Abstract
Abstract RNA binding proteins (RBPs) tightly control mRNA abundance, stability and translation while mutations or altered expression of specific factors can drive malignancy. Yet, the identity of the RBPs that govern myeloid stem cells remains poorly characterized. We and others have recently demonstrated that MUSASHI-2 (MSI2) is a central regulator of the cancer stem cell program in myeloid leukemia. Therefore, 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, non-deleted 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 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 depleted for SYNCRIP. 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 a 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 validated 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. Disclosures Melnick: Janssen: Research Funding. Levine:Novartis: Consultancy; Qiagen: Membership on an entity's Board of Directors or advisory committees. Järås:Cantargia AB: Equity Ownership.
