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Even though mutations in epigenetic regulators frequently occur in myeloproliferative neoplasms, their effects on the epigenome have not been well studied. Furthermore, even though primary myelofibrosis (PMF) has a markedly worse prognosis than essential thrombocytosis or polycythemia vera, the molecular distinctions between these subgroups are not well elucidated. We conducted the HELP (HpaII tiny fragment enriched by LM-PCR) assay to study genome-wide methylation in polycythemia vera, essential thrombocytosis, and PMF samples compared with healthy controls. We determined that polycythemia vera and essential thrombocytosis are characterized by aberrant promoter hypermethylation, whereas PMF is an epigenetically distinct subgroup characterized by both aberrant hyper- and hypomethylation. Aberrant hypomethylation in PMF was seen to occur in non-CpG island loci, showing further qualitative differences between the disease subgroups. The differentially methylated genes in polycythemia vera and essential thrombocytosis were involved predominantly in cell signaling pathways and were enriched for binding sites of GATA1 and other transcription factors. In contrast, aberrantly methylated genes in PMF were involved in inflammatory pathways and were enriched for NF1, LEF1, and other transcription factors. Within the PMF subgroup, cases with ASXL1 disruptions formed an epigenetically distinct subgroup with relatively increased methylation. Cases of myeloproliferative neoplasms (MPN) with TET2 mutations showed decreased levels of hydroxymethylation and distinct set of hypermethylated genes. In contrast, the JAK2V617F mutation did not drive epigenetic clustering within MPNs. Finally, the significance of aberrant methylation was shown by sensitivity of MPN-derived cell lines to decitabine. These results show epigenetic differences between PMF and polycythemia vera/essential thrombocytosis and reveal methylomic signatures of ASXL1 and TET2 mutations. Cancer Res; 73(3); 1076–85. ©2012 AACR.

Even though hematopoietic stem cell (HSC) dysfunction is presumed in myelodysplastic syndrome (MDS), the exact nature of quantitative and qualitative alterations is unknown. We conducted a study of phenotypic and molecular alterations in highly fractionated stem and progenitor populations in a variety of MDS subtypes. We observed an expansion of the phenotypically primitive long-term HSCs (lineage−/CD34+/CD38−/CD90+) in MDS, which was most pronounced in higher-risk cases. These MDS HSCs demonstrated dysplastic clonogenic activity. Examination of progenitors revealed that lower-risk MDS is characterized by expansion of phenotypic common myeloid progenitors, whereas higher-risk cases revealed expansion of granulocyte-monocyte progenitors. Genome-wide analysis of sorted MDS HSCs revealed widespread methylomic and transcriptomic alterations. STAT3 was an aberrantly hypomethylated and overexpressed target that was validated in an independent cohort and found to be functionally relevant in MDS HSCs. FISH analysis demonstrated that a very high percentage of MDS HSC (92% ± 4%) carry cytogenetic abnormalities. Longitudinal analysis in a patient treated with 5-azacytidine revealed that karyotypically abnormal HSCs persist even during complete morphologic remission and that expansion of clonotypic HSCs precedes clinical relapse. This study demonstrates that stem and progenitor cells in MDS are characterized by stage-specific expansions and contain epigenetic and genetic alterations.

Summary Homeobox domain-containing transcription factors are important regulators of hematopoiesis. Here, we report that increased levels of nonclustered H2.0-like homeobox (HLX) lead to loss of functional hematopoietic stem cells and formation of aberrant progenitors with unlimited serial clonogenicity and blocked differentiation. Inhibition of HLX reduces proliferation and clonogenicity of leukemia cells, overcomes the differentiation block, and leads to prolonged survival. HLX regulates a transcriptional program, including PAK1 and BTG1 , that controls cellular differentiation and proliferation. HLX is overexpressed in 87% of patients with acute myeloid leukemia (AML) and independently correlates with inferior overall survival (n = 601, p = 2.3 × 10 −6 ). Our study identifies HLX as a key regulator in immature hematopoietic and leukemia cells and as a prognostic marker and therapeutic target in AML.

Cellular and interpatient heterogeneity and the involvement of different stem and progenitor compartments in leukemogenesis are challenges for the identification of common pathways contributing to the initiation and maintenance of acute myeloid leukemia (AML). Here we used a strategy of parallel transcriptional analysis of phenotypic long-term hematopoietic stem cells (HSCs), short-term HSCs, and granulocyte-monocyte progenitors from individuals with high-risk (−7/7q−) AML and compared them with the corresponding cell populations from healthy controls. This analysis revealed dysregulated expression of 11 genes, including IL-1 receptor accessory protein (IL1RAP), in all leukemic stem and progenitor cell compartments. IL1RAP protein was found to be overexpressed on the surface of HSCs of AML patients, and marked cells with the −7/7q− anomaly. IL1RAP was also overexpressed on HSCs of patients with normal karyotype AML and high-risk myelodysplastic syndrome, suggesting a pervasive role in different disease subtypes. High IL1RAP expression was independently associated with poor overall survival in 3 independent cohorts of AML patients (P = 2.2 × 10−7). Knockdown of IL1RAP decreased clonogenicity and increased cell death of AML cells. Our study identified genes dysregulated in stem and progenitor cells in −7/7q− AML, and suggests that IL1RAP may be a promising therapeutic and prognostic target in AML and high-risk myelodysplastic syndrome.

Eltrombopag (EP) is a small-molecule, nonpeptide thrombopoietin receptor (TPO-R) agonist that has been approved recently for the treatment of thrombocytopenia in patients with chronic immune thrombocytopenic purpura. Prior studies have shown that EP stimulates megakaryopoiesis in BM cells from patients with acute myeloid leukemia and myelodysplastic syndrome, and the results also suggested that it may inhibit leukemia cell growth. In the present study, we studied the effects of EP on leukemia cell proliferation and the mechanism of its antiproliferative effects. We found that EP leads to a decreased cell division rate, a block in G1 phase of cell cycle, and increased differentiation in human and murine leukemia cells. Because EP is species specific in that it can only bind TPO-R in human and primate cells, these findings further suggested that the antileukemic effect is independent of TPO-R. We found that treatment with EP leads to a reduction in free intracellular iron in leukemic cells in a dose-dependent manner. Experimental increase of intracellular iron abrogated the antiproliferative and differentiation-inducing effects of EP, demonstrating that its antileukemic effects are mediated through modulation of intracellular iron content. Finally, determination of EP's antileukemic activity in vivo demonstrated its ability to prolong survival in 2 mouse models of leukemia.

Cell fate regulation is a central component of maintaining tissue homeostasis, yet the mechanisms instructing cell division diversity in tissue-specific stem cells have not been well understood. A new study uncovers a central role for microtubule motor–regulating protein Lis1 in hematopoietic stem cell fate determination and in leukemogenesis.

Owing to the discovery that rare leukemia-initiating cells (or leukemia stem cells, LSC) give origin to and propagate a hierarchical cellular organization of variably differentiated leukemic blasts, the analysis of precisely defined stem and progenitor cells have increasingly gained importance. Emergence of multi-parameter high-speed fluorescence-activated cell sorting (FACS) for the subfractionation of hematopoietic progenitor cells has allowed research on the biology of the cell-of-origin for LSCs and of LSCs as potential (or essential) therapeutic targets that may escape chemotherapy and consequently contribute to disease relapse. This review introduces the current state-of-the-art methods for the fractionation of hematopoietic stem and progenitor cells, with particular focus on myeloid malignancies. As many aspects of human normal and malignant hematopoiesis are frequently modeled in animal studies, we also provide an overview of hematopoietic stem and progenitor cell purification methods that are commonly utilized for research in murine models of disease.

A novel wingless gene was isolated from the marine colonial hydroid Hydractinia echinata. Alignments and Bayesian inference analysis clearly assign the gene to the Wnt5A group. In line with data found for the brachyury ortholog of Hydractinia, He-wnt5A is expressed during metamorphosis in the posterior tip of the spindle-shaped planula larva, suggesting that the tip functions as a putative organizer during metamorphosis. Additionally, the outermost cells of the posterior tip are omitted from apoptosis during metamorphosis. In order to investigate this putative organizer function, we transplanted the posterior tip of metamorphosing animals into non-induced larvae and into primary polyps 24 h and 48 h of age. In larvae, the tip induced formation of a secondary axis. In polyps the building of ectopic head structures was induced. Based on our data on axis formation, on gene expression similar to the organizers of other species, and the absence of regular apoptosis, we conclude that the posterior tip of the Hydractinia larva has organizing activity during metamorphosis.

Thrombocytopenia is a frequent symptom and clinical challenge in patients with myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Eltrombopag is a small molecule thrombopoietin receptor agonist that might be a new option to treat thrombocytopenia in these diseases, provided that it does not stimulate malignant hematopoiesis. In this work, we studied the effects of Eltrombopag on proliferation, apoptosis, differentiation, colony formation, and malignant self-renewal of bone marrow mononuclear cells of patients with AML and MDS. Malignant bone marrow mononuclear cells did not show increased proliferation, or increased clonogenic capacity at concentrations of Eltrombopag ranging from 0.1 to 30 μg/mL. On the contrary, we observed a moderate, statistically nonsignificant (P = .18), decrease of numbers of malignant cells (mean, 56%; SD, 28%). Eltrombopag neither led to increased 5-bromo-2-deoxyuridine incorporation, decreased apoptosis, an increase of malignant self-renewal, nor enhanced in vivo engraftment in xenotransplantations. Furthermore, we found that Eltrombopag was capable of increasing megakaryocytic differentiation and formation of normal megakaryocytic colonies in patients with AML and MDS. These results provide a preclinical rationale for further testing of Eltrombopag for treatment of thrombocytopenia in AML and MDS.

Presence of the JAK2V617F mutation in only 40% to 60% of patients with essential thrombocythemia (ET) underscores the heterogeneity of this myeloproliferative disorder (MPD). Several distinct mutations, either in JAK2 (exon 12) or in c-Mpl (W515L) have been described in subsets of other MPDs, polycythemia vera, and idiopathic myelofibrosis. Analogous to JAK2,V617F these mutations cause constitutive JAK2 and signal transducer and activation of transcription (STAT) activation. It has therefore been proposed that constitutive activation of the JAK/STAT pathway underlies the molecular etiology of all MPDs. We investigated the alternative hypothesis that distinct alterations, separate from the JAK/STAT signal transduction pathway, underlie a subset of JAK2V617F-negative ET.cDNA microarrays and quantitative reverse transcriptase polymerase chain reactions were used to compare gene expression in 40 ET patients with and without the JAK2V617F mutation.Unsupervised clustering of gene-expression patterns in ET patients revealed two distinct subclasses of patients. These subclasses differed in presence or absence of the JAK2V617F mutation. Patients lacking the JAK2V617F mutation displayed significantly lower expression of the JAK/STAT target genes Pim-1 and suppressor of cytokine signaling-2. In addition, JAK2V617F-negative patients showed lower levels of STAT3 phosphorylation.These data demonstrate that a large proportion of JAK2V617F-negative ET patients do not display constitutive JAK/STAT signaling. Hence, we propose that alterations in different signal transduction pathways can lead to the clinical phenotype of ET. Elucidation of novel ET-inducing changes will facilitate both a molecular classification of ET and development of rationally designed therapies.

Despite the established use of poly-chemotherapy, relapse continues to be the most common cause of death in AML and MDS and cure rates remain below 20%. AML/MDS arise following the accumulation of stepwise genetic and epigenetic changes in hematopoietic stem and progenitor cells (HSPC). Utilizing a novel strategy of parallel transcriptional analysis of sorted HSPC populations in distinct subtypes of AML, we compared the gene expression in AML HSPC with identical compartments from age-matched healthy controls and identified Interleukin 1 receptor accessory protein (IL1RAP) as one of the most significantly upregulated genes in HSPC in all examined subtypes of AML. Fluorescence in situ hybridization of sorted IL1RAP+ and IL1RAP- cells from patients with monosomy 7 AML (-7) indicated that the aberrant clone was restricted to IL1RAP+ cells, demonstrating that IL1RAP overexpression is a distinguishing feature of the -7 clone. Multivariate analysis of a large cohort of patients with normal karyotype AML showed that patients with high IL1RAP levels had inferior overall survival than patients with lower IL1RAP levels, suggesting an independent prognostic value for this molecule in AML. IL1RAP expression levels in MDS were found elevated on stem cells of patients with high risk disease, proposing a role of IL1RAP in higher risk MDS and progression to AML. Downregulation of IL1RAP expression by lentivirally expressed shRNAs decreased clonogenicity in cell lines and AML/MDS primary patient samples, induced apoptosis of AML cells, and reduced proliferation of AML cells and infiltration of hematopoietic organs in vivo. IL1RAP is a transmembrane protein required for signaling through several receptors of the IL1 family. Downregulation of IL1RAP expression in AML cells led to phosphorylation changes in several kinases and their substrates suggesting participation of IL1RAP in multiple signaling pathways and highlighting its potential as therapeutic target. We investigated whether inhibition of IL1RAP with pharmacological compounds is feasible and effective. Antibody-mediated inhibition of IL1RAP led to inhibition of AML cell growth in vitro. In addition, we designed peptides to interfere with IL1RAP-receptor interactions which lead to inhibition of AML cell growth. Both types of agents are being further tested and optimized. In summary, our study reveals IL1RAP as aberrantly expressed on HSPC of AML and high-risk MDS patients. Inhibition of IL1RAP is feasible and functionally effective, and thus has the potential to lead to novel therapies specifically directed at such stem cells. Beyond IL1RAP, our study provides a map of dysregulated transcripts in HSPC from patients with AML, which may offer further opportunities for therapeutic intervention. The strategy of comparative analysis of sorted stem and progenitor cells in cancer versus healthy controls may be applicable to other type of cancers with a suspected stem cell origin, and instrumental for the identification of targets for stem cell-directed therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C225. Citation Format: Laura Barreyro, Kelly Mitchell, Britta Will, Boris Bartholdy, Li Zhou, Tihomira Todorova, Robert Stanley, Susana Ben-Neriah, Cristina Montagna, Samir Parekh, Andrea Pellagatti, Jacqueline Boultwood, Elisabeth Paietta, Rhett Ketterling, Larry Cripe, Hugo Fernandez, Peter Greenberg, Jacob Rowe, Martin Tallman, Christian Steidl, Constantine Mitsiades, Amit Verma, Ulrich Steidl. IL1RAP as functionally relevant target for stem-cell directed therapy in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C225.

Mutations in the isocitrate dehydrogenase 1 (IDH1) gene are known driver mutations in acute myeloid leukemia (AML) and other cancer types. Patient outcomes in AML have remained poor, especially for patients above 60 years of age who typically do not tolerate high dose chemotherapy and stem cell transplantation, leading to cure rates below 20%. The development of novel targeted therapies for defined AML subtypes is urgently desired. Inhibitors of mutants of the closely related IDH2 gene as well as IDH1 have recently been described and show promising pre-clinical and early phase clinical activity. However, the specific molecular and functional effects of IDH1 inhibitors in AML, including in primary patients' cells, have not been reported yet. Here, we report the development of novel allosteric inhibitors of mutant IDH1 for differentiation therapy of acute myeloid leukemia. A high-throughput biochemical screen targeting an IDH1 heterodimer composed of R132H and WT IDH1 led to the identification of a tetrahydropyrazolopyridine series of inhibitors. Structural and biochemical analyses revealed that these novel compounds bind to an allosteric site that does not contact any of the mutant residues in the enzymes active site and inhibit enzymatic turnover. The enzyme complex locked in the catalytically inactive conformation inhibits the production of the oncometabolite 2-hydroxyglutarate (2-HG). In biochemical studies, we observed potent inhibition of several different clinically relevant R132 mutants in the presence or absence of the cofactor NADPH, accompanied by significant decrease in H3K9me2 levels. Allosteric inhibitor treatment of primary AML patients' cells with different clinically relevant R132 mutants of IDH1 ex vivo uniformly led to a decrease in intracellular 2-HG, abrogation of the myeloid differentiation block, increased cell death and induction of differentiation both at the level of leukemic blasts and immature stem-like cells. Allosteric inhibition of IDH1 also led to a decrease in blasts in an in vivo xenotransplantation model. At the molecular level, enhanced reduced representation bisulfite sequencing showed that treatment with allosteric IDH1 inhibitors led to a significant reversal of the DNA cytosine hypermethylation pattern induced by mutant IDH1, accompanied by gene expression changes of key sets of genes and pathways, including "Cell Cycle", "G1/S transition", "Cellular growth and proliferation", and "Cell death and survival". Taken together, our findings provide novel insight into the cellular and molecular effects of inhibition of mutant IDH1 in primary AML patients' cells. Furthermore, our study provides proof-of-concept for the molecular and biological activity of novel allosteric inhibitors for targeting of different mutant forms of IDH1 in leukemia, and opens new avenues for future investigations with these and other allosteric inhibitors for targeting mutant IDH1 in leukemia and other cancers. Disclosures Gao: GlaxoSmithKline: Employment. Pietrak:GlaxoSmithKline: Employment. Rendina:GlaxoSmithKline: Employment. Rominger:GlaxoSmithKline: Employment. Quinn:GlaxoSmithKline: Employment. Smallwood:GlaxoSmithKline: Employment. Wiggall:GlaxoSmithKline: Employment. Reif:GlaxoSmithKline: Employment. Schmidt:GlaxoSmithKline: Employment. Qi:GlaxoSmithKline: Employment. Zhao:GlaxoSmithKline: Employment. Joberty:GlaxoSmithKline: Employment. Faelth-Savitski:GlaxoSmithKline: Employment. Bantscheff:GlaxoSmithKline: Employment. Drewes:GlaxoSmithKline: Employment. Duraiswami:GlaxoSmithKline: Employment. Brady:GlaxoSmithKline: Employment. Concha:GlaxoSmithKline: Employment. Adams:GlaxoSmithKline: Employment. Schwartz:GlaxoSmithKline: Employment. McCabe:GlaxoSmithKline: Employment.

Genomic studies have shown that human cancer is rarely associated with a complete loss of transcripts; instead, acquired DNA alterations often occur within the non-coding part of the genome, are enriched in gene-regulatory regions, and cause only moderate transcriptional changes. It is currently not well understood how such moderate gene expression changes impact normal tissue function and how they contribute to malignant transformation. Acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) develop through a multi-step transformation process originating in hematopoietic stem cells (HSCs) and mainly present in the elderly (median age of >65 years at diagnosis). Although loss or near-complete loss of the hematopoietic transcription factor PU.1 induces AML in mice, a similar degree of PU.1 impairment is exceedingly rare in human AML; yet moderate PU.1 inhibition is common in AML patients. At the example of the Ets-family transcription factor PU.1, which is indispensable for HSC function and the differentiation of cells within the myeloid as well as lymphoid lineages, we tested the hypothesis that even moderate gene expression alterations of key regulators can drive malignant transformation. We assessed the effects of minimal PU.1 inhibition on hematopoiesis in a novel mouse model that co-models the genomic context found in aging human individuals and patients with MDS/AML. Mice lacking Msh2, the key component of the MutSα and MutSβ complexes mediating DNA mismatch repair, accumulate elevated numbers of point mutations, in particular C/G>T/A transitions and small insertions/deletions resembling the mutation spectrum acquired in HSCs in aging human individuals and patients with MDS and AML. We crossed Msh2-/- mice with animals carrying a heterozygous deletion of an upstream regulatory element of PU.1 (UREΔ/+). UREΔ/+Msh2-/- mice exhibited a significant, but very modest reduction of PU.1 expression on average by 26-37% in fractionated hematopoietic multipotent stem and myeloid progenitor cells. Strikingly, this minimal reduction of PU.1 led to the emergence of an aggressive, transplantable AML in more than two thirds of UREΔ/+Msh2-/- mice which was never observed in URE+/+Msh2-/- mice. Overt leukemia was preceded by a preleukemic phase hallmarked by an expanded population of multipotent murine hematopoietic stem cell enriched cells (HSPCs) that was myeloid-biased and less quiescent than their wild type counterpart. Longitudinal monitoring of preleukemic UREΔ/+Msh2-/- mice revealed a progressive increase in immature myeloid cells along with a gradual decrease in mature myeloid cells, as well as expansion of phenotypic HSPC compartments and multi-lineage dysplasia resembling human MDS. AML progression was accompanied by additional inhibition of a PU.1-cooperating factor, interferon responsive factor 8 (Irf8). Irf8 expression restoration rescued impaired expression of genes harboring PU.1/IRF consensus binding sites, led to the loss of aberrant self-renewal, promoted myeloid differentiation, and induced apoptosis in leukemic UREhetMsh2-/- cells demonstrating that Irf8 impairment functionally cooperates with minimally reduced PU.1 expression in our model. We also found evidence of disease-relevant joint PU.1/IRF8 inhibition in human myeloid leukemogenesis: (1) patients with MDS with a higher risk for the progression to AML had lower IRF8 levels; (2) lower IRF8 expression was detected specifically in AML patients with reduced PU.1 levels; (3) restoration of IRF8 expression induced differentiation in IRF8 low expressing AML cells, and (4) a positive correlation of PU.1 and IRF8 expression was found in human leukemia stem cells, but not in healthy HSCs. Strikingly, comparative pathway analysis revealed a genome-wide molecular resemblance of preleukemic and leukemic UREΔ/+Msh2-/- mice with gene expression profiles from human MDS and AML patients, respectively. Our study demonstrates that minimal reduction of a key lineage-specific transcription factor that commonly occurs in human disease is sufficient to initiate cancer development and provides mechanistic insight into the formation and progression of preleukemic stem cells in MDS and AML. Disclosures No relevant conflicts of interest to declare.

Hematopoietic stem cells (HSCs) maintain lifelong blood homeostasis. While many of the cell-intrinsic mechanisms regulating HSC function at steady state have been well characterized, the role of inflammatory cytokines and other environmental factors in tailoring blood production following physiological insults has become a topic of emerging interest. The cytokine interleukin-1 (IL-1) is a prototypical pro-inflammatory cytokine that plays a key role in host inflammatory responses to injury and infection, and is associated with elevated myeloid cell production. Importantly, IL-1 also drives a wide range of chronic inflammatory conditions such as diabetes, obesity, and arthritis that are often characterized by deregulated blood homeostasis. Here, we show at single-cell resolution using continuous tracking technology that IL-1 drives accelerated HSC cell division kinetics and myeloid differentiation via the rapid activation of a precocious PU.1-dependent myeloid gene program. Activation of this program requires direct IL-1R signaling and subsequent activation of IKK kinases, and instructively primes HSCs to adopt a myeloid fate. Strikingly, we demonstrate that IL-1 produced by myeloid cells and endothelial cells of the bone marrow (BM) niche exerts similar effects in vivo, and is required for efficient myeloid recovery following acute challenges such as transplantation or myeloablation. On the other hand, we find that chronic IL-1 exposure substantially remodels HSC blood output, resulting in myeloid overproduction and expansion of myeloid-biased multipotent progenitor (MPP) compartments at the expense of lymphoid and erythroid lineages. Critically, chronic IL-1 erodes HSC self-renewal, significantly impairing their regenerative capacity following transplantation. On the other hand, chronically exposed HSCs recover their function upon IL-1 withdrawal. Collectively, these findings identify IL-1 as a critical regulator of HSC fate and lineage specification via activation of a PU.1 circuit. They also demonstrate a role for IL-1 as a double-edged sword in HSC biology, promoting HSC regeneration in response to acute insults while severely disrupting HSC self-renewal and lineage output during chronic exposure, hence identifying IL-1 as an important and therapeutically targetable factor underwriting myeloid overproduction and other deregulations that contribute to the pathogenesis of a variety of chronic inflammatory diseases and blood disorders. Disclosures No relevant conflicts of interest to declare.

The marrow microenvironment contributes to the pathogenesis of ineffective hematopoiesis in Myelodysplastic Syndromes (MDS). Since mutations and cytogenetic alterations seen in the hematopoietic compartment are generally not present in marrow stromal cells, we hypothesized that epigenetic alterations may be responsible for altered stroma function in MDS. To elucidate the epigenome of MDS microenvironment, we used the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay to study cytosine methylation patterns of 50,000 CpGs loci covering 14,000 promoters in primary stromal cells. Global DNA methylation of 6 MDS marrow-derived stroma was analyzed by HELP assay and compared to 3 healthy controls. MDS stroma showed aberrant hypermethylation compared with controls (3626 hypermethylated vs 306 hypomethylated loci in untreated MDS stromal cells) that preferentially occurred outside of CpG islands (Test of Proportions, P value After demonstrating aberrant hypermethylation in MDS stroma, we next examined the functional role of these alterations by treatment with DNMT inhibitors. 5-Aza treatment of MDS stroma enhanced hematopoietic activity and erythroid differentiation from co-cultured healthy CD34+ cells. Significantly increased erythroid differentiation was seen in CD34+ cells cocultured with aza pretreated MDS stroma as shown by their glycophorin A positivity. Conclusions: These results reveal widespread aberrant epigenetic changes in the MDS marrow microenvironmentthat affect many important pathways. A recent report had shown that activation of beta-catenin in the bone marrow niche can trigger MDS/AML in vivo. Our results demonstrate that epigenetic silencing of WNT antagonists can potentially lead to activation of the WNT pathway in MDS stroma. Finally, we demonstrate that DNA methyl transferase inhibitors alter the epigenomic profiles of stromal cells and it raises the possibility that epigenetic modulation of the stroma by these drugs contributing to their therapeutic efficacy. Disclosures No relevant conflicts of interest to declare.

Acute Myeloid Leukemia (AML) and Myelodysplastic syndrome (MDS) arise from accumulation of multiple stepwise genetic and epigenetic changes in hematopoietic stem cells (HSC) and/or committed progenitors. A series of transforming events can initially give rise to pre-leukemia stem cells (pre-LSC) as well as fully transformed leukemia stem cells (LSC), both of which need to be targeted in strategies aimed at curing these diseases. We conducted parallel transcriptional and epigenetic analysis of highly fractionated stem and progenitor populations in individual patients of MDS and identified STAT3 upregulation in MDS HSCs. qRTPCR in an independent set of sorted MDS/AML HSCs (Lineage-negative, CD34+, CD38-) confirmed the significant increase in STAT3 expression in 60% of cases when compared to healthy controls. We further analyzed gene expression profiles of CD34+ cells from 183 MDS patients and found significant increased expression of STAT3 in MDS when compared to healthy controls (FDR Next, we studied the functional role of STAT3 by using Pyrimethamine as a clinically relevant inhibitor. Pyrimethamine is an FDA approved antifolate compound that was found to be a specific inhibitor of STAT3 activity in an initial screen of 1120 compounds. 3D modeling studies reveal that Pyrimethamine can occupy the SH2 domain of STAT3 protein with high avidity. It shows half-maximal activity (EC50) for STAT3 inhibition at approximately 1.5 μM, well within the plasma concentrations found with clinical use. We assessed the effect of Pyrimethamine on cellular proliferation of multiple leukemic cell lines (KG-1, KT-1 and CMK), and observed that these were significantly inhibited at 120 hours of exposure in a dose dependent fashion (t test, p value In summary, we have found significant demethylation and increased expression of STAT3 in sorted HSCs from AML and MDS patients. High STAT3 expression is a marker of adverse prognosis in a large cohort of MDS patients. In vitro studies show that Pyrimethamine can inhibit STAT3 activation and is able to inhibit proliferation by inducing apoptosis in leukemic cells. A phase II study evaluating Pyrimethamine for the treatment of intermediate/high-risk MDS patients that have relapsed or are refractory to azanucleoside therapy has been initiated. Disclosures Brown: Sanofi, Onyx, Vertex, Novartis, Boehringer, GSK, Roche/Genentech, Emergent, Morphosys, Celgene, Janssen, Pharmacyclics, Gilead: Consultancy.

Hematopoietic stem cells (HSCs) harbor extensive self-renewal capacity and the ability to commit to multilineage differentiation, thereby providing a life-long supply of mature peripheral blood cells. Cell fate decisions of self-renewal or differentiationcommitment are tightly regulated by the interplay of transcription factors instructing HSCs to function in a highly plastic manner during tissue maintenance, regeneration and repair. We have identified Special AT-rich sequence-binding protein 1 (Satb1) as a novel regulator integrating different HSC functions by promoting quiescence and repressing differentiation commitment through promoting symmetric self-renewal divisions of HSCs. In a Satb1-deficient murine model system we determined Satb1-dependent HSC function and molecular regulation utilizing congenic stem cell transplantation, cell cycle and division analyses, in vivo tracing of HSC commitment, asymmetric division assays, and clonogenic differentiation cultures. HSCs lacking Satb1 display defective self-renewal, were significantly less quiescent, and showed significantly increased and accelerated differentiation commitment caused by reduced symmetric self-renewal and increased symmetric differentiation divisions. Mechanistically, we found that Satb1 represses the expression of c-Myc and Numb, two factors known to be involved in HSC activation. Global DNA cytosine methylation analysis uncovered that Satb1 KO HSCs harbor premature differentiation-specific DNA methylation changes which closely resemble the pattern found in differentiation-committed multipotent progenitors. Our results also suggest that Satb1 can be retained at certain loci during mitosis, ensuring their appropriate transcriptional activity immediately upon cell division. Our findings uncover Satb1 as a novel transcriptional regulator in HSCs which promotes quiescence and represses differentiation commitment and thereby ensures the sustained self-renewal of HSCs.

Cell cycling is a tightly regulated process involving the structured expression modulation of various regulatory genes. This process is crucial for the maintenance of cell survival/proliferation in both normal and malignant hematopoietic cells. We have previously described the highly expressed CIITA-BX648577 gene fusion (Steidl C. et al., Nature 2011), involving the novel gene locus BX648577/FLJ27352 /hypothetical LOC 145788/C15orf65 and the Class II Transactivator (CIITA) in the Hodgkin’s lymphoma cell line KM-H2. While CIITA is well known to be involved in the regulation of immune responses, specifically through regulation of the Major Histocompatibility Complex (MHC)-II, nothing is known about the expression and function of the BX648577 locus. The objective of the current study was to (I) study RNA and protein expression of the putative full length gene encoded by the BX648577 (TIHL) gene locus, and (II) study its biological function in normal and malignant hematopoietic cells, including its effects on cell proliferation, clonogenicity and cell death. We detected robust endogenous TIHL RNA and protein expression in a variety of healthy and malignant hematopoietic cell types using quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analysis using a TIHL-specific antibody. At the functional level, we found that ectopic expression of the highly conserved full length TIHL protein in human NB4 leukemia cells and murine hematopoietic progenitor HPC-7 cells leads to enhanced clonogenicity and increased proliferative capacity with significant increases in the percentage of cells in S-phase of the cell cycle. Furthermore, we observed more aggressive leukemia and decreased survival of NSG mice following retro-orbital transplantation of TIHL-expressing compared to empty control-expressing NB4 cells. Interestingly, although we did not observe a change in the rate of cell proliferation or colony forming ability following TIHL overexpression in the ATRA-resistant cell line NB4.306, there was a significant alteration in its cell cycle distribution, with an increase in the fraction of cells in S-phase. The increase in S-phase cells was confirmed by 5-ethynyl-2’-deoxyuridine (EdU) incorporation assays and flow cytometry. Similarly, knockdown of TIHL using 2 independent lentiviral shRNAs, led to a significant decrease in the growth of both NB4 and acute myeloid leukemia KG1a cells in both suspension cultures and semi-solid media. Although we observed slightly increased apoptosis upon TIHL downregulation, the changes could be more significantly attributed to a decrease in the percentage of cells in S-phase within 2-3 days after transduction with the lentiviral shRNAs. Finally, in silico analysis of the TIHL promoter identified various predicted transcriptional regulators of TIHL, the majority of which are cell cycle specific transcription factors including Nuclear Receptor Subfamily 5 Group A Member 1 (NR5A1), the ets domain transcription factors ELF5 and ELF1, and Glioma-Associated Oncogene Homolog 1 (GLI-1). Our findings thus far strongly support a novel role for TIHL in cell cycle regulation/modulation in both normal and malignant hematopoiesis. Future directions include gene expression studies to identify downstream targets of TIHL following overexpression and knockdown and co-immunoprecipitation coupled to mass spectrometry analysis will be used to identify direct interacting protein partners of this novel gene. Disclosures: No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is an aggressive disease associated with poor clinical outcome. Less than one third of patients achieve durable remission with current treatment regimens, and prognostication and risk stratification are challenging. We have recently reported that the non-clustered homeobox gene, H2.0-like homeobox (HLX), is 2 to 16 fold overexpressed in more than 80% of patients with AML, across all major disease subtypes, and higher levels of HLX are associated with poor overall survival in AML. Inhibition of HLX in both murine and human AML cells has a significant anti-leukemic and differentiation-inducing effect suggesting HLX and its downstream targets as novel therapeutic targets in AML. In order to better understand the role of Hlx at the stem cell level and in myeloid differentiation in vivo, we generated knock-in mice conditionally overexpressing Hlx from the Rosa26 locus and bred them to mice that bear Cre recombinase under the control of the pIpC-inducible, hematopoietic specific promoter, Mx1. Animals overexpressing HLX exhibit elevated WBC counts and abnormal myeloid cells in the peripheral blood. Analysis of the bone marrow reveals expansion of the granulocyte-macrophage progenitor population (lin- ckit+ cd34+ CD16/32high) and expansion of immature myelocytes (ckit+ cd34+ CD16/32high Gr1int). Hlx knock-in bone marrow cells, and specifically immature granulocyte precursors, exhibit enhanced serial clonogenicity in methylcellulose colony assays, and a differentiation block and maintenance of immaturity in response to GM-CSF. Internal tandem duplications of FLT3 (FLT3-ITD) are seen in approximately 30% of all AML patients, and frequently co-occur with elevated HLX levels. Correlative analyses showed that AML patients with mutant FLT3 and low HLX have overall survival similar to WT FLT3 patients, and survive significantly longer than patients with mutant FLT3 and high HLX (p=0.005), demonstrating that FLT3 mutations confer poor prognosis only if HLX is highly expressed, and suggesting that HLX and mutant FLT3 functionally cooperate. We retrovirally co-expressed HLX and FLT3-ITD, or FLT3-ITD alone (plus an empty control), in primary Lin-Kit+cells and transplanted them into congenic recipient animals. Four weeks after transplantation, donor chimerism was 4-fold increased on average in the peripheral blood (PB) and bone marrow (BM), and by 12 weeks post-transplantation mice expressing FLT3-ITD and HLX developed AML with large numbers of leukemic blasts in the peripheral blood and bone marrow. We then crossed our new Hlx knock-in mouse model with previously generated FLT3-ITD knock-in mice. Strikingly, heterozygous double-transgenic mice expressing both the knock-in FLT3-ITD mutation and HLX develop acute myeloid leukemia after a latency of 2 months. Morphological and flow cytometric analysis revealed large numbers of blasts circulating in the peripheral blood and replacing the marrow, as well as substantial leukemic infiltrates in the spleen and liver. Our studies reveal a critical role for HLX in conferring a differentiation block and increased clonogenicity at the pre-leukemic stem and progenitor cell level in a genetic in vivo model. Furthermore, a novel compound knock-in mouse model of Hlx overexpression and FLT3-ITD demonstrates that Hlx can initiate AML in cooperation with FLT3-ITD in vivo. Disclosures: No relevant conflicts of interest to declare.

Abstract 651 Acute myeloid leukemia (AML) is an aggressive disease which is associated with poor clinical outcome. Less than one third of patients achieve durable remission with current treatment regimens, and prognostication and risk stratification are challenging. Identification and functional studies of genes and pathways which regulate leukemic transformation and maintenance is instrumental to understanding the pathogenesis of AML and for development of novel therapeutic strategies. Several members of the Hox (class I homeobox) family of transcription factors have been implicated in the regulation of normal hematopoiesis and leukemogenesis. Less is known about the role of non-clustered (class II) homeobox genes. We found that a new non-clustered homeobox gene, H2.0-like homeobox (HLX), regulates early hematopoiesis and promotes AML in mice and humans. HLX is 2 to 16 fold overexpressed in more than 80% of patients with AML, across all major disease subtypes. Higher levels of HLX are associated with poor overall survival in 3 different, large cohorts of AML patients (N=601, p=2.3×10−6), and HLX holds up as an independent prognostic factor in a multivariate analysis. ShRNA-mediated inhibition of HLX in both murine and human AML cells significantly inhibits leukemic growth and clonogenic capacity, and overcomes the differentiation block of AML cells. When we analyzed pre-leukemic hematopoietic stem and progenitor cells (HSPC) in a PU.1 URED/D AML mouse model, we found a 4-fold elevation of Hlx, suggesting that Hlx is involved in malignant transformation. Overexpression of HLX in wildtype HSPC in a competitive, congenic transplantation model led to near complete depletion of long-term HSC and 16-fold enrichment of myeloid progenitors with a surface phenotype slightly past the GMP stage (CD45+Kit−CD34−CD44highCD49bhighCD11bmid). Overexpression of HLX in HSPC in vitro led to a myeloid differentiation block and to formation of aberrant, CD34−Kit− progenitors with unlimited serial clonogenicity. The mechanism of action of Hlx is so far unknown. The presence of a C-terminal homeobox domain suggests Hlx may directly interact with DNA, however, no studies have shown DNA binding by Hlx or identified direct Hlx target genes. We find that mutation of only two residues of the Hlx homeodomain is sufficient to completely abrogate the differentiation block induced by HLX overexpression in HSPC, indicating Hlx is acting through the DNA-binding ability of its homeodomain. Furthermore, we have now identified direct HLX target genes in both HSPC and AML cells using a combination of expression microarrays and chromatin-immunoprecipitation (chIP). We find that HLX regulates a set of genes which mediate its leukemia-promoting functions, such as BTG1, and we have used chIP to identify a subset of these genes, including PAK1, that are direct targets of HLX. Internal tandem duplications of FLT3 (FLT3-ITD) are seen in approximately 25% of all AML patients, and confer a poor prognosis. Correlative analyses showed that AML patients with mutant FLT3 and low HLX have overall survival similar to WT FLT3 patients, and survive significantly longer than patients with mutant FLT3 and high HLX (p=0.005), demonstrating that FLT3 mutations confer poor prognosis only if HLX is highly expressed, and suggesting that HLX and mutant FLT3 functionally cooperate. We find that co-expression of HLX and FLT3-ITD leads to dramatically enhanced cytokine independent growth and clonogenicity of 32D cells as well as primary murine HSPC in vitro. When we retrovirally co-expressed HLX and FLT3-ITD, or FLT3-ITD alone (plus an empty control), in primary Lin−Kit+ cells and transplanted them into congenic recipient animals, we found that four weeks after transplantation donor chimerism was 4-fold increased on average in the peripheral blood (PB) and bone marrow (BM), and by 12 weeks post-transplantation mice expressing FLT3-ITD and HLX developed AML with large numbers of leukemic blasts in the PB and BM. We have generated knock-in mice conditionally overexpressing Hlx from the Rosa26 locus and ongoing studies include crossing these mice into FLT3-ITD knock-in animals. In summary, our studies have identified HLX as a novel key transcription factor involved in the regulation of early hematopoiesis and AML pathogenesis, and suggest HLX and downstream pathways as promising new therapeutic targets in AML. Disclosures: No relevant conflicts of interest to declare.

PU.1 is a hematopoietic transcription factor that is required for the development of myeloid and B cells. PU.1 is also expressed in erythroid progenitors, where it blocks erythroid differentiation by binding to and inhibiting the main erythroid promoting factor, GATA-1. However, other mechanisms by which PU.1 affects the fate of erythroid progenitors have not been thoroughly explored. Here, we used ChIP-Seq analysis for PU.1 and gene expression profiling in erythroid cells to show that PU.1 regulates an extensive network of genes that constitute major pathways for controlling growth and survival of immature erythroid cells. By analyzing fetal liver erythroid progenitors from mice with low PU.1 expression, we also show that the earliest erythroid committed cells are dramatically reduced in vivo. Furthermore, we find that PU.1 also regulates many of the same genes and pathways in other blood cells, leading us to propose that PU.1 is a multifaceted factor with overlapping, as well as distinct, functions in several hematopoietic lineages., Author Summary Cellular identities are established by master regulatory transcription factors that promote cell type–specific gene expression. In some instances such factors also inhibit differentiation of alternative, closely related lineages. PU.1 is an Ets family transcription factor that is required for myeloid and B cell development. PU.1 is also expressed in red blood cell progenitors where it blocks erythroid terminal differentiation. One mechanism used by PU.1 to block red blood cell differentiation is by binding to and inhibiting the erythroid master regulator GATA-1. Here, we describe another mechanism utilized by PU.1 in erythroid progenitors. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-Seq) combined with gene expression profiling of erythroid progenitors, we show that PU.1 controls a large gene network in immature erythroid cells, including genes in pathways involved in regulating growth, survival, and differentiation of these cells. We also find that PU.1 controls many of the same genes and pathways in other blood cells. The results indicate that, in addition to activating lineage-specific genes, master regulatory transcription factors, like PU.1, also control numerous, widely expressed genes in multiple cell lineages.

Expression of the transcription factor PU.1 is dependent on a highly conserved upstream regulatory element (URE) located 14 kilobases upstream of the transcriptional start site. The proximal promoter of PU.1 alone, without this enhancer, displays 100-fold reduced promoter activity and is not capable of driving PU.1 expression in transgenic mice. Targeted disruption of the URE reduces PU.1 expression by 80% and leads to AML in mice. However, the mechanisms mediating the long-range regulatory function of the URE are largely unknown. Here, we identified a binding site of the chromatin-remodeling special AT-rich sequence-binding protein 1 (SATB1). Utilizing EMSA and ChIP, we found that SATB1 binds to the URE in myeloid U937 cells. Luciferase assays demonstrated 7-fold reduced reporter activity upon disruption of the SATB1 site. Lentiviral overexpression of SATB1 in primary murine Lin−, Kit+ progenitor cells led to an upregulation of PU.1 expression. We did not observe this effect in URE−/− progenitors indicating that the PU.1 regulatory function of SATB1 is mediated by the URE. Inhibition of SATB1 by siRNA in U937 cells led to a decrease in PU.1 expression. This inhibitory effect was not seen in myeloid URE−/− cells. To address the question at which stages during myeloid development SATB1 regulates PU.1, we sorted KSL-HSC, CMP, GMP, and MEP of SATB1 knockout mice and determined PU.1 expression levels. Interestingly, PU.1 expression was 88% and 80% reduced in SATB1−/− GMP and MEP, while KSL-HSC and CMP did not show significantly changed PU.1 levels. This finding indicates a stage-specific regulatory function of SATB1 during myelopoiesis. When we sequenced the URE in human individuals with AML we identified a SNP that abates binding of SATB1 and leads to decreased PU.1 expression in GMP and MEP. While the overall frequency of the homozygous SNP was not significantly changed in AML patients compared with healthy controls, this SNP was 2.3-fold more frequent in patients with AML with complex karyotype than in AML with normal karyotype (p

The transcription factor Nuclear-Factor-Erythroid2 (NF-E2) is overexpressed in the vast majority of patients with polycythemia vera (PV). PV affects precisely those lineages expressing NF-E2: hematopoietic precursors, erythroid, megakaryocytic and granulocytic cells. In murine models, NF-E2 overexpression increases proliferation and promotes cellular viability in the absence of EPO. EPO-independent growth is a hallmark of PV. We therefore hypothesized that NF-E2 overexpression contributes to erythrocytosis, the pathognomonic symptom of PV. Consequently, we investigated the effect of NF-E2 overexpression in healthy CD34+ cells. NF-E2 overexpression lead to a delay in erythroid differentiation, manifested by a belated appearance of GlycophorinA-positive mature erythroid cells. Differentiation delay was similarly observed in primary PV patient erythroid cultures compared to healthy controls. Protracted differentiation lead to a significant increase in the accumulated number of erythroid cells both in PV cultures and in CD34+ cells overexpressing NF-E2. Similarly, NF-E2 overexpression altered erythroid colony formation, leading to an increase in BFU-E formation. These data argue that NF-E2 overexpression delays the early phase of erythroid differentiation, resulting in an expansion of erythroid progenitors, thereby increasing the number of erythrocytes derived from one CD34+ cell. These data propose a role for NF-E2 in mediating the erythrocytosis of PV.

Despite recent advances in characterizing molecular markers for the diagnosis of polcycythemia vera (PV), the aberrations leading to disease development remain unknown. We therefore used expression profiling to identify candidate genes involved in the pathophysiology of PV. RNA from purified granulocytes of 40 PV patients was analyzed by hybridizing individual samples to a pool of 50 healthy controls. Of the 7,496 genes represented in the cDNA array, 253 were upregulated more than 1.5 fold in PV compared to healthy controls (p< 0.01, FDR corrected). Promoters for 26 of the 253 genes overexpressed in PV are regulated by members of the Sp1 family of transcription factors. We have therefore hypothesized that altered activity of one or several Sp1-like transcription factors may contribute to the molecular etiology of PV. Here we report that one of the Sp1 target genes identified, the transcription factor NF-E2, is overexpressed in 37 of the 40 PV patients (92.5%) assayed by microarray. NF-E2 overexpression was confirmed by Northern Blot and quantitative RT-PCR analysis. Transcription factor overexpression varies from 2.3 to 40 fold, with a median increase of 7 fold in PV patients compared to healthy controls. The NF-E2 protein is readily detected in PV granulocytes by Western Blot whereas it is undetectable in healthy control cells. Immunohistochemistry revealed that in PV bone marrow, NF-E2 is overexpressed in megakaryocytes as well as erythroid and granulocytic precursors. Several published observations suggest that NF-E2 is an exceptionally promising candidate in the molecular etiology of PV. Firstly, the transcription factor is expressed in hematopoietic precursors as well as in erythroid, megakaryocytic and granulocytic cells, those lineages affected in PV. Secondly, Sayer et al. have shown that overexpression of NF-E2 in fetal liver cells leads to the development of Epo-independent erythroid colonies, analogous to the endogenous erythroid colonies (EECs) observed in PV patients. Furthermore, ectopic expression of NF-E2 results in the spontaneous emergence of morphologically mature erythroid cells in the absence of Epo and can reprogram monocytic cells towards erythroid and megakaryocytic differentiation. These data support the hypothesis that the concentration of an individual transcription factor can control lineage commitment. We thus propose that in PV patients elevated concentrations of NF-E2 alter the physiological transcription factor balance leading to an overproduction of erythroid and, in select patients, megakaryocytic cells/platelets. In this model the level of NF-E2 overexpression determines both the severity of erythrocytosis and the concurrent presence or absence of thrombocytosis.

Recurring deletions of chromosome 7 and 7q [−7/del(7q)] occur in myelodysplastic syndromes and acute myeloid leukemia (AML) and are associated with poor prognosis. However, the identity of functionally relevant tumor suppressors on 7q remains unclear. Using RNAi and CRISPR/Cas9 approaches, we show that an ~50% reduction in gene dosage of the mixed lineage leukemia 3 (MLL3) gene, located on 7q36.1, cooperates with other events occurring in −7/del(7q) AMLs to promote leukemogenesis. Mll3 suppression impairs the differentiation of HSPC. Interestingly, Mll3-suppressed leukemias, like human −7/del(7q) AMLs, are refractory to conventional chemotherapy but sensitive to the BET inhibitor JQ1. Thus, our mouse model functionally validates MLL3 as a haploinsufficient 7q tumor suppressor and suggests a therapeutic option for this aggressive disease.