Myelodysplastic Syndrome Mice Research Articles

Iron overload impairs normal hematopoietic stem and progenitor cells through reactive oxygen species and shortens survival in myelodysplastic syndrome mice.

There is increasing clinical evidence to suggest a suppressive effect on hematopoiesis in myelodysplastic syndrome patients with iron overload. However, how iron overload influences hematopoiesis in myelodysplastic syndrome (MDS) remains unknown. Here, the RUNX1S291fs-transduced bone marrow mononuclear cells were yielded and transplanted into lethally irradiated recipient mice together with radioprotective bone marrow cells to generate MDS mice. Eight weeks post transplantation, the recipient mice received an intraperitoneal injection of 0.2 mL iron dextran at a concentration of 25 mg/mL once every other day for a total of 8 times to establish an iron overload model. In the present study, we show that iron overload impairs the frequency and colony-forming capacity of normal hematopoietic stem and progenitor cells, especially in erythroid, in MDS mice, which is due, at least in part, to growth differentiation factor 11-induced reactive oxygen species, shortening survival of MDS mice. Given that we are the first to construct an iron overload model in MDS mice, we hope this model will be helpful for further exploring the influence and mechanism of iron overload on MDS.

Bone-targeting nanoparticle to co-deliver decitabine and arsenic trioxide for effective therapy of myelodysplastic syndrome with low systemic toxicity

Myelodysplastic syndromes (MDS) are a diverse group of bone marrow disorders and clonal hematopoietic stem cell disorders characterized by abnormal blood cells, or reduced peripheral blood cell count. Recent clinical studies on combination therapy of decitabine (DAC) and arsenic trioxide (ATO) have demonstrated synergy on MDS treatment, but the treatment can cause significant side effects to patients. In addition, both drugs have to be administered on a daily basis due to their short half-lives. In addressing key issues of reducing toxic side effects and improving pharmacokinetic profiles of the therapeutic agents, we have developed a new formulation by co-packaging DAC and ATO into alendronate-conjugated bone-targeting nanoparticles (BTNPs). Our pharmacokinetic studies revealed that intravenously administered BTNPs increased circulation time up to 3days. Biodistribution analysis showed that the BTNP facilitated DAC and ATO accumulation in the bone, which is 6.7 and 7.9 times more than untargeted NP. Finally, MDS mouse model treated with BTNPs showed better restoration of complete blood count to normal level, and significantly longer median survival as compared to free drugs or untargeted NPs treatment. Our results support bone-targeted co-delivery of DAC and ATO for effective treatment of MDS.

Altered follicular helper T cell impaired antibody production in a murine model of myelodysplastic syndromes.

Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic diseases which have a high risk of progressing to acute myeloid leukemia. MDS patients have immunologic deficiency, including T and B cells dysfunction. Follicular T helper cells (Tfh, CD4+CXCR5+) are an important subset of helper T cells which help to the formation of germinal centers and B cells differentiation. In this study, we investigated the proportion and function of Tfh using NUP98-HOXD13 transgenic (NHD13) mice model with MDS phenotype. The proportion of Tfh from bone marrow and spleen of NHD13 mice decreased compared with wild type (WT) mice tested by flow cytometry. In NHD13 mice spleens, there were decreased CXCR5+ cells and increased PD-1+ cells using immunohistochemistry. The active markers (ICOS, CD40L and OX40) expressed on Tfh of NHD13 mice were decreased. In contrast, PD-1 expression on Tfh of NHD13 mice was higher than that of WT mice. After coculture with Tfh from NHD13 mice, IgG and IgM production of B cells were decreased. In conclusion, the proportion and function of Tfh in the MDS mice model were altered. The dysfunction and reduction of Tfh may inhibit B cells differentiation and antibody production. Abnormal Tfh might contribute to the immune tolerance promoting the progression of MDS.

Inhibition of WNT signaling in the bone marrow niche prevents the development of MDS in the Apcdel/+ MDS mouse model

AbstractThere is accumulating evidence that functional alteration(s) of the bone marrow (BM) microenvironment contribute to the development of some myeloid disorders, such as myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). In addition to a cell-intrinsic role of WNT activation in leukemia stem cells, WNT activation in the BM niche is also thought to contribute to the pathogenesis of MDS and AML. We previously showed that the Apc-haploinsufficient mice (Apcdel/+) model MDS induced by an aberrant BM microenvironment. We sought to determine whether Apc, a multifunctional protein and key negative regulator of the canonical β-catenin (Ctnnb1)/WNT-signaling pathway, mediates this disease through modulating WNT signaling, and whether inhibition of WNT signaling prevents the development of MDS in Apcdel/+mice. Here, we demonstrate that loss of 1 copy of Ctnnb1 is sufficient to prevent the development of MDS in Apcdel/+ mice and that altered canonical WNT signaling in the microenvironment is responsible for the disease. Furthermore, the US Food and Drug Administration (FDA)-approved drug pyrvinium delays and/or inhibits disease in Apcdel/+ mice, even when it is administered after the presentation of anemia. Other groups have observed increased nuclear CTNNB1 in stromal cells from a high frequency of MDS/AML patients, a finding that together with our results highlights a potential new strategy for treating some myeloid disorders.

Hematopathological alterations of major tumor suppressor cascade, vital cell cycle inhibitors and hematopoietic niche components in experimental myelodysplasia

Myelodysplastic syndrome (MDS) is a poorly understood dreadful hematopoietic disorder that involves maturational defect and abnormalities in blood cell production leading to dysplastic changes and peripheral blood pancytopenia. The present work aims in establishing the mechanistic relationship of the expressional alterations of major tumor suppressor cascade, vital cell cycle inhibitors and hematopoietic microenvironmental components with the disease pathophysiologies. The study involves the development of N-N′ Ethylnitrosourea (ENU) induced mouse model of MDS, characterization of the disease with blood film and bone marrow smear studies, scanning electron microscopic observation, mitochondrial membrane potential determination, flowcytometric analysis of osteoblastic and vascular niche components along with the expressional study of cleaved caspase-3, PCNA, Chk-2, p53, Ndn, Gfi-1, Tie-2, Sdf-1, Gsk-3β, p18 and Myt-1 in the bone marrow compartment. Dysplastic features were found in peripheral blood of MDS mice which seemed to be the consequence of three marrow pathophysiological conditions viz; aberrant rise of cellular proliferation, increased apoptosis and crowding of abnormal blast population. Expressional decline of the p53 cascade involving Chk-2, p53, Ndn, Gfi-1 along with the downregulation of major cell cycle inhibitors seemed to be associated with the hyper-proliferative nature of bone marrow cells during MDS. Moreover the disruption of osteoblastic niche components added to the decreased hematopoietic quiescency. Increased marrow vascular niche components signified the pre-malignant state of MDS. Elevated cellular apoptosis and rise in the blast burden were also found to be associated with the p53 expression dependent collapsing of mitochondrial membrane potential and upregulation of Tie-2 respectively. The study established the mechanistic correlation between the alterations of the mentioned signaling components and hematopoietic anomalies during MDS which may be beneficial for the development of therapeutic strategies for the disease.

Genetic Investigation of the Role of GDF11 in the Treatment of β-Thalassemia and MDS

The current treatment of β-thalassemia only partially mitigates the phenotype of the disease, making the need for novel therapeutic agents imperative. The investigational drug Luspatercept (ACE-536) is a ligand trap that contains the modified extracellular domain of activin receptor IIB (ACVR2B) and induces red blood cell production in an erythropoietin independent fashion. ACE-536 binds with high affinity to members of the transforming growth factor (TGF) β superfamily and therefore alters activin/GDF signaling through the intracellular SMAD complex. In search of the specific ligands, recent studies in a mouse model of β thalassemia intermedia identified growth differentiation factor 11 (GDF11) as a possible target of the drug. It has been proposed that GDF11 is overexpressed in thalassemic erythroblasts and inhibits terminal erythroid maturation via SMAD complex phosphorylation. A negative role of GDF11 in erythropoiesis has been postulated also in myelodysplastic syndrome (MDS).We recapitulate, by a genetic approach, the phenotype of thalassemic and MDS mice treated with RAP-536, the murine counterpart of ACE-536. We generated and analyzed animals with GDF11 deletion in erythroid cells (Hbbth3/+ Gdf11fl/flEpoR-Cre and NUP98-HOXD13 Gdf11fl/flEpoR-Cre) and in all hematopoietic tissues (Hbbth3/+Gdf11fl/flVav-Cre and NUP98-HOXD13 Gdf11fl/flVav-Cre). We did not detect any changes in red blood cell number, reticulocyte count, hemoglobin or hematocrit levels compared to thalassemic or MDS mice in absence of the floxed gene. Focusing on thalassemic mice, administration of RAP-536 significantly improved the anemia and other hematopoietic parameters in the peripheral blood, decreased spleen size and ameliorated ineffective erythropoiesis as indicated by an increased ratio of mature to immature splenic erythroblasts analyzed by flow cytometry. Similar endpoints were seen comparing floxed and non-floxed animals treated with RAP-536. Therefore, the lack of GDF11 in erythroid or bone marrow derived cells did not prevent a response to the drug. To assess the effect of a pancellular absence of GDF11, we are currently in process of generating a model of β-thalassemia with total Gdf11 deletion.To investigate the possible effects of RAP-536, we treated erythroid cells derived from normal or thalassemic patients with the drug. Erythroid cell viability, number, differentiation and cell cycle remained unvaried. Of note, we did not detect significant expression of GDF11/Gdf11 in human and mouse erythroid cells. To investigate the effect of an exogenous source of GDF11 production on erythroid cells, we treated murine erythroleukemia (MEL) cells with recombinant GDF11. Upon treatment we observed phosphorylation of the SMAD2/3 complex by western blot. This effect was hindered by co-treatment of GDF11 with RAP-536.We further assessed the effects of RAP-536 on the mouse erythroid transcriptome, using RNA seq analysis in splenic erythroid populations. After administration of a single dose of RAP-536, thalassemic mice were euthanized. We used flow cytometry to identify possible alterations on differentiating erythroid populations in the spleen. Notably, between 60 and 72h we observed reduced numbers of basophilic and increased numbers of polychromatophilic erythroblasts. Analysis at 60h revealed that signal transducer and activator of transcription 5a (Stat5a), cyclin-dependent kinase 6 (Cdk6) and other cell cycle-related and metabolic genes were increased in the basophilic erythroid progenitors treated with RAP-536. This effect suggests that RAP-536 promotes proliferation and/or differentiation of erythroblasts.Thus, our genetic analyses suggest that lack of GDF11 may be required but not sufficient to improve erythropoiesis. Furthermore, erythroid cells do not produce but can respond to exogenous GDF11, likely synthesized by non-erythroid cells and under conditions of ineffective erythropoiesis. Even though we detected in vitro effects, these may not mimic physiological effects, as experimental conditions may not correlate with GDF11 concentrations in vivo. As additional ligands have been proposed (such as GDF8 and Activin B), our future studies will focus on the potential role of these molecules. Altogether, these results reveal a potential alternative target of action for ACE-536 and may lead to the discovery of new therapeutic molecules. DisclosuresSuragani:Acceleron Pharma: Employment, Equity Ownership. Kumar:Acceleron Pharma: Employment, Equity Ownership.

Analysis of hematopathology and alteration of JAK1/STAT3/STAT5 signaling axis in experimental myelodysplastic syndrome

Hematological disorders like myelodysplastic syndrome (MDS) may arise due to cumulative dysregulation of various signalling pathways controlling proliferation, differentiation, maturation and apoptosis of bone marrow cells. This devastating bone marrow condition can be due to consequential abnormalities in haematopoiesis as well as its supportive microenvironment. Although mutations related to JAK/STAT pathway are common in myeloproliferative neoplasms, further studies are required to fully explore the myelodysplastic scenario regarding the concerned pathway. In this study, we have investigated the JAK-STAT signalling pathway which inevitably plays a crucial role in haematopoiesis. MDS was mimicked in a mouse model with an induction of ENU in adult mice. The bone marrow of the control and MDS groups of animals were subjected to a variety of tests, including cell morphology study in peripheral blood and bone marrow, cytochemistry and histochemistry of bone marrow smears, karyotyping and flowcytometric expression analysis of the phosphorylated forms of proteins like JAK1, STAT3 and STAT5 (denoted as pJAK1, pSTAT3 and pSTAT5) and the phenotypic expression of proteins like CD45 and CD71. The results revealed that the morphology of the blood and bone marrow cells were dysplastic compared to the affected blast populations of different lineages. The expression of common leucocyte antigen CD45 was less in comparison to the expression of transferrin receptor CD71 which was increased in the ENU induced MDS mouse model. Moreover, we have observed an upregulated expression of JAK1 followed by STAT5. Therefore, we can conclude that downregulation of CD45 may have helped in the upregulation of JAK-STAT signaling and CD71 expression. This aberrant signaling may be among one of the activated signaling axes that lead to affected hematopoietic lineages in Myelodysplastic syndrome.

Impact of combinatorial dysfunctions of Tet2 and Ezh2 on the epigenome in the pathogenesis of myelodysplastic syndrome

Somatic inactivating mutations in epigenetic regulators are frequently found in combination in myelodysplastic syndrome (MDS). However, the mechanisms by which combinatory mutations in epigenetic regulators promote the development of MDS remain unknown. Here we performed epigenomic profiling of hematopoietic progenitors in MDS mice hypomorphic for Tet2 following the loss of the polycomb-group gene Ezh2 (Tet2KD/KDEzh2Δ/Δ). Aberrant DNA methylation propagated in a sequential manner from a Tet2-insufficient state to advanced MDS with deletion of Ezh2. Hyper-differentially methylated regions (hyper-DMRs) in Tet2KD/KDEzh2Δ/Δ MDS hematopoietic stem/progenitor cells were largely distinct from those in each single mutant and correlated with transcriptional repression. Although Tet2 hypomorph was responsible for enhancer hypermethylation, the loss of Ezh2 induced hyper-DMRs that were enriched for CpG islands of polycomb targets. Notably, Ezh2 targets largely lost the H3K27me3 mark while acquiring a significantly higher level of DNA methylation than Ezh1 targets that retained the mark. These findings indicate that Ezh2 targets are the major targets of the epigenetic switch in MDS with Ezh2 insufficiency. Our results provide a detailed trail for the epigenetic drift in a well-defined MDS model and demonstrate that the combined dysfunction of epigenetic regulators cooperatively remodels the epigenome in the pathogenesis of MDS.

Chronic Iron Overload Damage the Erythropoiesis in MDS Mouse By Increasing GDF11 and ROS Levels

Iron overload (IO) is a kind of disease with an excess of iron depositing in the body's organs and tissues, resulting in the dysfunction of these organs and tissues, which contains hereditary hemochromatosis and secondary IO such as ineffective hematopoiesis in aplastic anemia and myelodysplastic syndrome (MDS) and blood transfusion. Numerous researches and our previous study found that IO can damage the hematopoietic function in bone marrow. MDS is one of the most common IO diseases and IO can further injure the hematopoietic function especially on erythropoiesis in MDS.However, it is indeterminacy on the mechanism behind this stiuation. This study is to explore the possible mechanism of IO on the injury of MDS erythropoiesis though establishing an IO MDS mice model.In this study, we first established an IO mice model by administering iron dextran(25 mg/ml) by intraperitoneal injection every 3 days for 2 months in wild and MDS mouse(NUP98-HOXD13 transgenic mouse). The mouse were devided into four grups: the wild mouse without IO(Ctrl group), the wild mouse with IO (IO group), MDS mouse without IO(MDS group), the MDS mouse with IO(MDS+IO group). After 6 months, the establishment of IO mice models were confirmed by both the iron deposits staining of bone marrow(BM), liver, spleen and the labile iron pool level of bone marrow mononuclear cells( BMMNCs).We next found that IO damage the erythropoiesis in MDS mice. We detected the proportion of different stages BM erythroblasts by flow cytometry plots of Ter119+ CD71+ cells (R1, basophilic erythroblasts, poly red cells, immature red blood cells) and Ter119+ CD71- cells (R2, late erythroblast and reticulocyte ). It was found that the hematopoietic colony-forming unit (CFU-E, BFU-E) in MDS+IO group were less than that in other groups. The proportion of R1(10.39±1.43) and R2(1.58±0.54) in MDS+IO group were the least compared with Ctrl group(R1 17.36±3.45,R2 4.59±1.57),IO group(R1 14.53±2.65,R2 2.56±0.76) and MDS group(R1 13.64±3.27,R2 2.35±0.78). And the reticulocytes proportion in MDS+IO group(11.36±3.65) was the highest compared with Ctrl group(5.159±1.537), IO group(7.61±1.36)) and MDS group(7.35±1.39).Then we further explored the mechanism of IO damage on MDS erythropoiesis. It was reported that TGF-β family and oxidative stress (ROS) played an important role on erythropoiesis. Our previous studies also had shown that the ROS and GDF11(a member of TGF-β family) levels were raised in BMMNCs on C57BL/6 mice with IO. Similarly, we detected them on the BM erythroblast in this study. Firstly, we sorted the R1 and R2 erythroblasts,then we performed RT-PCR to analyze mRNA levels of the TGF-β family. We found that the mRNA levels of GDF11, GDF15, Activin B, Acvr2b and ALK5 in MDS+IO group were the highest compared with that of other groups, while the mRNA levels of Activin A, ALK4 and BMP8 had no significant change in these groups. As we found that the change of GDF11 level was the most significant in MDS+IO group compared with other groups, we focused on the study of GDF11 in the following experiment. The concentration of GDF11 in peripheral serum was detected by ELISA method. It was found that the concentration of GDF11 was the highest in MDS+IO group compared with other groups. And also, the GDF11 related signal pathway proteins (Smad 2, Smad 3, p-Smad 2 and p-Smad 3) were significantly elevated in R1 and R2 erythroblast in MDS+IO group compared with other groups,which indicated that GDF11 related signal pathway was involved in the deficient of BM erythropoiesis in MDS+IO mice. Furthermore, We found that the ROS levels in Ter119+ cells in MDS+IO group, MDS group and IO group were increased 4.15 folds, 2.16 folds , 2.63 folds compared with Ctrl group. RT-PCR was used to detect the mRNA expression levels of the gene NOX4 related to ROS generation and GPX1 related with ROS clearance. It was found that the NOX4 expression levels were the highest in MDS+IO group compared with other groups,while the GPX1 mRNA level was the least in MDS+IO group.In conclusion, our study indicated that chronic IO damage the erythropoiesis in MDS mouse by increasing GDF11 and ROS levels which would provide an theoretical basis to find new targets for the treatment of IO in MDS patients with erythropoiesis dysfunction. DisclosuresNo relevant conflicts of interest to declare.

Effect of Azacytidine on the Hematopoietic Stem and Progenitor Cell Compartments of MDS Mouse Models: Unveiling the Mechanisms of Remission and Relapse

Myelodysplastic syndromes (MDS) are a group of heterogeneous hematopoietic malignancies characterized by the defective production of mature blood cells and the risk of progression to myeloid leukemia. Hypomethylating agents (HMA) are currently the first-line therapy for high-risk MDS and induce hematological improvement in 50% of the patients. However, complete responses occur in less than 20% of these patients, and a majority of MDS patients treated with HMA will eventually relapse, with a subsequently fatal prognosis. Although the presence of mutations in certain genes, such as Tet2, has been reported to predict response to HMA, the causes of resistance and relapse have yet to be described.MDS research has been traditionally a slow-moving field owing to the lack of well-established MDS cell lines and animal models. This landscape recently changed with the development of transgenic mouse models that mirror the phenotype of high-risk MDS patients. In mice from the 4th/5th generations (G4/G5) of our group's telomere-dysfunctional TERTER/ER mouse model, the DNA-damage response triggered by telomere erosion causes profound cell-intrinsic abnormalities in hematopoietic stem and progenitor cells (HSPC) that lead to myeloid-skewed hematopoiesis. These mice exhibit peripheral blood (PB) cytopenias, age-related granulocytosis and myeloid infiltration of the BM and the spleen (Colla et al. Cancer Cell 2015). Similarly, the conditional deletion of Tet2 in mouse hematopoietic cells generates a phenotype of HSC expansion and myeloid skewing of hematopoiesis. These defects, which are also cell-intrinsic, induce progressive PB neutrophilia and granulocytosis, with myeloid infiltration of the bone marrow (BM) and spleen and severe splenomegaly (Moran-Crusio et al. Cancer Cell 2011).Using the TERTER/ER and Tet2-/- mouse models as a platform for cellular and molecular studies, we have investigated the mechanism of action of HMA in MDS, with a focus on the characterization of response by the different HSPC populations and the mechanisms involved in resistance and relapse. Our results show that the administration of the HMA azacytidine (AZA) for 7 days induces in both mouse models a 20-40% drop in white blood cell counts and a 17-24% decrease in hemoglobin levels that mimic patient responses to AZA. After the last day of treatment and during the 3 last weeks of the cycle, cell counts and hemoglobin levels returned to normal values. These changes in PB were explained by an 80-86% decrease in the number of BM common myeloid progenitors (CMP; n = 8, P = 0.0046) and granulo-monocytic progenitors (GMP; n = 8, P <0.0001) after 1 week of AZA treatment, while megakaryocytic-erythroid progenitors remained largely unaffected. However, the number of CMP and GMP in the BM of both G5 and Tet2-/- mice recovered by the end of each cycle, and the characteristic myeloid skewing was not overcome after 2 cycles of treatment, which indicates that AZA cannot reverse the cell-intrinsic defects of these progenitors. Most importantly, BM analysis in both mouse models after one week of treatment and during the recovery period revealed that AZA has no effect on long and short-term HSC populations.To study the effect of AZA in a BM context that mimicked the coexistence of MDS and normal cells in patients, we performed competitive transplantation experiments in both models. Our preliminary results indicate that, in mixed populations, AZA induces a 2.6-fold expansion of the wild type long-term HSC compartment (n = 4, P = 0.0018). The fact that AZA gives a repopulation advantage to normal HSC suggest that, after several cycles, there is a shift in the abundance of normal versus MDS cells in patients BM, and this explains why patients enter morphologic remission. However, in both transgenic mouse models, AZA failed to decrease the size of the MDS-like HSC compartment. This indicates that, although this drug allows a temporary hematologic recovery, it does not eliminate the cell populations that maintain the disease, and therefore it cannot eliminate MDS.Ongoing unbiased transcriptomic and methylomic analyses in the sorted MDS-like HSC that survived AZA treatment will identify the HSC signature of resistance to HMA, and the integration of these results with similar analyses in MDS patients will provide valuable insights into the mechanistic bases of relapse and resistance to HMA and facilitate the design of better combination therapies for high-risk MDS. DisclosuresNo relevant conflicts of interest to declare.

HIF-1a Pathway, As a Signal Funnel for Genetic, Epigenetic, and Metabolic Aberrations, Is Sufficient and Essential for MDS Development

Myelodysplastic Syndromes (MDS) are heterogeneous clonal hematopoietic disorders, which are characterized by ineffective hematopoiesis and uni- or multi-lineage dysplasia. Despite the fact that a variety of genetic, epigenetic, and metabolic aberrations have been identified, the clinical features for MDS remain similar, indicating that there is a common underlying mechanism for MDS pathogenesis. Accumulating clinical and research evidence has shown the involvement of systemic inflammation and activated immune signaling in MDS pathogenesis. Hypoxia inducible factor-1α (HIF-1α) is a critical transcription factor for the hypoxic response, angiogenesis, and cancer development. Importantly, HIF-1α is also a key regulator for immune cell activation.To determine whether MDS patients have an activated HIF-1α signature, we analyzed a public gene expression array data set of CD34+ BM cells from a large cohort of MDS patients (n = 183). Although HIF-1α mRNA remains unchanged, some HIF-1α target genes relating with survival/cell growth, glucose metabolism, and invasion/metastasis were significantly activated in the broad spectrum of MDS. We further determined HIF-1α expression in the BM biopsies from MDS patients (n = 39) using immunohistochemistry staining. We found a high frequency of HIF-1α expressed cells in both low- and high-risk IPSS groups. Consistent with this human data, deficiency of frequently mutated genes in MDS resulted in an activated HIF-1α signature in mice. We found that the levels of Hif-1α protein in the c-Kit+ BM cells from Dnmt3a-KO, Runx1-KO, and Mll-partial tandem duplication (PTD) knock-in (MllPTD/WT) mice were significantly upregulated compared with those from WT controls. Although the expression level of Hif-1α protein in the c-Kit+ BM cells from Tet2-KO mice was not changed, they had an activated HIF-1α signature. IDH1/2 mutations are also known to elevate Hif-1α proteins. These results suggest that the HIF-1α pathway is widely activated in MDS patients by MDS-genic mutations through different mechanisms.To determine whether HIF-1α is sufficient for developing MDS phenotypes, we generated blood specific inducible HIF-1α transgenic mice. Using Vav1-Cre/Rosa26-loxP-Stop-loxP (LSL) rtTA driver, stable HIF-1α can be induced in a doxycycline dependent manner. HIF-1α-induced mice developed thrombocytopenia, leukocytopenia, and macrocytic anemia. We found micromegakaryocyte and multi-segmented megakaryocyte formations which are a major diagnostic criteria for MDS. In the BM of anemic mice, we found significant reduction of basophilic and polychromatic erythroblasts. The central macrophages are known to form erythroblastic islands that play a critical role in the late stage of erythropoiesis. Both the frequency and absolute number of F4/80+ Ter119+ BM erythroblastic islands were significantly decreased in HIF-1α-induced mice compared to the control mice. We also found activation of both innate and adaptive immunity in HIF-1α-induced mice. Lineage specific HIF-1α induction revealed the cell-intrinsic effect of HIF-1α on aberrant megakaryocytopoiesis and cell-extrinsic effect of HIF-1α on macrocytic anemia development.We have previously shown that MllPTD/WT mice develop several MDS-associated features, such as increased self-renewal and apoptosis in HSPCs, expansion of myeloid progenitors, and skewed myeloid differentiation. Synergistic effects between MLL-PTD and RUNX1 mutant (S291fs) or Runx1 deletion, further accumulated the Hif-1α protein and caused a variety of MDS features that phenocopy the human MDS. To understand the role of HIF-1α in the context of MDS pathogenesis, we took genetic and pharmacologic approaches. Hif-1α deletion significantly abrogated disease development and rescued macrocytic anemia and thrombocytopenia. This is not due to the elimination of donor derived cells. After the deletion of Hif-1α, a majority of donor derived cells in PB were still CD11b+ cells. HIF-1α inhibition significantly reduced colony formation of MllPTD/WT/RUNX1-291fs cells and MllPTD/WT/Runx1D/D cells in vitro, and prolonged survival of MDS mice in vivo.In conclusion, we identified the sufficient and essential role of HIF-1α in developing MDS. These findings implicate that HIF-1α pathway may be the common underlying mechanism of MDS pathophysiology and could be an effective therapeutic target for a broad spectrum of MDS patients. DisclosuresShih:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

Pathophysiological Scenario of Hematopoietic Disorders: A Comparative Study of Aplastic Anemia, Myelodysplastic Syndrome and Leukemia in Experimental Animals

The conversion of physiology to pathophysiology in hematological disorders viz: aplastic anemia, myelodysplastic syndrome (MDS) and leukemia in murine models was the subject of study in the present programme. Peripheral blood hemogram, spleno-somatic index, bone marrow smear study, cytochemical staining of marrow, cell release kinetics study during marrow explants culture, hematopoietic niche assessment, chromosomal aberration study, plasma membrane stability study of marrow cells, lysosomal membrane and mitochondrial membrane stability study and innate immune parameters were performed in the aplastic anemia, leukemia and MDS mouse model. In bone marrow aplasia, peripheral blood pancytopenia, marrow hypocellularity, decreased marrow cellular viability, deterioration of bone marrow hematopoiesis as well as hematopoietic microenvironment and extramedullary hematopoiesis were noticed. In addition, disruption of mitochondrial and lysosomal membrane integrity along with reduction of innate immune parameters were found in the hematopoietic suppressed condition. Surprisingly, no noticeable chromosomal aberration was found in the aplastic condition. Ineffective marrow hematopoiesis together with the disruption of hematopoietic microenvironment was observed in MDS. Also, extramedullary hematopoiesis, increased marrow cellular death, chromosomal aberration and loss of innate immunity were the common events. During leukemia, the number of functionally and structurally immature cells in the peripheral blood and bone marrow was increased together with malignant conversion of hematopoietic cells in the presence of malignancy supportive stromal microenvironment. Chromosomal aberration, decrease of cell mediated immunity with least mitochondrial apoptotic damage were also found in leukemic condition as well.

Role of p21 in a Mouse Model of NUP98-HOXD13 Fusion Driven Myelodysplastic Syndromes (MDS)

The myelodysplastic syndromes (MDS) are clonal stem cell disorders, characterized by ineffective hematopoiesis leading to cytopenias and a high rate of progression to acute myeloid leukemia (AML). The NUP98-HOXD13 (NHD13) fusion has been found in patients with MDS and AML. A transgenic (Tg) mouse model, generated by Peter Aplan’s group, which utilizes Vav 1 regulatory elements to direct expression of the NHD13 transgene in hematopoietic tissues, displays the phenotypic features of MDS including a chronic phase of cytopenias followed by transformation to AML (Lin et al., 2005). We previously reported that loss of one or both alleles of p53 did not rescue the MDS phenotype in NHD13+ Tg mice, but rather exacerbated the MDS phenotype and accelerated the development of AML (Xu et al., 2012). Expression of p21WAF1/CIP1(p21) was increased in the Lin−Sca-1+c-Kit+ (LSK) cells isolated from NHD13+ Tg mice, so we generated and analyzed NHD13+p21+/– and NHD13+p21–/– mice to further investigate whether the accelerated MDS and AML that occurs in the absence of p53 relates to the defective expression of the p53 target gene. Deletion of p21 significantly altered the fate of the NHD13+ Tg mice. All of the NHD13+p21–/– mice died of AML, rather than MDS. Only 18% (4 out of total 22 mice) of the NHD13+p21+/– mice developed MDS with a median survival of 289 d; in contrast 31% (9 out of total 29 mice) of NHD13+ Tg mice died from MDS, with a median survival of 230 d (p<0.05). We examined the peripheral blood counts of the “clinically healthy” NHD13+p21–/– mice at 3 to 5 months, and found increased white blood cell (WBC) and neutrophil (NE) counts, compared to the age matched NHD13+ Tg mice. Clearly the deletion of one or two alleles of p21 increases the median survival of NHD13+ Tg mice with MDS, and the complete loss of p21 rescues the fatal MDS induced by NHD13 fusion gene. However the deletion of one or two p21 alleles does not significantly affect the development of AML, which in 82% of NHD13+p21+/– mice resulting in a median survival of 291 d, AML in the NHD13+p21–/– mice was with a median survival of 320 d, and 69% of NHD13+ Tg mice showed AML with a median survival of 315 d. p21 is important for maintaining a normal-sized HSPC pool (Cheng et al., 2000), and both p21 and p53 have been shown to be involved in the determination of asymmetric vs symmetric cell divisions of epithelial cells (O’Brien et al., 2012; Cicalese et al., 2009). To determine whether the symmetric division of NHD13+ HSCs is affected by the loss of p21 or p53 in vitro, we performed paired daughter cell assays. Single LSKCD34–Flt3–CD150+ cells isolated from wild type (WT), NHD13+, NHD13+p21–/– and NHD13+p53–/– mice bone marrow were sorted into 96-well plates one cell/well. After the first cell division, the two daughter cells were split into two wells for 12 days in culture. We examined the ability of sorted single LSKCD34–Flt3–CD150+ cells to generate daughter cells that retain multipotent lineage differentiation potential and found that NHD13+p53–/– CD150+CD34–Flt3–LSK cells underwent symmetric self-renewal divisions 85% of the time (both daughter cells are multipotent), with 15% asymmetric divisions (only one daughter cell is multipotent); the NHD13+p21–/– CD150+CD34–Flt3–LSK cells produced 13% symmetric self-renewal divisions, 50% asymmetric divisions and 37% symmetric commitment divisions (both daughter cells are not multipotent); the NHD13+ CD150+CD34–Flt3–LSK cells produced 50% symmetric self-renewal divisions, 40% asymmetric divisions and 10% symmetric commitment divisions; and the WT CD150+CD34–Flt3–LSK cells produced 27% symmetric self-renewal divisions, 42% asymmetric divisions and 31% symmetric commitment divisions. These data indicate that loss of p53 increases symmetric self-renewal divisions of NHD13+ HSCs, and loss of p21 increases asymmetric self-renewal divisions of NHD13+ HSC in vitro. Collectively, our data indicate that loss of p21 maintains the survival of MDS driven by NUP98-HOXD13 fusion, which is independent of the function of p53; and the increased asymmetric self-renewal divisions of NHD13+p21–/– HSCs may contribute to the increased survival observed in NHD13+p21–/– MDS mice. DisclosuresArmstrong:Epizyme : Consultancy.

The Biological Function of DNA Hypermethylation in Murine MDS Model Lacking Tet2 and Ezh2

Mutations of epigenetic regulators are often found in patients with myelodysplastic syndrome (MDS). Furthermore, DNA methylation inhibitors have a therapeutic impact on MDS. However, it remains unknown how altered DNA methylation promotes the development of MDS. We have shown that concurrent depletion of Tet2 and Ezh2 in hematopoietic cells significantly promotes the development of MDS in vivo by utilizing hypomorphic Tet2 (Tet2KD/KD) mice and Ezh2 conditional knockout mice (Cre-ERT;Ezh2fl/fl)(Muto T, et al. J Exp Med 2014).In order to determine how DNA methylation contributes to the formation of MDS in Tet2KD/KDEzh2Δ/Δ mice, we transplanted wild type (WT), Tet2KD/KD, Cre-ERT;Ezh2fl/fl, and Cre-ERT;Tet2KD/KDEzh2fl/fl fetal liver cells in lethally irradiated CD45.1+ recipient mice, and deleted Ezh2 at 4 weeks post-transplantation. We then performed reduced representation bisulfite sequence (RRBS) in Lin-Sca1+Kit+ (LSK) cells isolated from Tet2KD/KD and Ezh2Δ/Δ mice at 3 and 7 months post-deletion and WT and Tet2KD/KDEzh2Δ/Δ mice at 5 months post-deletion. We defined ≥10% difference of methylation in test cells compared with that in WT cells as hyper- or hypo-differentially methylated regions (DMRs) (p-value<0.01). We found that Ezh2Δ/Δ LSK cells gain significantly larger number of hyper-DMRs at CpG islands (CGIs) over time compared with Tet2KD/KD LSK cells. At 8 months post-deletion, Tet2KD/KD and Ezh2Δ/Δ LSK cells gained hyper-DMRs in 196 and 366 out of 12677 promoters, and 106 and 306 out of 11504 CGIs, respectively. Tet2KD/KDEzh2Δ/Δ-MDS LSK cells at 5 months post-deletion had hyper- and hypo-DMRs in 109 and 51 out of 12677 promoters, and 124 and 27 out of 11504 CGIs, respectively. Hyper-CGI DMRs of Tet2KD/KDEzh2Δ/Δ-MDS LSK cells largely overlapped with those of Tet2KD/KD and Ezh2Δ/Δ LSK cells (p<1.00E-16). Nonetheless, a significant number of hyper-CGI DMRs were newly generated in Tet2KD/KDEzh2Δ/Δ-MDS LSK cells, suggesting unique impact of combined loss of Tet2 and Ezh2 on epigenome. Of note, hyper-DMRs in this mouse MDS model significantly overlapped with those in human CD34+ cells of MDS patients (Maegawa S, et al. Genome Research 2014) (40 genes, p=0.0001). As reported in solid tumor and aging stem cells, polycomb-group (PcG) protein targets were significantly enriched among hyper-CGI DMRs in LSK cells of all three genotypes; Tet2KD/KD, Ezh2Δ/Δ, and Tet2KD/KDEzh2Δ/Δ-MDS LSK cells. Indeed, hypermethylated CGIs were enriched for genes encoding DNA binding transcription factors (p=8.96E-05) and cell differentiation (p=3.98E-05) in GO analysis, suggesting that DNA methylation involves the PcG targets such as developmental regulator genes that regulate hematopoietic cell differentiation in the pathogenesis of MDS.To determine an association between levels of differential DNA methylation and transcription of genes, we next performed DNA microarray analysis by using LSK cells. As several studies reported, we observed no significant association between levels of DNA methylation and gene expression (R2=0.003). However, 27 hypermethylated genes showed reduced levels of gene expression in Tet2KD/KDEzh2Δ/Δ-MDS LSK cells. Those included key transcription factors and epigenetic modifiers for hematopoiesis, such as Gata2, Gata3, Evi1, and Bcor.Finally, we examined whether decitabine, a demethylating agent, reverses the DNA hypermethylation of Tet2KD/KDEzh2Δ/Δ-MDS LSK cells in vivo. Although the administration of decitabine into secondary recipients of MDS BM cells did not improve the survival of mice, platelet counts were significantly increased in decitabine-treated MDS mice, compared to the control MDS mice. After 4 weeks of treatment, a partial restoration of DNA hypermethylation was observed in the decitabine-treated MDS mice (34 out of 124 CGIs; p<1.00-16E). These regions were again enriched in DNA binding transcription factors (p=1.72E-05) and cell differentiation (p=2.25E-07) in GO analysis.Taken together, our work unveiled the biological function of DNA hypermethylation in the pathogenesis of MDS, and will give a clue to understanding how demethylating agents improve the outcome of patients with MDS. DisclosuresNo relevant conflicts of interest to declare.

Function of Growth Factor Independence 1 (GFI1) in the Polarization AML -Associated Stroma

The growth of various solid tumors, lymphoma and leukemias is not only the result of cell-specific changes at the genetic and epigenetic level, but is also affected by the surrounding stroma and the cells therein. It has been shown that tumor cells induce surrounding immune cells to express various cytokines and other factors, which promote further growth and spread of tumor cells. Most studies have been conducted with solid tumors, however, not much is known about the role of stroma cells with regard to haematopoietic tumors. In addition, our knowledge is limited with regard to the cell intrinsic factors governing the polarization of stroma cells. We intended to study the role of stroma cells in acute myeloid leukemia (AML), a malignant disease of the myeloid lineage and in myelodysplastic syndrome (MDS), a disease which is characterized by disturbed function of the bone marrow, which can often progress to AML.We focused our studies on two different components of the stroma: mesenchymal stroma cells (MSC) and monocytes.Bone marrow derived MSCs and monocytes originating from AML patients better supported the growth of human leukemia cells in vitrothan MSCs and monocytes from control healthy persons. Interestingly, after achieving a remission, MSCs and monocytes from the cured patients did not any more support the growth of leukemia cells to the same extent as monocytes and MSCs from leukemic patients.To better understand the mechanism behind this observation, we used different murine MDS and AML models. Similar to the finding in human patients, bone marrow derived MSCs and monocytes from MDS and AML mice better supported the growth of leukemic cells in vitro than monocytes and MDSc from healthy mice. In addition, we observed a higher number of non-malignant MSCs and monocytes in the bone marrow of leukemic mice than in the bone marrow of healthy mice. Furthermore, the higher the number of monocytes was in the bone marrow of the mice, the more aggressive was the course of the leukemia. On a molecular level we found that the transcription factor Gfi1 is 3-4 fold upregulated in the MSCs and monocytes of leukemic mice. To verify whether Gfi1 is indeed required for the polarization of MSCs and monocytes we used leukemic Gfi1-deficient mice. In these mice the accumulation of monocytes was less pronounced than in the bone marrow of Gfi1 wildtype mice. In addition MSCs and monocytes from Gfi1-deficient mice did not support to the same extent the growth of leukemic cell lines in vitro, as did the monocytes and MSCs of Gfi1 wildtype leukemic mice. Thus, we have first indications that MSCs and monocytes play an important role in the support of leukemic cells and that Gfi1 is involved in the polarization of these cells and thus Gfi1 could serve as an additional approach to treat leukemia and MDS. DisclosuresNo relevant conflicts of interest to declare.

Gfi136N As a Novel Marker and Therapeutic Target of MDS and AML

Growth Factor Independence 1 (GFI1) is a hematopoietic transcription factor that plays a crucial role in the development of myeloid precursor cells. Its variant single nucleotide polymorphism called GFI136N (whereby Serine at position 36 is replaced by Asparagine) has been described to play a role in acute myeloid leukemia (AML) development. The prevalence of this variant is about 5-7% in the healthy Caucasian populations.Patients with myelodysplastic syndrome (MDS) show disturbed bone marrow function and are at risk to develop AML. Identifying prognostic markers and potential targets is essential for improved MDS therapy.We explored how GFI136N influences onset and progression of MDS and investigated the biological mechanisms behind our findings.To examine the role of GFI136N with regard to progression of MDS to AML, we characterized the status of GFI1 in 201 German-, 350 US- and 86 Dutch MDS patients. Our results revealed that 11-13% of patients were heterozygous for GFI136N compared to 5-7% in the healthy population. Patients carrying GFI136N also showed a 2-3-fold higher risk of AML-development and inferior leukemia-free survival than patients carrying the common variant GFI136S. The onset of MDS and AML tended to be at younger ages for GFI136N carriers and they did not respond to demethylating therapy to the same extent as GFI136S homozygous MDS patients. Whole exome sequencing revealed that patients carrying GFI136N had mutations in epigenetic modifiers such as EZH2 and expressed higher levels of different oncogenes such as HOXA9.To dissect the mechanisms behind these findings and to study the role of GFI136N in AML development, we used GFI136N and GFI136S knock-in mice that expressed either of the two human variants instead of murine Gfi1. We transduced hematopoietic progenitor cells from GFI136S or GFI136N expressing mice with retroviral vectors overexpressing oncofusion proteins AML1-ETO9a or MLL-AF9, which can be recurrently found in AML patient cohorts. GFI136N expressing cells that had been transduced with either AML1-ETO9a or MLL-AF9 generated significantly more cells and colonies than GFI136S controls.In order to confirm our findings in vivo, we used three different AML and MDS mouse models crossed to mice carrying either GFI136N or the common GFI136S.As a first in vivoapproach, we used a mouse strain that expresses the human oncofusion protein CBFB-MYH11 (inv(16)) frequently found in AML patients. Mice carrying GFI136N and expressing CBFB-MYH11 showed a significantly accelerated onset of AML compared to mice carrying GFI136S (p=0.004). We found similar results in a second AML mouse model expressing MLL-AF9. Using the NUP98-HOXD13 MDS mouse model a marginally significant decrease in leukemia-free survival was observed in mice carrying GFI136N.To investigate molecular mechanisms that could cause the observed effects of GFI136N, we used ChipSeq and RNASeq. Our results revealed that GFI136N lineage negative cells show a genome-wide higher degree of H3K4 methylation and H3K9 acetylation than cells carrying GFI136S. GFI1 recruits among others LSD1 and HDAC to demethylate H3K4 and deacetylate H3K9 and thus GFI136N fails to initiate epigenetic changes previously shown for GFI1, leading to higher expression of various oncogenes such as HoxA9 and Kras. A number of MDS and AML patients are treated with HDAC and LSD1 inhibitors, but based on the above data we would predict that this therapy would likely fail in GFI136N patients and that therapy with histone acetylase (HAT) inhibitors or histone methyltransferase inhibitors would be more beneficial for these patients.To test this hypothesis in vitro, we treated the above-described GFI136N and GFI136S expressing AML1-ETO9a or MLL-AF9 leukemic cells with HAT or HDAC inhibitors. HAT inhibitors inhibited growth of preleukemic GFI136N expressing cells more efficiently than HDAC inhibitors.Similarly in vivo, we transplanted irradiated mice with GFI136N or GFI136S expressing MLL-AF9 leukemic cells. We found that HAT inhibitors tended to prolong the survival of mice transplanted with GFI136N leukemic cells more than mice transplanted with GFI136S leukemic cells.In summary, GFI136N is a novel prognostic marker for MDS patients that predisposes to AML likely via epigenetic changes at different oncogenes. We also suggest that HAT inhibitors could be more beneficial to GFI136N carrier patients thus enabling a more individualized therapy. DisclosuresPlatzbecker:TEVA: Research Funding, Speakers Bureau.

Ezh2 loss promotes development of myelodysplastic syndrome but attenuates its predisposition to leukaemic transformation.

Loss-of-function mutations of EZH2, a catalytic component of polycomb repressive complex 2 (PRC2), are observed in ~\n10% of patients with myelodysplastic syndrome (MDS), but are rare in acute myeloid leukaemia (AML). Recent studies have shown that EZH2 mutations are often associated with RUNX1 mutations in MDS patients, although its pathological function remains to be addressed. Here we establish an MDS mouse model by transducing a RUNX1S291fs mutant into hematopoietic stem cells and subsequently deleting Ezh2. Ezh2 loss significantly promotes RUNX1S291fs-induced MDS. Despite their compromised proliferative capacity of RUNX1S291fs/Ezh2-null MDS cells, MDS bone marrow impairs normal hematopoietic cells via selectively activating inflammatory cytokine responses, thereby allowing propagation of MDS clones. In contrast, loss of Ezh2 prevents the transformation of AML via PRC1-mediated repression of Hoxa9. These findings provide a comprehensive picture of how Ezh2 loss collaborates with RUNX1 mutants in the pathogenesis of MDS in both cell autonomous and non-autonomous manners.

C-Terminal-Truncating ASXL1 Mutations Induce MDS Via Inhibition Of PRC2

Recurrent mutations of ASXL1 (Additional sex combs-like1) are found in various hematological malignancies including myelodysplastic syndromes (MDS), chronic myelomonocytic leukemia, and acute myeloid leukemia (AML) with myelodysplasia-related changes. Additionally, ASXL1 mutations are linked with adverse survival in a variety of myeloid malignancies. A previous study demonstrated that loss of ASXL1 in mice promotes myeloid transformation by impairing polycomb repressive complex 2 (PRC2)-mediated gene repression at a number of critical loci and leads to myeloid transformation. However, most ASXL1 mutations are heterozygous and located in the 5’ region of the last exon, indicating a dominant-negative or gain-of-function feature of a truncated ASXL1 protein. Therefore, we investigated if the C-terminal truncated form of ASXL1 (ASXL1-MT) contributes to the development of myeloid malignancies. To this end, we examined the effects of ASXL1-MT using in vitro and in vivo experiments. In in vitro experiment, expression of ASXL1-MT inhibited G-CSF-induced myeloid differentiation of 32Dcl3 cells. In a mouse bone marrow transplantation (BMT) model, ASXL1-MT induced multilineage dysplasia, differentiation block, slowly progressive pancytopenia, BM hyperplasia and splenomegaly. The transduced mice died of severe anemia after a long latency (median survival, 400.5 days), and some of the mice progressed to overt leukemia. Thus, the current model displays all of the features of human MDS. In addition, ASXL1-MT collaborated with N-RAS-G12V, which confers a proliferative advantage, in inducing progression of N-RAS-G12V-induced myeloproliferative neoplasm (MPN) to AML, suggesting that ASXL1-MT contributes to leukemic transformation by inhibiting differentiation of MPN cells. To clarify the molecular mechanism for differentiation block and MDS development induced by ASXL1-MT, we performed expression profiles of 32Dcl3 cells transduced with ASXL1-MT and BM cells of the MDS mice. Of note, gene set enrichment analysis (GSEA) of BM cells of the MDS mice indicated that ASXL1-MT induced an expression profile which inversely correlated with known PRC target genes. In fact, ASXL1-MT remarkably derepressed expression of posterior Hoxa genes, including Hoxa5, Hoxa9 and Hoxa10, which are epigenetically silenced by PRC2 in mature cells. In consistent with this, H3K27me3 was globally reduced in ASXL1-MT transduced cells. We also found ASXL1-MT as well as wild type ASXL1 (ASXL1-WT) can bind to EZH2 and, importantly, co-expression of ASXL1-MT with ASXL1-WT efficiently inhibited the binding between ASXL1-WT and EZH2, suggesting a dominant-negative role of ASXL1-MT against the PRC2 function. Using a chromatin immunoprecipitation (ChIP) assay, we confirmed that H3K27me3 and Ezh2-bindig profoundly decreased around the promoter regions of Hoxa5, Hoxa9, and Hoxa10 in the MDS mice, correlating with the upregulation of their mRNA expression. On the other hand, we found that ASXL1-MT reduced the expression of Clec5a, a type 2 transmembrane receptor and that this reduction was associated with differentiation block of the 32Dcl3 cells. Moreover, utilizing an shRNA or a mutant form of Clec5a, we identified that Clec5a plays essential roles in myeloid differentiation of 32Dcl3 cells. Lastly, we searched for microRNAs deregulated by ASXL1-MT since a large subset of microRNAs are found to be transcriptionally regulated by PRC2. Among upregulated microRNAs related to myeloid malignancies, we found that miR-125a targeted 3’UTR of Clec5a gene, repressed Clec5a expression and inhibited granulocytic differentiation in vitro. Intriguingly, H3K27me3 and Ezh2-bindig greatly decreased around the miR-125a gene in the BM cells of the MDS mice, similar to the results of ChIP assays around Hoxa genes. The present results indicate that ASXL1-MT which results in a truncated protein product may (1) inhibit PRC2-function by impairing the interaction of EZH2 with the ASXL1-WT and (2) promote myeloid transformation through impaired PRC2-mediated repression of posterior HOXAs and miR-125a, and subsequent suppression of CLEC5A. HOXA9 and CLEC5A expression were shown to be high and low, respectively, in MDS patients with ASXL1-MT. Our data provide evidence for a novel axis in MDS pathogenesis and implicate both mutant forms of ASXL1 and miR-125a as therapeutic targets in MDS. Disclosures: No relevant conflicts of interest to declare.

Concurrent Loss Of Ezh2 and Tet2 Cooperates In The Pathogenesis Of Myelodysplastic Disorders,

Inactivating somatic mutations in polycomb-group (PcG) genes such as EZH2 and ASXL1occur frequently in patients with myelodysplastic syndromes (MDS), myeloproliferative neoplasm (MPN) and MDS/MPN overlap disorders. While these mutations suggest a tumor suppressor function of polycomb repressive complex 2 (PRC2)-related genes in these diseases, both the impact of each PcG mutation and its interplay with coinciding mutations remain largely unknown. To understand the contribution of inactivating PcG mutations to the development of myeloid malignancies, genomic DNA from 119 patients with MDS and related neoplasms were analyzed for mutations in EZH2, ASXL1 and TET2 by high-throughput sequencing. Inactivating mutations in EZH2 and ASXL1 were detected in 8.4 and 16.8 % of patients, respectively. Moreover, 3.4 % of patients had deletion of EZH2 (located at 7q36) associated with -7 and 7q- chromosomal abnormalities. Notably, 57.1 % of these EZH2 mutations coexisted with TET2 mutations. Conversely, 34.8 % of patients with TET2 mutations had coexisting EZH2mutations. In order to understand the impact of inactivating EZH2 mutations and concurrent EZH2 and TET2 mutations on hematopoiesis, we crossed Cre-ERT;Ezh2fl/fl mice and Tet2 gene trap mice (Tet2KD/KD). Due to the early time of death in Tet2KD/KD mice and a necessity to exclude the influence of the loss of Tet2 and Ezh2 in BM niche cells, we transplanted E14.5 fetal liver cells from Cre-ERT control (WT), Cre-ERT;Tet2KD/KD, Cre-ERT;Ezh2fl/fl and Cre-ERT;Tet2KD/KDEzh2fl/fl CD45.2 mice into lethally irradiated CD45.1 recipient mice and deleted Ezh2 by intraperitoneal injection of tamoxifen at 4 weeks post-transplantation. During a long observation period, we found that Ezh2Δ/Δ mice developed MDS/MPN and half of the mice died by 10 months post-transplantation. They showed myeloproliferative features characterized by extramedullary hematopoiesis in the spleen as evident from splenomegaly with a marked increase in LSK cells. They were anemic and showed increased apoptosis in Ter119highCD71high erythroblasts in the BM, suggesting ineffective erythropoiesis, a feature compatible with myelodysplastic disorders. Ezh2Δ/Δ mice also showed dysplasia of myeloid cells, including a pseudo Pelger-Huët anomaly. To our surprise, concurrent deletion of Tet2 and Ezh2 significantly shortened the latency of disease development of not only MDS/MPN but also MDS, and all of the compound mice died of pneumonia by 10 months. Tet2KD/KDEzh2Δ/Δ MDS/MPN mice showed myeloproliferative features, including monocytosis and/or splenomegaly with extramedullary hematopoiesis. In contrast, Tet2KD/KDEzh2Δ/Δ MDS mice did not show obvious myeloproliferative features, but showed a trend of pancytopenia. The proportion of Annexin V+ cells in CD71highTer119high erythroblasts was significantly higher in both MDS/MPN and MDS mice compared to their WT counterparts, implicating enhanced apoptosis as a cause of anemia. Furthermore, myeloid dysplasia was more pronounced in these mice compared to Ezh2Δ/Δmice. Gene set enrichment analysis with microarray data showed that the Myc module was significantly enriched in Ezh2Δ/Δ LSK cells and became highly enriched in Tet2KD/KDEzh2Δ/Δ LSK cells during the development of MDS/MPN and MDS in Tet2KD/KDEzh2Δ/Δ mice. As expected, all of the PRC2 gene sets (Ezh2 targets and Ezh1 targets) showed a trend of positive enrichment in Ezh2Δ/Δ and Tet2KD/KDEzh2Δ/Δ LSK cells. Notably, however, Ezh1 targets became negatively enriched in Tet2KD/KDEzh2Δ/Δ LSK cells during the development of myelodysplastic disorders. ChIP-seq and microarray analysis data showed that upon deletion of Ezh2, a series of potential PcG related target oncogenes, such as Hmga2 and Pbx3, became derepressed in LSK cells. On the other hand, key developmental regulator genes, such as genes encoding homeobox, paired-box, T-box, forkhead and Gata family transcription factors and zinc finger DNA-binding proteins, were kept transcriptionally repressed by the compensatory action of Ezh1. Our findings provide the first evidence of the tumor suppressor function of EZH2 and demonstrate the cooperative effect of concurrent gene mutations in the pathogenesis of myelodysplastic disorders. These two models represent novel, genetically accurate models of myelodysplastic disorders amenable to epigenomic as well as preclinical therapeutic studies. Disclosures: No relevant conflicts of interest to declare.

Unveiling the paradoxical nature of myelodysplastic syndromes (MDS): Why hypercellular marrow strongly favors accelerated apoptosis

The pathogenesis of bone marrow failure in myelodysplastic syndromes (MDS) is an unresolved mystery. MDS causes peripheral blood cytopenias and increased bone marrow cellularity. This apparent paradox has been interpreted as a sign of intramedullary destruction of a substantial portion of the developing hematopoietic cells by apoptosis. The present study aimed to delineate the exact mechanistic relationship between the bone marrow hypercellularity and the accelerated apoptosis in an N-ethyl-N-nitrosourea (ENU)-induced experimental MDS mouse model. The observations made so far clarify the quantitative and qualitative changes that occur in the bone marrow microenvironment through cell cycle analysis, especially involving the telomerase reverse transcriptase (TERT) and p53 expression patterns. The survival fate of the bone marrow cells were observed by measuring the expression level of some intracellular protein molecules like apoptosis signal-regulating kinase 1 (ASK-1), c-Jun N-terminal kinase (JNK), and cleaved caspase-3 of the extrinsic pathway toward apoptosis. We found myelodysplasia damage occurs within one or more multipotent progenitor populations resulting in uncontrolled cellular proliferation within the MDS bone marrow. Then, due to homeostatic balance, this high cellular burden is minimized by activating the apoptosis pathway. As a result, the peripheral blood suffers cellular deprivation. This study can throw some light on the mechanism of disease progression and also help to reveal the paradoxical nature of the disease.