formation. Mechanistically, we showed that SYNCRIP regulates translation of HOXA9 without affecting HoxA9 mRNA stability. Overall, we provide a strategy for interrogating the functional RNA binding network in leukemia using shRNA screening. Additionally, we validate SYNCRIP as a novel RBP that controls the leukemia stem cell program and propose that targeting these functional complexes might provide a novel therapeutic strategy in myeloid leukemia.
Citation Format: Ly Vu, Camila Prieto, Eliana M. Amin, Gerard Minuesa, Sagar Chhangawala, Maria C. Vidal, Andrei Krivtsov, Timothy Chou, Arthur Chow, Trevor Barlowe, James Taggart, Patrick Tivnan, Raquel P. Deering, Lisa P. Chu, Mithat Gonen, Maria E. Figueroa, Elisabeth Paietta, Martin S. Tallman, Ari Melnick, Ross Levine, Fatima Al-Shahrour, Marcus Jaras, Nir Hacohen, Alexia Hwang, Ralph Garippa, Christopher Lengner, Scott Armstrong, Glenn S. Cowley, David Root, John Doench, Leandro Cerchietti, Christina Leslie, Benjamin L. Ebert, Michael G. Kharas. RNA regulators and the control of self-renewal [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr IA13.