Supp Fig 1 : SFRP1 methylation with coculture Supp Fig 2 : Validation of methylation changes with stroma and leukemic cell coculture Supp Fig 3: FRZB upregulation in MDS stroma after 5-Azacytidine treatment SuppFig 4: FRZB knockdown leads to partial inhibition of erythroid differentiation induced by treatment of MDS stromawith 5-Aza SuppFig 5: The expression of WNT signature genes in MDS and control marrow derived CD34+ cells

Sickle cell disease, a complex disorder with known pulmonary complications, has the potential to confound the diagnosis of pulmonary embolism. We hypothesized that when the choice of imaging is guided by chest radiographic results, CT pulmonary angiography (CTPA) and ventilation–perfusion (V/Q) scintigraphy have comparable diagnostic performance in sickle cell disease. Methods: A retrospective cohort of adults with sickle cell disease who were imaged for suspected pulmonary embolism with either CTPA or V/Q, from 2000 to 2016 at our institution, was established. To reduce radiation exposure, our practice recommends V/Q for stable patients with normal chest radiographs. Results of index pulmonary embolism imaging, 90-d follow-up, and results of chest radiography were recorded. Results: Two hundred forty-five adults with sickle cell disease comprised the cohort. The mean age (±SD) was 33 ± 10.5 y, and 58% (141) were men. Index imaging was V/Q in 62.9% (n = 154) and CTPA in 37.1% (n = 91). Chest radiographs, performed in 96.3% (n = 236), were normal in 72.9% (n = 172). Imaging results for pulmonary embolism were negative in 88.2% (n = 216), positive in 4.1% (n = 10), and indeterminate in 7.8% (n = 19) with no difference between V/Q and CTPA (P = 0.63). Reimaging within 90 d occurred in 9.8% (n = 24), 14.7% (20/136) after initial V/Q, and 5% (4/109) after initial CTPA (P = 0.08). Reimaging revealed a pulmonary embolism diagnosis after negative/indeterminate results in 0.7% (1/149) of V/Qs and 1.2% of (1/86) CTPAs (P = 0.69). Over the 17-y study period, 47% (114/245) underwent repeated imaging, and 11% (27/245) were diagnosed with pulmonary embolism at least once. Conclusion: In sickle cell disease patients with suspected pulmonary embolism, positive imaging rates were low for any given clinical presentation, but 11% of the cohort was diagnosed with pulmonary embolism over the 17-y study period. CTPA and V/Q performed comparably for pulmonary embolism diagnosis when the choice of imaging was guided by results of chest radiography. Hence, V/Q is a reasonable first choice for sickle cell disease patients with normal chest radiographs.

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.

Abstract The bone marrow microenvironment influences malignant hematopoiesis, but how it promotes leukemogenesis has not been elucidated. In addition, the role of the bone marrow stroma in regulating clinical responses to DNA methyltransferase inhibitors (DNMTi) is also poorly understood. In this study, we conducted a DNA methylome analysis of bone marrow–derived stromal cells from myelodysplastic syndrome (MDS) patients and observed widespread aberrant cytosine hypermethylation occurring preferentially outside CpG islands. Stroma derived from 5-azacytidine–treated patients lacked aberrant methylation and DNMTi treatment of primary MDS stroma enhanced its ability to support erythroid differentiation. An integrative expression analysis revealed that the WNT pathway antagonist FRZB was aberrantly hypermethylated and underexpressed in MDS stroma. This result was confirmed in an independent set of sorted, primary MDS-derived mesenchymal cells. We documented a WNT/β-catenin activation signature in CD34+ cells from advanced cases of MDS, where it associated with adverse prognosis. Constitutive activation of β-catenin in hematopoietic cells yielded lethal myeloid disease in a NUP98–HOXD13 mouse model of MDS, confirming its role in disease progression. Our results define novel epigenetic changes in the bone marrow microenvironment, which lead to β-catenin activation and disease progression of MDS. Cancer Res; 77(18); 4846–57. ©2017 AACR.

The identity of the RNA-binding proteins (RBPs) that govern cancer stem cells remains poorly characterized. The MSI2 RBP is a central regulator of translation of cancer stem cell programs. Through proteomic analysis of the MSI2-interacting RBP network and functional shRNA screening, we identified 24 genes required for in vivo leukemia. Syncrip was the most differentially required gene between normal and myeloid leukemia cells. SYNCRIP depletion increased apoptosis and differentiation while delaying leukemogenesis. Gene expression profiling of SYNCRIP-depleted cells demonstrated a loss of the MLL and HOXA9 leukemia stem cell program. SYNCRIP and MSI2 interact indirectly though shared mRNA targets. SYNCRIP maintains HOXA9 translation, and MSI2 or HOXA9 overexpression rescued the effects of SYNCRIP depletion. Altogether, our data identify SYNCRIP as a new RBP that controls the myeloid leukemia stem cell program. We propose that targeting these RBP complexes might provide a novel therapeutic strategy in leukemia.

Tushar D. Bhagat, Si Chen, Matthias Bartenstein, A.,Trevor Barlowe, Dagny Von Ahrens, Gaurav S. Choudhary, Patrick Tivnan, Elianna Amin, Mario Marcondes, Mathijs A. Sanders, Remco M. Hoogenboezem, Suman Kambhampati, Nandini Ramachandra, , Iaonnis Mantzaris, Vineeth Sukrithan, Remi Laurence, Robert Lopez, Prafullla Bhagat, Orsi Giricz, Davendra Sohal, Amittha Wickrema, Cecilia Yeung, Kira Gritsman, Peter Aplan, Konrad Hochedlinger, Yiting Yu, Kith Pradhan, Jinghang Zhang, John M. Greally, Siddhartha Mukherjee, Andrea Pellagatti, Jacqueline Boultwood, Britta Will, Ulrich Steidl, Marc H.G.P. Raaijmakers, H. Joachim Deeg, Michael G. Kharas*, Amit Verma

Sickle cell disease (SCD) has been associated with an increased incidence of pulmonary embolism (PE) but not an increased incidence of deep vein thrombosis (DVT). Because of this and other chest complications associated with SCD, these individuals undergo frequent imaging. Computerized tomographic pulmonary angiography (CTPA) is the most common diagnostic imaging test for PE but repeated CTPAs in a chronically ill, younger population with potential renal dysfunction may not be optimal. Ventilation perfusion (VQ) scintigraphy is an alternate imaging modality for PE with a generally lower radiation exposure. However, the diagnostic utility of VQ in SCD is uncertain as ventilation/perfusion mismatches from other diseases, such as acute chest syndrome, may be misdiagnosed as PE. With this in mind, we investigated the performance of CTPA and VQ for the diagnosis of PE in patients with SCD.

Myelodysplastic syndromes (MDS) are driven by complex genetic and epigenetic alterations. The MSI2 RNA-binding protein has been demonstrated to have a role in acute myeloid leukaemia and stem cell function, but its role in MDS is unknown. Here, we demonstrate that elevated MSI2 expression correlates with poor survival in MDS. Conditional deletion of Msi2 in a mouse model of MDS results in a rapid loss of MDS haematopoietic stem and progenitor cells (HSPCs) and reverses the clinical features of MDS. Inversely, inducible overexpression of MSI2 drives myeloid disease progression. The MDS HSPCs remain dependent on MSI2 expression after disease initiation. Furthermore, MSI2 expression expands and maintains a more activated (G1) MDS HSPC. Gene expression profiling of HSPCs from the MSI2 MDS mice identifies a signature that correlates with poor survival in MDS patients. Overall, we identify a role for MSI2 in MDS representing a therapeutic target in this disease.

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.

Deregulated epigenetic program is found in many cancers, and genetic aberrations of histone methyltransferases contribute to transformation in myeloid leukemias. Post-transcriptional regulation in leukemia has recently been highlighted as a novel way for maintaining the leukemia stem cell (LSC) program. We have recently demonstrated that Msi2 is required for LSC function in a murine MLL-AF9 leukemia model. We determined that MSI2 maintains the mixed-lineage leukemia (MLL) self-renewal program by interacting and retaining efficient translation of critical MLL regulated transcription factors including Hoxa9, Myc and Ikzf2. Despite extensive studies implicating Myc and Hoxa9 in leukemia, the role for Ikzf2 in myeloid leukemia is not known.

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.

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.

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.

Hematopoiesis is a tightly regulated process in which different cell lineages of the blood and immune system are generated from hematopoietic stem cells (HSC). HSCs can self renew and also give rise to more differentiated progenitor cells through symmetric and asymmetric cell division respectively. Progenitors can further differentiate into more committed cells that can generate the mature lymphoid and myeloid compartments. In order to support a normal hematopoietic system HSCs must maintain normal cell fate decisions between symmetric and asymmetric divisions. Recent studies from our group and others have implicated Msi2 as a regulator of HSCs. Nevertheless, the exact role for Msi2 in HSCs and critical pathways regulated by Msi2 in these stem cells remains unclear. In addition to its high expression in normal HSC, MSI2 is upregulated in patients with poor clinical prognosis in acute myeloid leukemia and in the blast crisis phase of chronic myelogenous leukemia. To understand the role of Msi2 in normal HSCs, we are characterizing mice with a conditional deletion of Msi2 (Mx1-Cre::Msi2flox/flox mice). We previously demonstrated that the conditional ablation of Msi2 results in the failure of HSC maintenance due to a loss of quiescence and increased commitment divisions. Although Msi2 is critical for HSC engraftment, we observed a preferential requirement for Msi2 in the myeloid biased HSCs (My-HSCs). Based on the surface markers LSK CD34- CD150 high we found a ∼3 fold reduction in the frequency of My-HSCs and no differences in the frequency of the unbiased HSCs or in common lymphoid progenitors. Consistent with these results, we observed a dramatic decrease in chimerism in the myeloid versus lymphoid compartment of the Msi2 deleted cells in mice that were transplanted. In order to understand why hematopoietic stem and progenitors cells require Msi2, we previously performed global transcriptome profiling and RNA target analysis using high throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). This overlap analysis implicated a variety of pathways including RNA translation, HSC self-renewal and TGF-β. The TGF-β pathway has been linked to the maintenance of both normal and leukemia stem cell self-renewal, and the control of myeloid biased HSCs. We examined the output of the TGF-β pathway in HSCs in the presence and absence of Msi2. We found reduced phosphorylation of Smad2/Smad3 in Msi2D/D HSCs grown in vitro or directly isolated from bone marrow. Recently, it has been reported that the loss of p57 in HSCs results in reduced quiescence and compensatory up-regulation of p27. Consistent with reduced signaling output of TGF-β, we observed a significant decrease in p57 expression and up-regulation of p27 in Msi2D/D LSKs. The effect of TGF-β treatment is biphasic in hematopoietic stem cells, with high levels of TGF-β blocking proliferation and low levels of this pathway activation leading to increased proliferation. We therefore examined the functional response of Msi2D/D HSCs to biphasic TGF-β signaling in vitro. Msi2D/D HSCs responded normally to high dose of exogenous TGF-β resulting in potent growth suppression. In contrast, when exposed to lower levels of TGF-β that has been shown to expand normal HSCs, Msi2D/D HSCs failed to respond, consistent with diminished sensitivity to the proliferative effects of TGF-β. These results demonstrate that the proliferative signals downstream of TGF-β signaling are impaired in the Msi2 deleted HSCs. Most intriguingly, our data suggests that Msi2 is required to maintain the response to TGF-β signaling and this contributes to the maintenance and fate of the My-HSCs. It will be interesting to find out if Msi2 is involved in the prevalence of My-HSCs during aging. Taken together, these results have important implications for understanding how the activity of RNA binding proteins contribute to HSC biology that govern normal tissue homeostasis. Disclosures: No relevant conflicts of interest to declare.

Abstract Abstract 2324 Hematopoietic stem cells (HSC) must maintain normal cell fate decisions between symmetric and asymmetric divisions as alterations can lead to hematopoietic malignancies. The MSI2 RNA binding protein is upregulated in patients with a poor clinical prognosis in acute myeloid leukemia and in the blast crisis phase of chronic myelogenous leukemia. The related RNA-binding protein Msi1 has been shown to block translation of specific target mRNAs by interacting with the 3'UTR. To understand the role of Msi2 in both normal and leukemic contexts, we are characterizing mice with a conditional deletion of Msi2 in the hematopoietic compartment. Msi2 conditional knockouts have reduced overall number of HSCs in the bone marrow and have decreased engraftment capacity in congenic transplants, however the molecular mechanisms through which Msi2 elicits these effects remains unclear. To understand how Msi2 alters HSC self renewal, we utilized Mx1-Cre::Msi2flox/flox conditional mice, enabling Msi2 inactivation via poly(I):poly(C) injection. We first tested if Msi2 deleted HSCs or MPPs had an intrinsic defect in cell fate decision and proliferation. We performed proliferation and colony assays on sorted HSCs and MPPs and found reduced cellular numbers in both the HSCs and MPPs with an increase in the frequency of more differentiated cells based on Mac/Gr1 staining after seven days. These reduced overall cell numbers in vitro may have resulted from a defect in maintaining the stem cell population or a decrease in proliferative capacity. To assess if there was a defect in the initial HSC cell division, we examined Numb protein levels and distribution as a surrogate readout for asymmetric cell division. Numb mRNA is a known target of Msi binding and translational repression. Surprisingly, in the Msi2 null HSC or MPPs we observed no difference in the overall staining of Numb indicating that Msi2 deficiency does not globally alter Numb levels. However, we did observe a decrease in the percentage of cells that underwent asymmetric Numb segregation in the MPPs and an increase in the percentage of cells that showed increased Numb staining in some of the daughter pairs in both HSCs and MPPs, indicating increased commitment away from the hematopoietic stem and progenitors. Although controversial, Notch signaling has been implicated in self renewal of HSCs and as a critical downstream target of the Msi family through Numb inactivation. However, the Notch signaling pathway was not noted to be significantly altered in gene set analysis from microarrays performed on Msi2 deleted HSC enriched populations. Moreover using quantitative PCR for Notch target genes in sorted Linlow, c-kit+ and Sca+(LSK) cells we found no statistical difference in the expression of Notch targets (Notch1, Notch2, Hes1 or Myc). These interesting negative results prompted us take a more global approach in characterizing the direct targets of Msi2 in hematopoietic cells. Due to the requirement for a large number of cells, we utilized K562 cells overexpressing a FLAG-tagged version of MSI2 to identify its direct RNA-binding targets. Using recently developed technologies, we performed experiments with UV-cross-linked and immunoprecipitated MSI2 which was then followed by RNA-sequencing (HITS-CLIP) to identify the global direct binding. Our analysis characterized the distribution of binding across the genome. Additionally, gene set enrichment analysis (GSEA) indicated a positive correlation of genes that were upregulated in the Msi2 deleted LSKs and the top 3-fold bound RNA targets (2,713 genes). Using the entire RNA target list we identified gene set signatures including “Cell Cycle”, “Self-renewal” and “HSC to CMP” that matched our results in the microarray from Msi2 deleted LSKs. In summary, direct RNA target analysis for MSI2 in human leukemia cells overlapped with self renewal and differentiation gene sets in mouse HSC enriched populations and correlated with the phenotypes we observed in isolated HSCs grown in vitro lacking Msi2. These results uncover the complexity of MSI2 RNA binding targets and have important implications for both normal and leukemic stem cell biology. Disclosures: Ebert: Celgene: Consultancy; Genoptix: Consultancy.