Musashi-2 controls cell fate, lineage bias, and TGF-β signaling in HSCs

Hematopoietic stem cells (HSCs) are maintained through the regulation of symmetric and asymmetric cell division. We report that conditional ablation of the RNA-binding protein Msi2 results in a failure of HSC maintenance and engraftment caused by a loss of quiescence and increased commitment divisions. Contrary to previous studies, we found that these phenotypes were independent of Numb. Global transcriptome profiling and RNA target analysis uncovered Msi2 interactions at multiple nodes within pathways that govern RNA translation, stem cell function, and TGF-β signaling. Msi2-null HSCs are insensitive to TGF-β–mediated expansion and have decreased signaling output, resulting in a loss of myeloid-restricted HSCs and myeloid reconstitution. Thus, Msi2 is an important regulator of the HSC translatome and balances HSC homeostasis and lineage bias

Msi2 Maintains the MLL Leukemia Stem Cell Regulatory Program

Abstract Leukemia stem cells (LSCs) are found in most aggressive myeloid diseases and contribute to therapeutic resistance. LSCs are characterized by their gain of a self-renewal program that is normally associated with hematopoietic stem cells (HSCs). Previously we have shown that the RNA binding protein, Msi2 contributes to both HSC and myeloid leukemia function. Elevated MSI2 expression predicts a poor prognosis in a variety of leukemias and shRNA-mediated depletion in human AML cell lines reduces proliferation, increases differentiation and induces apoptosis. Despite these in vitroand correlative studies, MSI2’s molecular mechanism is not known and its role in LSC function has not been assessed. To elucidate MSI2’s role in LSC function, we utilized the MLL-AF9 leukemia mouse model. Initially we found MSI2 was elevated in the LSC enriched compartment (c-KitHigh cells) compared to non-LSCs (c-KitLow cells) based on flow cytometric intracellular staining. Therefore, to establish a model to study Msi2 and its contribution to myeloid LSCs, we have utilized the Msi2 conditional knockout mice that we previously crossed (Msi2f/f) into an Mx1-Cre background to generate the Msi2Δ/Δallele (injection of polyinositol-polycytosine; pIpC). In order to test if Msi2 is critical for MLL-AF9 mediated initiation, we transduced control Msi2f/f and Msi2Δ/ΔLin- Sca1+ c-Kit+cells (LSKs) with MLL-AF9 expressing retroviruses co-expressing GFP. Msi2 deleted LSKs or granulocyte-monocyte progenitors (GMPs) transduced with MLL-AF9 demonstrated delayed leukemogenesis with dramatically reduced diseased burden. Msi2 deficient leukemias were found to have a 4-fold reduced phenotypic LSC population and were more differentiated based on cellular morphology. Msi2 deficient leukemias failed to transplant into secondary recipients demonstrating that Msi2 is required for maintaining LSCs. Deletion of Msi2after leukemia engraftment led to a delay in leukemogenesis indicating that Msi2 is also important for leukemia maintenance. Gene expression profiling of the Msi2 ablated LSCs resulted in a loss of the HSC/LSC program and an increase in differentiation gene sets. The gene signature from the Msi2 deleted murine LSCs (121 genes) was overlapped and subjected to unsupervised clustering with the gene expression profiles from 336 AML patients (ECOG1900 dataset). This analysis resulted in distinct clusters that had differential MSI2 expression and the MSI2“high” cluster predicted a worse clinical outcome when compared to the other clusters. Overlapping of the differential transcriptional analysis of the Msi2 deleted murine LSCs with our global MSI2 direct mRNA targets (HITS-CLIP) led us to identify that MSI2 binds to transcripts that are associated with the downstream MLL self-renewal program, including Myc and Ikzf2. Ikzf2 is a member of the Ikaros transcription factor family and is known to regulate lymphocyte development by controlling regulatory T-cell function and chromatin remodeling. Ikzf2 shRNA mediated depletion resulted in reduced colony formation, decreased proliferation and increased apoptosis. The MLL associated targets were also reduced, which included Bcl-2 and Hoxa9. In contrast to its tumor suppressor role in hypodiploid B-ALL, these results suggest that Ikzf2 contributes to MLL leukemia cell maintenance. Thus, we provide evidence that MSI2 maintains the oncogenic LSC epigenetic program and the rationale for clinically targeting MSI2 in myeloid leukemia. Disclosures No relevant conflicts of interest to declare

Rapid TCR:Epitope Ranker (RAPTER): a primary human T cell reactivity screening assay pairing epitope and TCR at single cell resolution

Abstract Identifying epitopes that T cells respond to is critical for understanding T cell-mediated immunity. Traditional multimer and other single cell assays often require large blood volumes and/or expensive HLA-specific reagents and provide limited phenotypic and functional information. Here, we present the Rapid TCR:Epitope Ranker (RAPTER) assay, a single cell RNA sequencing (scRNA-SEQ) method that uses primary human T cells and antigen presenting cells (APCs) to assess functional T cell reactivity. Using hash-tag oligonucleotide (HTO) coding and T cell activation-induced markers (AIM), RAPTER defines paired epitope specificity and TCR sequence and can include RNA- and protein-level T cell phenotype information. We demonstrate that RAPTER identified specific reactivities to viral and tumor antigens at sensitivities as low as 0.15% of total CD8+ T cells, and deconvoluted low-frequency circulating HPV16-specific T cell clones from a cervical cancer patient. The specificities of TCRs identified by RAPTER for MART1, EBV, and influenza epitopes were functionally confirmed in vitro. In summary, RAPTER identifies low-frequency T cell reactivities using primary cells from low blood volumes, and the resulting paired TCR:ligand information can directly enable immunogenic antigen selection from limited patient samples for vaccine epitope inclusion, antigen-specific TCR tracking, and TCR cloning for further therapeutic development

Musashi2 sustains the mixed-lineage leukemia–driven stem cell regulatory program

Leukemia stem cells (LSCs) are found in most aggressive myeloid diseases and contribute to therapeutic resistance. Leukemia cells exhibit a dysregulated developmental program as the result of genetic and epigenetic alterations. Overexpression of the RNA-binding protein Musashi2 ( MSI2 ) has been previously shown to predict poor survival in leukemia. Here, we demonstrated that conditional deletion of Msi2 in the hematopoietic compartment results in delayed leukemogenesis, reduced disease burden, and a loss of LSC function in a murine leukemia model. Gene expression profiling of these Msi2 -deficient animals revealed a loss of the hematopoietic/leukemic stem cell self-renewal program and an increase in the differentiation program. In acute myeloid leukemia patients, the presence of a gene signature that was similar to that observed in Msi2 -deficent murine LSCs correlated with improved survival. We determined that MSI2 directly maintains the mixed-lineage leukemia (MLL) self-renewal program by interacting with and retaining efficient translation of Hoxa9 , Myc , and Ikzf2 mRNAs. Moreover, depletion of MLL target Ikzf2 in LSCs reduced colony formation, decreased proliferation, and increased apoptosis. Our data provide evidence that MSI2 controls efficient translation of the oncogenic LSC self-renewal program and suggest MSI2 as a potential therapeutic target for myeloid leukemia

RNA Binding Protein Syncrip Regulates the Leukemia Stem Cell Program

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

Abstract IA13: RNA regulators and the control of self-renewal

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