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Notch signaling is important for tumor angiogenesis induced by vascular endothelial growth factor A. Blockade of the Notch ligand Dll4 inhibits tumor growth in a paradoxical way. Dll4 inhibition increases endothelial cell sprouting, but vessels show reduced perfusion. The reason for this lack of perfusion is not currently understood. Here we report that inhibition of Notch signaling in endothelial cell using an inducible binary transgenic system limits VEGFA-driven tumor growth and causes endothelial dysfunction. Neither excessive endothelial cell sprouting nor defects of pericyte abundance accompanied the inhibition of tumor growth and functional vasculature. However, biochemical and functional analysis revealed that endothelial nitric oxide production is decreased by Notch inhibition. Treatment with the soluble guanylate cyclase activator BAY41-2272, a vasorelaxing agent that acts downstream of endothelial nitric oxide synthase (eNOS) by directly activating its soluble guanylyl cyclase receptor, rescued blood vessel function and tumor growth. We show that reduction in nitric oxide signaling is an early alteration induced by Notch inhibition and suggest that lack of functional vessels observed with Notch inhibition is secondary to inhibition of nitric oxide signaling. Coculture and tumor growth assays reveal that Notch-mediated nitric oxide production in endothelial cell requires VEGFA signaling. Together, our data support that eNOS inhibition is responsible for the tumor growth and vascular function defects induced by endothelial Notch inhibition. This study uncovers a novel mechanism of nitric oxide production in endothelial cells in tumors, with implications for understanding the peculiar character of tumor blood vessels. Cancer Res; 74(9); 2402–11. ©2014 AACR.

PDF file - 3357K, Figure S1. DnMAML-mediated EC-specific Notch blockade inhibits larger vessel function. Figure S2. Flow cytometry analysis of PDGFRβ in LLC-VEGFA tumors of control and double Tg. Figure S3. NO production in the microvasculature of LLC-VEGFA tumors in response to EC-specific Notch inhibition. Figure S4 Effect of the NO-independent soluble guanylyl cyclase agonist BAY41-2272 on tumor growth in EC-specific Notch-inhibited mice. Figure S3. NO production in the microvasculature of LLC-VEGFA tumors in response to EC-specific Notch inhibition. Figure S5. dnMAML-mediated EC Notch blockade inhibits growth, functional blood vessels and eNOS activation of B16F10-VEGFA tumors. Figure S6. Expression analysis of vasodilatory genes Nts and Adm in response to EC-specific Notch inhibition in tumors. Table S1. RT-PCR analysis of Ace2, Edn2, Agt and Ednra in bulk and sorted endothelial cells (EC) from LLC-VEGFA tumors grown in control and double Tg VE-cadherin-tTA x TetOS-dnMAML-GFP.

Interstitial deletion of the long arm of chromosome 5 (del(5q)) is the most common structural genomic variant in myelodysplastic syndromes (MDS)1. Lenalidomide (LEN) is the treatment of choice for patients with del(5q) MDS, but half of the responding patients become resistant2 within 2 years. TP53 mutations are detected in ~20% of LEN-resistant patients3. Here we show that patients who become resistant to LEN harbour recurrent variants of TP53 or RUNX1. LEN upregulated RUNX1 protein and function in a CRBN- and TP53-dependent manner in del(5q) cells, and mutation or downregulation of RUNX1 rendered cells resistant to LEN. LEN induced megakaryocytic differentiation of del(5q) cells followed by cell death that was dependent on calpain activation and CSNK1A1 degradation4,5. We also identified GATA2 as a LEN-responsive gene that is required for LEN-induced megakaryocyte differentiation. Megakaryocytic gene-promoter analyses suggested that LEN-induced degradation of IKZF1 enables a RUNX1-GATA2 complex to drive megakaryocytic differentiation. Overexpression of GATA2 restored LEN sensitivity in the context of RUNX1 or TP53 mutations by enhancing LEN-induced megakaryocytic differentiation. Screening for mutations that block LEN-induced megakaryocytic differentiation should identify patients who are resistant to LEN.

The myelodysplastic syndrome (MDS) are a group of hematological malignancies with the propensity to develop into either acute myeloid leukemia (AML) or bone marrow failure (BMF). Dysregulation of immune and inflammatory responses has been implicated in MDS and other BMF disorders. There is significant evidence for IFNγ playing a key role in MDS and BMF syndromes. However, there are conflicting theories regarding the mechanism by which IFNγ promotes BMF. There is also very little information on the triggers that underlie upregulation of IFNγ in these BMF syndromes. Interstitial deletion of chromosome 5q is the most common cytogenetic abnormality observed in MDS, accounting for approximately 10% of all cases. Our lab has previously shown that miR-145, which is located on the minimally deleted region of chromosome 5q, targets Toll/Interleukin-1 receptor domain containing adaptor protein (TIRAP) - an innate immune adaptor protein. However, the role of TIRAP in marrow failure has not been well elucidated. In this study, we identify a novel role for TIRAP in dysregulating normal hematopoiesis through activation of Ifnγ. Using bone marrow transplants in wild-type mice, we showed that constitutive expression of TIRAP in wild-type hematopoietic stem and progenitor cells (HSPC) caused peripheral blood cytopenia, suppressed the bone marrow endothelial niche and significantly reduced overall survival of the mice (median survival 9 weeks post-transplant) compared to controls (p < 0.0001). RNA-seq analysis of TIRAP expressing HSPC identified several proinflammatory cytokines to be significantly overrepresented. Geneset enrichment analysis (GSEA) identified the Ifnγ response as the single most significantly enriched pathway of the Hallmark genesets. To test the functional role of Ifnγ in TIRAP-mediated BMF, wild-type recipient mice were transplanted with TIRAP- HSPC from Ifnγ-/- donor mice. Mice that received TIRAP-transduced Ifnγ-/- HSPC were rescued from BMF, as evidenced by normalized blood cell counts and improved median survival (median survival 48.6 weeks) (Ifnγ-/- TIRAP vs. wild-type TIRAP: p = 0.0004). Interestingly, in our model of TIRAP induced BMF, myeloid rather than the conventional T and NK cells were the cells most responsible for the increased production of Ifnγ. Further, when we transplanted TIRAP expressing wild-type HSPC into NSG recipient mice, which are deficient in functional B, T and NK cells, the NSG mice developed BMF with pancytopenia in a similar time-frame as wild-type mice. This suggested that T and NK cells are not central for the development of TIRAP induced BMF. Delving deeper into the mechanism by which the TIRAP-Ifnγ axis causes BMF, we saw that while Ifnγ played a direct role in suppressing erythropoiesis and megakaryopoiesis, it played an indirect, Ifnγ receptor independent role on myelopoiesis. TIRAP-induced activation of Ifnγ released the alarmin, Hmgb1, which suppressed the marrow endothelial niche, which in turn promoted myeloid suppression. Overexpression of TIRAP in Ifnγ -/- background blocked Hmgb1 release. Further, blocking Hmgb1 in presence of TIRAP expression was sufficient to reverse the marrow endothelial defect and restore myelopoiesis in vivo. Our findings highlight a novel, non-canonical effect of aberrant TIRAP expression via the Ifnγ-Hmgb1 axis on the endothelial cell component of the marrow microenvironment and hematopoiesis. Further understanding of this pathway would open up avenues for developing new therapies for BMF. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Activation of inflammatory pathways is associated with bone marrow failure syndromes, but how specific molecules impact on the marrow microenvironment is not well elucidated. We report a novel role for the miR-145 target, Toll/Interleukin-1 receptor domain containing adaptor protein (TIRAP), in driving bone marrow failure. We show that TIRAP is overexpressed in various types of myelodysplastic syndromes (MDS), and suppresses all three major hematopoietic lineages.. Constitutive expression of TIRAP in hematopoietic stem/progenitor cells (HSPC) promotes upregulation ofIfnγ, leading to bone marrow failure. Myelopoiesis is suppressed through Ifnγ-Ifnγr-mediated release of the alarmin, Hmgb1, which disrupts the marrow endothelial niche. Deletion ofIfnγor Ifnγr blocks Hmgb1 release and is sufficient to reverse the endothelial defect and prevent myelosuppression. In contrast, megakaryocyte and erythroid production is repressed independently of the Ifnγ receptor. Contrary to current dogma, TIRAP-activated Ifnγ-driven marrow suppression is independent of T cell function or pyroptosis. In the absence of Ifnγ, TIRAP drives myeloproliferation, implicating Ifnγ in suppressing the transformation of bone marrow failure syndromes to myeloid malignancy. These findings reveal novel, non-canonical roles of TIRAP, Hmgb1 and Ifnγ function in the marrow microenvironment,and provide insight into the pathophysiology of preleukemic syndromes.Graphical Abstract: Model of proposed mechanism of TIRAP-induced BMFConstitutive TIRAP expression in marrow cells releases Ifnγ, which directly impacts on megakaryocyte and erythroid production, but indirectly suppresses myelopoiesis through the release of the alarmin, Hmgb1, which disrupts the marrow endothelial compartment.

Interstitial deletion of the long arm of chromosome 5 (del(5q)) is the most common structural genomic variant in myelodysplastic syndromes (MDS)

Summary: The sterile alpha motif (SAM) and SRC homology 3 (SH3) domain containing protein 1 (Sash1) acts as a scaffold in TLR4 signaling. We generated Sash1−/− mice, which die in the perinatal period due to respiratory distress. Constitutive or endothelial-restricted Sash1 loss leads to a delay in maturation of alveolar epithelial cells causing reduced surfactant-associated protein synthesis. We show that Sash1 interacts with β-arrestin 1 downstream of the TLR4 pathway to activate Akt and endothelial nitric oxide synthase (eNOS) in microvascular endothelial cells. Generation of nitric oxide downstream of Sash1 in endothelial cells affects alveolar epithelial cells in a cGMP-dependent manner, inducing maturation of alveolar type 1 and 2 cells. Thus, we identify a critical cell nonautonomous function for Sash1 in embryonic development in which endothelial Sash1 regulates alveolar epithelial cell maturation and promotes pulmonary surfactant production through nitric oxide signaling. Lung immaturity is a major cause of respiratory distress and mortality in preterm infants, and these findings identify the endothelium as a potential target for therapy. : Surfactant deficiency due to lung immaturity is a major cause of respiratory distress in premature newborns. Coulombe et al. show that endothelial SAM and SH3 domain containing protein 1 (Sash1) drives perinatal lung maturation via nitric oxide signaling to alveolar cells. Sash1 interacts with β-arrestin1 to activate Akt-eNOS and induce alveolar epithelial cell maturation and surfactant synthesis. Keywords: TLR4, Sash1, surfactant, endothelium, alveolar type 2 cells, respiratory distress, lung development, β-arrestin, nitric oxide

Acute myeloid leukemia (AML) is the most common adult leukemia, and AML patients have a 5-year survival rate of We performed quantitative tandem mass spectrometric analysis of FBXO11 co-immunoprecipitating proteins in FBXO11 CRISPR/Cas9 knockout (KO) and control clones from K562 cells to identify FBXO11-regulated targets. The top target, ABCF1, regulates METTL3 expression, which is required for maintaining leukemic state, and enhances protein synthesis. Accordingly, we found increased global protein synthesis in FBXO11 KO clones. FBXO11 targets are enriched in glutathione metabolism-related proteins, which are frequently dysregulated in CD34+ AML cells. We found FBXO11 KO cells have increased sensitivity to inhibition of glutathione redox reactions by piperlongumine. FBXO11 loss likely has pleiotropic effects that contribute to leukemogenesis. We identified novel candidate targets of FBXO11. With the commonality of SCF-FBXO11 perturbations in AML, this could lead to development of new and widely applicable therapeutic options.

The immunomodulatory drug lenalidomide (LEN) is the treatment of choice for del(5q) MDS patients. LEN has been shown to trigger the specific degradation of CSNK1A1 and IKZF1 proteins after binding the E3-ligase substrate adaptor CRBN. When brought below a certain expression threshold, CSNK1A1 deficiency activates a p53-dependent apoptotic response. Thus, the unique sensitivity of del(5q) cells to LEN is explained by CSNK1A1 haploinsufficiency in del(5q) MDS patients. Despite its efficacy, 50% of LEN-treated patients eventually relapse within an interval of 2-3 years after treatment. Treatment failure is associated to low platelet counts and occurrence of additional mutations, such as TP53. To identify novel genetic determinants of LEN resistance, we have compared whole genome sequencing data of paired samples from six del(5q) patients who have been treated with LEN and eventually became resistant to the treatment. We identified 2 patients with mutations in TP53. The remaining four presented RUNX1 alterations: two patients had protein coding mutations in RUNX1 and two had a significant reduction in RUNX1, but not TP53, transcript levels. As a model of sensitivity, we studied the response to LEN in two human del(5q) cell lines, MDS-L and KG-1a. RUNX1 protein levels are postranscriptionally upregulated upon exposure to LEN, accompanied by increased levels of RUNX1 activity. Deletion of CRBN expression cancelled these effects. RUNX1 overexpression inhibited clonogenic growth and induced apoptosis. We then generated RUNX1 knock-out (KO) clones derived from MDS-L cells using CRISPR/Cas9 system. RUNX1 KO cells presented increased proliferation, increased colony growth and reduced apoptosis in the presence of LEN compared to wild-type (WT) control clones. These results were validated with different shRNAs against RUNX1. Genetic rescue experiments showed that RUNX1 mutants were unable to restore sensitivity to the drug compared to RUNX1 WT. Finally, modeling RUNX1 loss-of-function (LOF) in CSNK1A-depleted human CD34+ cells abrogated the effects of LEN on colony forming cell assays. Thus, RUNX1 function is required for the elimination of del(5q) cells by LEN. To understand the molecular mechanisms underlying the resistant phenotype, we performed RNA-seq on MDS-L cells treated with LEN for 24h. We observed a significant upregulation of Platelet specific genes (ITGB3, ITGA2B, VWF, THBD, SELP, TREML1, GATA1) coupled to downregulation of Cell Cycle genes (E2F2, E2F1, MCM5, CDKN1A), suggesting that LEN induces differentiation in to the Megakaryocytic (Meg) lineage. We found a significant upregulation of CD41+/CD61+ double positive cells after LEN exposure in vitro and in vivo, associated to the appearance of multinucleated cells. Importantly, the apoptotic response was associated to the emergence of the differentiating population. At the molecular level, CRBN is required for LEN-induced differentiation. Further downstream we identified IKZF1 degradation as key trigger, as IKZF1 overexpression restrained Meg differentiation and a IKZF1 dominant negative isoform enhanced it. In contrast, CSNK1A overexpression did not alter differentiation after LEN, but did reduce apoptotic induction. Moreover, we identified GATA2 targets enriched in LEN-regulated genes and showed that GATA2 overexpression or downregulation using shRNAs significantly increased or reduced LEN induced differentiation respectively. Finally, gene expression analysis after LEN exposure showed that Meg signatures were not enriched in resistant RUNX1 KO cells compared to WT control. Accordingly, RUNX1 KO cells did not undergo differentiation upon LEN exposure. RUNX1 LOF in CSNK1A-depleted primary human CD34+ cells blocked CFU-Mk growth in LEN treated cells. GATA2 overexpression was unable to restore LEN effects in RUNX1 deficient cells, suggesting a cooperative mechanism between both transcription factors. Luciferase assays using the human CD41 promoter showed that RUNX1 mutants reduced promoter transactivation compared to RUNX1 WT. Remarkably, we observed a similar phenotype for LEN-resistant TP53 KO cells. As a conclusion, our results suggest that GATA2, RUNX1 and TP53 cooperate to drive Meg differentiation after LEN-mediated degradation of IKZF1 protein. Loss of function mutations affecting RUNX1 or TP53 alter the activity of GATA2 transcriptional complex, rendering del(5q) cells unresponsive to LEN. Disclosures Platzbecker: Celgene: Research Funding.

Background.Clostridium difficileis a major cause of gastrointestinal illness. Epidemic NAP1 strains contain toxins A and B, a deletion in repressortcdC, and a binary toxin.Objectives. To determine the molecular epidemiology ofC. difficilein British Columbia and compare between two time points in one region.Methods.C. difficileisolates from hospital and community laboratories (2008) and one Island Health hospital laboratory (2013) were characterized by pulsed-field gel electrophoresis, PCR-ribotyping, toxin possession,tcdCgenotype, and antimicrobial susceptibility.Results. In 2008, 42.7% of isolates had NAP1 designation. Hospital-collected isolates were associated with older patients and more NAP1 types. Unlike other isolates, most NAP1 isolates possessed binary toxin and a 19 bp loss intcdC. All isolates were susceptible to metronidazole and vancomycin. A 2013 follow-up revealed a 28.9% decrease in NAP1 isolates and 20.0% increase in isolates without NAP designation in one region. Then, community-associated cases were seen in younger patients, while NAP types were evenly distributed. Isolates without NAP designation did not cluster with a PFGE pattern or ribotype.Conclusions. Evaluation ofC. difficileinfections within British Columbia revealed demographic associations, epidemiological shifts, and characteristics of strain types. Continuous surveillance ofC. difficilewill enable detection of emerging strains.

Pericytes are perivascular support cells, the origin of which in tumor tissue is not clear. Recently, we identified a Tie1(+) precursor cell that differentiates into vascular smooth muscle, in a Notch-dependent manner. To understand the involvement of Notch in the ontogeny of tumor pericytes we used a novel flow immunophenotyping strategy to define CD146(+)/CD45(-)/CD31(-/lo) pericytes in the tumor stroma. This strategy combined with ex vivo co-culture experiments identified a novel pericyte progenitor cell population defined as Sca1(hi)/CD146(-)/CD45(-)/CD31(-). The differentiation of these progenitor cells was stimulated by co-culture with endothelial cells. Overexpression of the Notch ligand Jagged1 in endothelial cells further stimulated the differentiation of Sca1(hi)/CD146(-)/CD45(-)/CD31(-) cells into pericytes, while inhibition of Notch signaling with a γ-secretase inhibitor reduced this differentiation. However, Notch inhibition specifically in Tie1-expressing cells did not change the abundance of pericytes in tumors, suggesting that the pericyte precursor is distinct from the vascular smooth muscle cell precursor. Transplant experiments showed that the bone marrow contributes minimally to tumor pericytes. Immunophenotyping revealed that Sca1(hi)/CD146(-)/CD45(-)/CD31(-) cells have greater potential to differentiate into pericytes and have increased expression of classic mesenchymal stem cell markers (CD13, CD44, Nt5e and Thy-1) compared to Sca1(-/lo)/CD146(-)/CD45(-)/CD31(-) cells. Our results suggest that a local Sca1(hi)/CD146(-)/CD45(-)/CD31(-) pericyte progenitor resides in the tumor microenvironment and requires Notch signaling for differentiation into mature pericytes.

The histone methyltransferase EZH2 is frequently mutated in germinal center-derived diffuse large B-cell lymphoma and follicular lymphoma. To further characterize these EZH2 mutations in lymphomagenesis, we generated a mouse line where EZH2(Y641F) is expressed from a lymphocyte-specific promoter. Spleen cells isolated from the transgenic mice displayed a global increase in trimethylated H3K27, but the mice did not show an increased tendency to develop lymphoma. As EZH2 mutations often coincide with other mutations in lymphoma, we combined the expression of EZH2(Y641F) by crossing these transgenic mice with Eµ-Myc transgenic mice. We observed a dramatic acceleration of lymphoma development in this combination model of Myc and EZH2(Y641F). The lymphomas show histologic features of high-grade disease with a shift toward a more mature B-cell phenotype, increased cycling and gene expression, and epigenetic changes involving important pathways in B-cell regulation and function. Furthermore, they initiate disease in secondary recipients. In summary, EZH2(Y641F) can collaborate with Myc to accelerate lymphomagenesis demonstrating a cooperative role of EZH2 mutations in oncogenesis. This murine lymphoma model provides a new tool to study global changes in the epigenome caused by this frequent mutation and a promising model system for testing novel treatments.

Notch signaling is important for tumor angiogenesis induced by vascular endothelial growth factor A. Blockade of the Notch ligand Dll4 inhibits tumor growth in a paradoxical way. Dll4 inhibition increases endothelial cell sprouting, but vessels show reduced perfusion. The reason for this lack of perfusion is not currently understood. Here we report that inhibition of Notch signaling in endothelial cell using an inducible binary transgenic system limits VEGFA-driven tumor growth and causes endothelial dysfunction. Neither excessive endothelial cell sprouting nor defects of pericyte abundance accompanied the inhibition of tumor growth and functional vasculature. However, biochemical and functional analysis revealed that endothelial nitric oxide production is decreased by Notch inhibition. Treatment with the soluble guanylate cyclase activator BAY41-2272, a vasorelaxing agent that acts downstream of endothelial nitric oxide synthase (eNOS) by directly activating its soluble guanylyl cyclase receptor, rescued blood vessel function and tumor growth. We show that reduction in nitric oxide signaling is an early alteration induced by Notch inhibition and suggest that lack of functional vessels observed with Notch inhibition is secondary to inhibition of nitric oxide signaling. Coculture and tumor growth assays reveal that Notch-mediated nitric oxide production in endothelial cell requires VEGFA signaling. Together, our data support that eNOS inhibition is responsible for the tumor growth and vascular function defects induced by endothelial Notch inhibition. This study uncovers a novel mechanism of nitric oxide production in endothelial cells in tumors, with implications for understanding the peculiar character of tumor blood vessels. Cancer Res; 74(9); 2402–11. ©2014 AACR.

Valvuloseptal defects are the most common congenital heart defects. Notch signaling-induced endothelial-to-mesenchymal transition (EMT) in the atrioventricular canal (AVC) cushions at murine embryonic day (E)9.5 is a required step during early valve development. Insights to the transcriptional network that is activated in endocardial cells (EC) during EMT and how these pathways direct valve maturation are lacking.We show that at E11.5, AVC-EC retain the ability to undergo Notch-dependent EMT when explanted on collagen. EC-Notch inhibition at E10.5 blocks expression of known mesenchymal genes in E11.5 AVC-EC. To understand the genetic network and AVC development downstream of Notch signaling beyond E9.5, we constructed Tag-Seq libraries corresponding to different cell types of the E11.5 AVC and atrium in wild-type mice and in EC-Notch inhibited mice. We identified 1,400 potential Notch targets in the AVC-EC, of which 124 are transcription factors (TF). From the 124 TFs, we constructed a transcriptional hierarchy and identify 10 upstream TFs within the network.We validated 4 of the upstream TFs as Notch targets that are enriched in AVC-EC. Functionally, we show these 4 TFs regulate EMT in AVC explant assays. These novel signaling pathways downstream of Notch are potentially relevant to valve development.

Myelodysplastic syndromes (MDS) are a collection of hematopoietic malignancies in which genomic abnormalities within the hematopoietic stem cell (HSC) compartment results in dysplasia of the marrow cells and ineffective hematopoiesis. As a result, the primary cause of mortality in these patients is eventual bone marrow failure although MDS patients also have a significantly increased risk of transformation to acute myeloid leukemia (AML). The most common karyotypic change in MDS is an interstitial deletion of the long arm of chromosome 5, del(5q) MDS. Patients with an isolated interstitial deletion of chromosome 5q are referred to as having 5q- syndrome. Mapping of the commonly deleted region (CDR) within 5q- syndrome has identified a 1.5-megabase region on band 5q32. MicroRNA (miRNA) -143 and -145 are located within the CDR of del(5q) MDS and have been implicated in the pathogenesis of the disease. However, their functional role in myelodysplastic syndromes has not been well studied. To investigate the role of miR-143 and miR-145, we utilized a gene-targeted mouse model containing deletion of miR-143 and miR-145. Here we show that mouse marrow lacking miR-143 and miR-145 have a decrease in short-term repopulating HSC and progenitors of the myeloid lineage by flow cytometry, as well as of hematopoietic progenitor activity using colony forming assays. Additionally, we performed a limiting dilution assay of miR-143-/-145-/- bone marrow and observed significantly fewer functional HSCs compared to wildtype marrow. To explore the molecular mechanism behind this defect, we performed Ingenuity Pathway Analysis of the predicted targets of miR-143 and miR-145. We identified the transforming growth factor-beta (TGFβ)-signaling pathway as a common target of these two miRNAs. Gene Set Enrichment Analysis of del(5q) using mRNA expression of MDS patient CD34+ marrow cells show an enriched TGFβ-signature compared to healthy controls. In addition, the defect in hematopoietic progenitor activity in miR-143-/-145-/- marrow can be rescued by inhibiting Smad3 using the chemical inhibitor SIS3. We validated the TGFβ pathway adaptor protein, Disabled-2 (DAB2), as a target of miR-145 and show that TGFβ signaling is activated upon loss of miR-145 or enforced expression of DAB2. Enforced expression of DAB2 in mouse marrow is able to recapitulate many of the features of miR-143-/-145-/- mice. DAB2 overexpressing marrow formed significantly fewer colonies in progenitor assays, and in competitive transplants, vector-transduced marrow was able to out compete DAB2-overexpressing marrow in both primary transplants as well as in secondary limiting dilution assays. Interestingly, compared to wildtype mice, aged miR-143-/-145-/- mice showed decreased hemoglobin and platelet counts with elevated white blood cell counts. This phenotype was also observed in a subset of mice with enforced DAB2 expression where a proportion of mice developed a transplantable myeloproliferative disorder. Together, our data identifies a role for miR-143 and miR-145 in the pathogenesis of del(5q) MDS where their loss results in a defect in HSC activity. We observe that the TGFβ signaling pathway is activated in patient marrow and we validate DAB2 as a direct target of miR-145. We provide evidence that the defect observed in miR-143-/-145-/- marrow is mediated in part by DAB2 where its enforced expression leads to a defect in HSC self-renewal but contributes to myeloproliferation. Disclosures Karsan: Celgene: Research Funding.

The Myelodysplastic Syndromes (MDS) are the most common hematological malignancies arising from stem/progenitor cells. MDS is characterized by ineffective hematopoiesis in one or more lineages of the bone marrow, resulting in peripheral cytopenias and the propensity to progress to either acute myeloid leukemia (AML) or bone marrow failure (BMF). The most common cytogenetic aberration associated with MDS is deletion of the long arm of chromosome 5. Many of the molecular events involved in the development of del(5q) MDS have been elucidated including haploinsufficiency of the gene encoding the ribosomal protein RPS14, responsible for the anemia observed, and haploinsufficency of the miRNAs miR-145 and miR-146a, which together target the innate immune signaling pathway, specifically, the Toll-like receptor-4 (TLR-4)signalling pathway. It has been demonstrated that overexpression of a target of miR-146a,TRAF6, in mouse bone marrow can recapitulate the phenotype of del(5q) MDS including the cytopenias and progression to BMF or AML. However, enforced expression of TIRAP, a miR-145 target gene, results in rapid BMF independent of TRAF6. The molecular and cellular mechanisms responsible for the differential outcome of overexpression of two genes that act within the same signalling pathway remain to be fully understood. We have identified several differentially expressed cytokines, including interferon gamma (IFNγ) and interleukin-10 (IL-10), following TIRAP overexpression compared with TRAF6 overexpression. Promoter methylation analysis has shown hypermethylation of key adaptors and signal transducers that lie between TIRAP and TRAF6 in the TLR-4 signalling pathway, suggesting activation of different pathways by TIRAP and TRAF6 overexpression. Indeed, blockade of TRAF6 and MyD88 did not inhibit TIRAP induced expression of these cytokines, suggesting that IFNγ and IL-10 production occurs in a TRAF6 and MyD88 independent manner. We identified IFNγ as the critical effector cytokine responsible for TIRAP mediated marrow failure. Gene set enrichment analysis has shown an enrichment of an IFNγ signature in MDS patients with a low risk of transformation to AML compared to healthy controls. Furthermore, interferon signatures were highly enriched in MDS patients compared to patients with AML, suggesting an important role for IFNγ signaling in driving MDS progression toward marrow failure as opposed to leukemic progression. IFNγ has been shown to inhibit components of the bone marrow niche by blocking RANK signalling in stromal cells such as osteoclast progenitors. Using coculture of TIRAP expressing bone marrow cells with the RAW264.7 monocyte cell line, a cell line that is capable of differentiation into osteoclasts, we found an inhibition in the ability of these cells to form osteoclasts compared to control. This provides the first line of evidence suggesting that immune signalling defects arising from genetic perturbations in the hematopoietic stem cell compartment can result in stem cell niche dysfunction leading to marrow failure. Disclosures No relevant conflicts of interest to declare.

The histone methyl-transferase EZH2 is frequently affected by gain-of-function mutations in germinal-center lymphoma. To further test if these EZH2 mutations can be driver mutations in lymphomagenesis, we have previously generated and characterized a mouse line transgenic for EZH2Y641F. This mouse model leads to an acceleration of lymphoma development in combination with Myc. We have now further investigated this model system and have used RNAseq and ChIPseq to characterize the effects of mutated EZH2 on gene expression and the epigenome. We have previously shown that in contrast to lymphomas observed with Emu-Myc alone, all of the lymphomas observed in mice transgenic for both, Myc and EZH2Y641F, present with a B220+IgM+phenotype. We have now characterized the accumulating B cell subset by performing in-depth immunophenotyping. This analysis showed that the cells are IgDlo, CD21-, CD23- and partially express AA4.1. This marker combination is consistent with the transitional stage (T1) of B cell development. For the global gene expression and histone methylation analysis, splenic B cells (B220+) were isolated from one Eμ-Myc and one Eμ-Myc/EZH2Y641F mouse before the onset of disease symptoms. The samples were then processed for RNA sequencing and ChIP sequencing. ChIP was performed using validated antibodies for H3K4me3 and H3K27me3. Of the 22,137 genes studied we observed that 1,112 (412) genes were down-regulated and 788 (209) genes were up-regulated in Eμ-Myc/EZH2Y641F mice with p-value 0.05 and a minimum number of reads of 30 were considered. An integrated analysis of the RNA sequencing data with the ChIP sequencing data for H3K27me3 and H3K4me3 was performed, and indicated that the altered epigenome of the Eμ-Myc/EZH2Y641F mouse impacted protein-coding gene expression. This analysis showed that genes down-regulated in the Eμ-Myc/EZH2Y641F mouse have increased H3K27me3 marks at their transcription start site indicative of a significant fraction of these genes being regulated by this mark. In contrast, genes up-regulated in the Eμ-Myc/EZH2Y641Fmouse mainly exhibited increased H3K4Me3 marks at their transcription start site. We then used DAVID/KEGG and the MGSA package to identify pathways associated with mutated EZH2 and identified GO terms that were enriched by the differentially expressed genes. Interestingly this analysis returned many important pathways in B cell regulation and immune function. The identified pathways include the B cell receptor signalling pathway and the JAK-STAT signaling pathway. We previously observed an increase in the proliferation rate of splenic B cells in Eμ-Myc/EZH2Y641Fmice. While cell-cycle genes were not specifically enriched, there were several of these genes that were found deregulated (including e. g. CyclinD1). In summary, we were able to identify several key pathways that may be contributing to the acceleration of lymphoma development observed with EZH2Y641F and may also be important for the understanding of pathogenesis of EZH2 mutated lymphoma. Disclosures: No relevant conflicts of interest to declare.

Maintenance of blood homeostasis depends on the balance between self-renewal of hematopoietic stem cells (HSCs) and their differentiation into blood cell progenitors. A variety of different intrinsic or extrinsic regulators, including multiple microRNA (miRNA) species, have been described to play a role in the regulation of these processes. Disruption of any of these regulators could lead to stem cell exhaustion or increased risk of leukemogenesis. Given recent reports of the role of miR-146a in malignant hematopoiesis, we evaluated its role in hematopoietic stem progenitor cell (HSPC) function. We show that miR-146a is highly expressed in HSCs and its expression decreases in committed progenitors. miR-146a- deficient HSCs had dramatically reduced self-renewal capacity as measured by serial competitive bone marrow transplantation assays. The lower self-renewal capacity was accompanied by decreased quiescence in miR-146a-deficient cells, as revealed by decreased proportion of miR-146a-/- HSPCs (Lin- Sca-1+ c-kit-, LSK) in G0 of the cell cycle (Ki-67- negative), and their increased proliferation, measured by BrdU incorporation. We further showed that increased proliferation of HSPCs is cell intrinsic. By sorting EPCR+ CD48- CD150+ (ESLAM) HSCs and examining cell division kinetics at the single cell level, we found that miR-146a-/- HSC undergo cell division earlier and differentiate more rapidly than wild-type HSCs, thereby producing larger colonies containing more differentiated (Lin+) cells. Our data provide evidence that miR-146a loss attenuates HSC quiescence and impairs their self-renewal ability, leading to hyperproliferation of progenitor cells. The phenotype seen is cell autonomous and the findings suggest that miR-146a plays a critical role in maintaining long term HSC function.

Abstract 555 The Myelodysplastic Syndromes (MDS) are the most common hematological malignancies arising from stem/progenitor cells. MDS is characterized by ineffective hematopoiesis in one or more lineage of the bone marrow, resulting in peripheral cytopenias and the propensity to develop into either acute myeloid leukemia (AML) or bone marrow failure (BMF). The molecular and cellular events involved in MDS development are yet to be elucidated. Large scale cytogenetic aberrations such as del(5q) have been associated with many types of MDS. Previous work from our lab has shown that the chromosomal band 5q32–33 harbours microRNAs (miR-145 and miR-146a), haploinsufficiency of which recapitulates the megakaryocytic dysplasia and neutropenia associated with del(5q) MDS. Ingenuity pathway analysis predicts that innate immune signaling is one of the most highly deregulated pathways by loss of these two miRNAs. Two innate immune signaling molecules, TNF receptor associated factor 6 (TRAF6) and Toll/interleukin-1 receptor associated protein (TIRAP), are targets ofmiR-146a and miR-145 respectively. While the role of TRAF6 in del(5q) MDS has been investigated, little is known about the role of TIRAP in MDS pathogenesis. To investigate the role of TIRAP in aberrant hematopoiesis, we transplanted lethally irradiated mice with bone marrow cells expressing TIRAP or vector control. TIRAP transplanted mice develop an MDS-like bone marrow failure characterized by anemia, neutropenia, and thrombocytopenia as early as 4 weeks post-transplant (unlike TRAF6 transplanted mice which succumb to BMF or AML approximately 4 months post-transplant). The discrepancy between TIRAP and TRAF6 disease progression models suggests that TRAF6 independent signaling originating from TIRAP may be responsible for the rapid bone marrow failure onset. Furthermore, TIRAP transplanted mice display hypocellular marrows characterized by increased apoptosis as measured by Annexin V/PI staining, similar to low risk MDS patients. In MDS, normal hematopoiesis is blocked in the non-disease fraction of bone marrow by autoimmunity associated cytokines. To identify factors that may be responsible for suppression of normal hematopoiesis in our BMF model, we performed cytokine expression profiling of TIRAP expressing marrow. Quantitative RT-PCR showed increased expression of both IL-10 and IFNγ in TIRAP expressing marrow compared to vector control. Interestingly, IFNγ is known to suppress hematopoiesis has been shown to be overexpressed in BM mononuclear cells in MDS and other BMF conditions. Also, IL-10 levels have been shown to be elevated in high risk MDS patients. To investigate the role of IL-10 and IFNγ in TIRAP mediated marrow failure, we transduced IL-10 −/− and IFNγ −/− marrow with TIRAP or vector control, and transplanted wildtype leathally irradiated mice. Loss of IFNγ but not IL-10 partially rescues the pancytopenia phenotype and prevents early death due to bone marrow failure. Interestingly however, mice transplanted with TIRAP expressing IFNγ −/− bone marrow succumb to a myeloproliferative disorder at later time points, suggesting that the factors responsible for myelosuppression are the same ones responsible for progression to AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 227 The histone methyl-transferase EZH2 is frequently mutated in germinal-center derived diffuse-large B cell lymphoma (DLBCL) and follicular lymphoma. We and others have demonstrated that these mutations in tyrosine 641 of EZH2 represent a dominant gain-of-function, enhancing the capability to trimethylate H3K27 in combination with wildtype activity (Yap et al, Blood 2011; Sneeringer et al, PNAS 2010). To further test if these EZH2 mutations can be driver mutations in lymphomagenesis we have now generated and characterized a mouse line transgenic for EZH2 Y641F (the most commonly observed mutant form). Transgenic mice were created on the C57/Bl6 background by pronuclear microinjection of a construct where EZH2 Y641F is expressed from a lymphoid specific Emu-lck promoter. Spleen cells isolated from the transgenic mice displayed a global increase in H3K27me3 when analyzed by Western Blot. However none of the mice showed signs of lymphoma even when observed for prolonged periods of time. As EZH2 mutations often coincide with other mutations in lymphoma, we also combined the expression of EZH2 Y641F with the over-expression of Myc by crossing one of the transgenic lines with Emu-myc transgenic mice. In this combination model, we observed a dramatic acceleration of lymphoma development with the combination of Myc and EZH2 Y641F as compared to Myc alone (Myc alone (n=26) 137.5 days, Myc+EZH2 Y641F (n=20) 51 days, p < 0.0001 Log-Rank (Mantel-Cox) Test). The resulting lymphomas are characterized by a generalized massive lymphadenopathy, splenomegaly (spleen weight 0.416 +/− 0.133 g), bone marrow infiltration (72 +/− 8 %) and lymphocytosis (WBC 138.5 +/− 77.1 ×1000/μl). They show histological features of high-grade disease, and initiate disease in secondary recipients. In contrast to lymphomas observed with Emu-myc alone, all of the lymphomas observed in mice transgenic for both, Myc and EZH2 Y641F, presented with a mature B cell phenotype (B220+IgM+). This shift in differentiation can also be observed in the bone marrow even before development of overt lymphomas. While Myc transgenics mainly show an increase in the B220+IgM−compartment, this is shifted towards B220+IgM+ in double transgenics. Cell cycle analysis by in vivo Brdu incorporation before disease onset shows a drastic increase in the fraction of cells in cycle in the splenic B cells of double transgenic mice (43.3 and 44.9 % cells in S phase) as compared to mice transgenic for Myc alone (10.6 % and 14.2 % cells in S phase). Southern Blot analysis using a probe specific for JH4 showed more than one clonal rearrangement in a majority of the lymphomas in the double-transgenic mice. In summary, EZH2 Y641F can collaborate with Emu-myc in high efficiency lymphoma induction. This new murine lymphoma model now provides a powerful tool to study global changes in the epigenome caused by this frequent mutation and may be a useful system for testing novel treatments. Disclosures: No relevant conflicts of interest to declare.