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Myelodysplastic syndrome (MDS) is a hematopoietic stem-cell disorder characterized by trilineage dysplasia and susceptibility to acute myelogenous leukemia (AML). Analysis of molecular basis of MDS has been hampered by the heterogeneity of the disease. Recently, mutations of the transcription factor AML1/RUNX1 have been identified in 15% to 40% of MDS–refractory anemia with excess of blasts (RAEB) and MDS/AML. We performed mouse bone marrow transplantation (BMT) using bone marrow cells transduced with the AML1 mutants. Most mice developed MDS and MDS/AML-like symptoms within 4 to 13 months after BMT. Interestingly, among integration sites identified, Evi1 seemed to collaborate with an AML1 mutant harboring a point mutation in the Runt homology domain (D171N) to induce MDS/AML with an identical phenotype characterized by marked hepatosplenomegaly, myeloid dysplasia, leukocytosis, and biphenotypic surface markers. Collaboration between AML1-D171N and Evi1 was confirmed by a BMT model where coexpression of AML1-D171N and Evi1 induced acute leukemia of the same phenotype with much shorter latencies. On the other hand, a C-terminal truncated AML1 mutant (S291fsX300) induced pancytopenia with erythroid dysplasia in transplanted mice, followed by progression to MDS-RAEB or MDS/AML. Thus, we have developed a useful mouse model of MDS/AML that should help in the understanding of the molecular basis of MDS and the progression of MDS to overt leukemia.

The AML1 transcription factor complex is the most frequent target of leukemia-associated chromosomal translocations. Homeodomain-interacting protein kinase 2 (HIPK2) is a part of the AML1 complex and activates AML1-mediated transcription. However, chromosomal translocations and mutations of HIPK2 have not been reported. In the current study, we screened mutations of the HIPK2 gene in 50 cases of acute myeloid leukemia (AML) and in 80 cases of myelodysplastic syndrome (MDS). Results indicated there were two missense mutations (R868W and N958I) in the speckle-retention signal (SRS) domain of HIPK2. Subcellular localization analyses indicated that the two mutants were largely localized to nuclear regions with conical or ring shapes, and were somewhat diffused in the nucleus, in contrast to the wild type, which were mainly localized in nuclear speckles. The mutations impaired the overlapping localization of AML1 and HIPK2. The mutants showed decreased activities and a dominant-negative function over wild-type protein in AML1- and p53-dependent transcription. These findings suggest that dysfunction of HIPK2 may play a role in the pathogenesis of leukemia.

A histone H3 Lysine 27 (H3K27)-methyltransferase, enhancer of zeste homolog 2(EZH2) is known as a tumor-associated gene. Physiological role of EZH2 is an enzymatic component of polycomb repressive complex 2 (PRC2) to inhibit expression of target genes. While EZH2 plays oncogenic roles by repressing the expression of tumor suppressors in solid tumors and some lymphomas, it plays rather tumor-suppressive roles in myeloid malignancies. We have generated a short-form EZH2 that lacks the catalytic SET domain (EZH2-dSET). Using this EZH2 mutant we could produce serially transplantable MDS-like diseases. Microarray analysis using the MDS-like bone marrow cells enabled us to identify novel targets of EZH2 in MDS tumorigenesis, including ATP-binding cassette (ABC) transporters. Derepression of Abcg2 via decreased H3K27-trimethylation was confirmed. Retroviral transduction of EZH2-dSET to MDS-like cell lines increased surface ABCG2-high populations and those cells functioned to exclude anticancer drugs as expected. Intriguingly, with Abcg2 expression alone, primary bone marrow cells could produce an MDS-like cytopenic disease in our BMT model. In our clinical specimens, ABCG2 high expressions were observed in MDS samples but not in de novo AML and CML samples. In two MDS patients, ABCG2 expression decreased along with leukemic transformation. Interestingly, two out of 33 MDS patients with extremely high expression of ABCG2 harbored the same U2AF1 mutation (Q157P). In addition, somatic mutations of EZH2 and those of either U2AF1 or SRSF2 were mutually exclusive in all investigated cases. Interestingly, U2AF1 mutants (S34F and Q157P) reduced EZH2 expression, leading the derepression of ABCG2 via decreased H3K27-trimethylation. These results indicate a link between U2AF1 mutations and ABCG2 expression via disrupted EZH2. In conclusion, different mechanisms are supposed to converge at dysregulated EZH2 in MDS. And a short form of EZH2 upregulates ABCG2 expression resulting in MDS advancing to secondary leukemia. Thus, either mutations affecting the EZH2 function or mutations of EZH2 itself could play an important role in MDS, and one of the downstream targets of EZH2 suppression in MDS pathogenesis is aberrant expression of ABCG2. Disclosures No relevant conflicts of interest to declare.

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. Disclosures Shih: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

RUNX1/AML1 mutations have been frequently detected in patients with myeloid neoplasms, especially myelodysplastic syndromes (MDS) and chronic monocytic leukemia (CMML). Although the mutations have been analyzed thoroughly, its expression level has not been investigated. Therefore, we attempt to clarify the expression of RUNX1 in the pathogenesis of myeloid neoplasms. The study was approved by the institutional review board and patients gave written informed consent for the study, according to the Declaration of Helsinki. Several isoforms of RUNX1 mRNA are known and we analyzed RUNX1a (including exon 7a which has stop codon) and RUNX1b (skipping exon 7a and including exon 7b and 8). Expression levels of full length isoform (RUNX1b) and short isoform (RUNX1a which has a dominant negative effect on RUNX1b) in CD34+ cells from patients with myeloid neoplasms were examined. A part of patients with MDS or myelodysplastic syndrome / myeloproliferative neoplasms (MDS/MPN) including CMML showed RUNX1a overexpression. Average of relative RUNX1a expression level in MDS patients (n=34) and MDS/MPN patients (n=20) was 7.4-fold and 8.6-fold of the level in normal bone marrow (BM), respectively, whereas most of these patients showed almost same or slight increase of expression level of RUNX1b compared with normal BM. Interestingly, some patients showed high expression of RUNX1a and repression of RUNX1b. In both disease categories, patients with excess blasts displayed a significantly higher expression level of RUNX1a compared with normal BM and patients without excess blasts. During the disease progression in a single patient with MDS or MDS/MPN, the expression of RUNX1a became higher, while azacitidine treatment reduced RUNX1a expression. Genomic mutations of RUNX1 were also examined. RUNX1 mutations were detected in 16% of MDS and 35% of MDS/MPN. Surprisingly, a part of patients had both RUNX1 gene mutation and RUNX1a overexpression, and they showed rapid progression of disease. To evaluate the effects of RUNX1a overexpression, RUNX1a was transduced into CD34+ cells from MDS patients with low expression level of RUNX1a. RUNX1a-transduction resulted in cell proliferation on MS5 stromal cells. These results indicate that overexpression of RUNX1a may add growth advantage to CD34+ cells in patients with MDS or MDS/MPN. We next analyzed the mechanism of RUNX1a overexpression. Gene mutations affecting exon recognition were examined in the patients. Splicing factor mutations, SRSF2 and U2AF1, were detected frequently in MDS (15%) and MDS/MPN (50%). Patients with splicing factor mutations showed higher RUNX1a expression than patients without the mutations. To confirm that the splicing factor mutations affect the expression of RUNX1a, we performed enforced expression of SRSF2 p.P95H mutant using pMYs.IRES.EGFP retrovirus vector in a MDS-derived cell line, TF-1. After a single cell sorting, independent 13 expanding clones were analyzed. Most of the clones demonstrated higher expression of RUNX1a than mock cells, whereas RUNX1b expression was reduced in all clones. Increase of RUNX1a expression in SRSF2 mutant-transduced TF-1 cells was also confirmed by Western blot. Moreover, the clones with higher GFP intensity showed higher expression level of RUNX1a, suggesting that SRSF2 p.P95H expression level may affect the expression level of RUNX1a. Furthermore, SRSF2 mutant-transduced TF-1 cells showed phenotypic changes of higher CD11b and CD14 than mock TF-1 cells, suggesting that SRSF2 mutant may induce monocytic differentiation via RUNX1a overexpression. Gene mutations of RUNX1 in intron 6 and exon 7a were also analyzed. A 5' splice site change just after exon 6 was detected in a CMML patient with RUNX1a overexpression, which may be another mechanism of RUNX1a overexpression. Mutations of exon 7a or changes in 3' splice site just before exon 7a have not been detected yet. In conclusion, our data suggest that overexpression of RUNX1a may play a critical role in the progression of MDS and MDS/MPN, in addition to RUNX1 mutations. Splicing factor mutations are suspected to contribute to the mechanism of the dysregulation of RUNX1. Disclosures No relevant conflicts of interest to declare.

Two types of CCAAT-enhancer-binding protein α (C/EBPα) mutants are found in acute myeloid leukemia (AML) patients: N-terminal frame-shift mutants (C/EBPα-N m ) generating p30 as a dominant form and C-terminal basic leucine zipper domain mutants (C/EBPα-C m ). We have previously shown that C/EBPα-K304_R323dup belonging to C/EBPα-C m , but not C/EBPα-T60fsX159 belonging to C/EBPα-N m , alone induced AML in mouse bone marrow transplantation (BMT) models. Here we show that various C/EBPα-C m mutations have a similar, but not identical, potential in myeloid leukemogenesis. Notably, like C/EBPα-K304_R323dup, any type of C/EBPα-C m tested (C/EBPα-S299_K304dup, K313dup, or N321D) by itself induced AML, albeit with different latencies after BMT; C/EBPα-N321D induced AML with the shortest latency. By analyzing the gene expression profiles of C/EBPα-N321D- and mock-transduced c-kit + Sca-1 + Lin − cells, we identified Csf1r as a gene downregulated by C/EBPα-N321D. In addition, leukemic cells expressing C/EBPα-C m exhibited low levels of colony stimulating factor 1 receptor in mice. On the other hand, transduction with C/EBPα-N m did not influence Csf1r expression in c-kit + Sca-1 + Lin − cells, implying a unique role for C/EBPα-C m in downregulating Csf1r . Importantly, Csf1r overexpression collaborated with C/EBPα-N321D to induce fulminant AML with leukocytosis in mouse BMT models to a greater extent than did C/EBPα-N321D alone. Collectively, these results suggest that C/EBPα-C m -mediated downregulation of Csf1r has a negative, rather than a positive, impact on the progression of AML involving C/EBPα-C m , which might possibly be accelerated by additional genetic and/or epigenetic alterations inducing Csf1r upregulation.

Myelodysplastic syndrome (MDS) is a clonal disorder of hematopoietic stem cells characterized by ineffective and inadequate hematopoiesis. Because MDS is a heterogeneous disorder, specific gene abnormalities implicated in the pathogenesis of MDS have been difficult to identify. Cytogenetic abnormalities are seen in half of the MDS patients and generally consist of partial or complete chromosome deletion or addition, whereas balanced translocations are rare. Although point mutations of critical genes had been demonstrated to contribute to the development of MDS, there was no strong correlation between these mutations and clinical features. Recently, we reported the high incidence of somatic mutations in the AML1/RUNX1 gene (which is a critical regulator of definitive hematopoiesis and the most frequent target for translocation of acute myeloid leukemia [AML]) in MDS, especially refractory anemia with excess blasts (RAEB), RAEB in transformation (RAEBt), and AML following MDS (defined here as MDS/AML). The MDS/AML patients with AML1 mutations had a significantly worse prognosis than those without AML1 mutations. Most AML1 mutants lose trans-activation potential, which leads to a loss of AML1 function. These data indicate that AML1 point mutation is one of the major causes of MDS/AML, and "MDS/AML with AML1 mutation" represents a distinct clinicopathologic-genetic entity.

A 26-year-old man with idiopathic hypereosinophilic syndrome (HES) was treated with imatinib mesylate following a 5-year history of prednisolone therapy. The patient had hypereosinophilia (absolute eosinophil counts1500/microL) occurring in cyclic oscillations as well as histologically diagnosed eosinophilic vasculitis, bursitis, and periodic soft-tissue swellings. Laboratory data revealed high levels of serum tryptase and increased numbers of mast cells in the bone marrow, but serum interleukin 5 levels were within the normal range. The disease initially responded well to 100 mg/day of imatinib mesylate but recurred 8 weeks later. Thereafter, a daily 200-mg dose was temporarily effective. Despite the response to imatinib, the FIP1L1-PDGFRA fusion gene was not detected by fluorescence in situ hybridization analysis. Additional molecular and cytogenetic studies showed neither translocations of platelet-derived growth factor receptor (PDGFR) genes nor mutations in the c-KIT or the PDGFR genes. Although imatinib mesylate is a choice of treatment for patients with HES, its precise molecular mechanism in individual cases remains to be clarified.

A high incidence of somatically acquired point mutations in the AML1/RUNX1 gene has been reported in poorly differentiated acute myeloid leukemia (AML, M0) and in radiation-associated and therapy-related myelodysplastic syndrome (MDS) or AML. The vast majority of AML1 mutations identified in these diseases were localized in the amino (N)–terminal region, especially in the DNA-binding Runt homology domain. In this report, we show that AML1 point mutations were found in 26 (23.6%) of 110 patients with refractory anemia with excess blasts (RAEB), RAEB in transformation (RAEBt), and AML following MDS (defined these 3 disease categories as MDS/AML). Among them, 9 (8.2%) mutations occurred in the carboxy (C)–terminal region, which were exclusively found in MDS/AML and were strongly correlated with sporadic MDS/AML. All patients with MDS/AML with an AML1 mutation expressed wild-type AML1 protein and had a significantly worse prognosis than those without AML1 mutations. Most AML1 mutants lost trans-activation potential, regardless of their DNA binding potential. These data suggested that AML1 point mutation is one of the major driving forces of MDS/AML, and these mutations may represent a distinct clinicopathologic-genetic entity.

We describe here the pharmacology of (E)-N-[6-methoxy-5-(2-methoxyphenoxy)[2,2'-bipyrimidin]-4-yl]-2-phenylethenesulfonamide monopotassium salt (YM598), a novel selective endothelin ET(A) receptor antagonist synthesized through the modification of the ET(A)/ET(B) non-selective antagonist, bosentan. YM598 inhibited [125I]endothelin-1 binding to cloned human endothelin ET(A) and ET(B) receptor, with K(i) of 0.697 and 569 nM, and inhibited endothelin-1-induced increases in intracellular Ca(2+) concentration in human and rat endothelin ET(A) receptor. YM598 also inhibited endothelin-1-induced vasoconstriction in isolated rat aorta with a pA(2) value of 7.6. In vivo, YM598 inhibited the pressor response to big endothelin-1, a precursor peptide of endothelin-1. DR(2) values of YM598 in pithed rats were 0.53 mg/kg, i.v. and 0.77 mg/kg, p.o., and its antagonism in conscious rats was maintained for more than 6.5 h at 1 mg/kg, p.o. In contrast, YM598 had no effect on the sarafotoxin S6c-induced depressor or pressor responses. YM598 showed not only superior antagonistic activity and higher-selectivity for endothelin ET(A) receptor in vitro, but at least a 30-fold higher potency in vivo than bosentan. In conclusion, YM598 is a potent and orally active selective endothelin ET(A) receptor antagonist.

CD34++ cells from 45 patients with myelodysplastic syndrome (MDS) and MDS-acute myeloid leukaemia (MDS-AML) were observed by flow cytometry for the expression of granulocyte colony-stimulating factor receptor (G-CSFR). Ten patients had a significantly reduced expression of G-CSFR. Late stages of disease showed a higher proportion of either high or low G-CSFR expression than earlier stages. In MDS refractory anaemia (RA), G-CSFR was inversely related to CD33 expression. Most patients (9/10) with low G-CSFR expression had neutropenia of the peripheral blood. Neutropenia was less common in the normal group, but also occurred in the high expression group. No neutrophil response was observed following G-CSF administration to MDS-AML patients (6/6) with low G-CSFR expression. In the high expression group, patients (3/3) showed a response to G-CSF while, in the normal group (1/2), the response was minor. In the normal- or high-receptor-expressing groups, the receptors were functionally active in terms of apoptosis but not proliferation and clonogenic growth, although no clear correlation to receptor expression was observed. The G-CSFR signal transduction pathway in the normal and high group was not deficient of messenger RNA for either janus kinases (Jaks) or signal transducers and activators of transcription (Stats). These findings suggest that the lowered expression of G-CSFR may cause neutropenia in MDS and MDS-AML patients and, therefore, may partially explain the neutropenia in myelodysplastic patients.

Somatically acquired point mutations of AML1/RUNX1 gene have been recently identified in rare cases of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). Moreover, germ line mutations of AML1 were found in an autosomal dominant disease, familial platelet disorder with predisposition to AML (FPD/AML), suggesting that AML1 mutants, as well as AML1 chimeras, contribute to the transformation of hematopoietic progenitors. In this report, we showed that AML1 point mutations were found in 6 (46%) of 13 MDS patients among atomic bomb (A-bomb) survivors in Hiroshima. Unlike acute or chronic leukemia patients among A-bomb survivors, MDS patients exposed relatively low-dose radiation and developed the disease after a long latency period. AML1 mutations also were found in 5 (38%) of 13 therapy-related AML/MDS patients who were treated with alkylating agents with or without local radiation therapy. In contrast, frequency of AML1 mutation in sporadic MDS patients was 2.7% (2 of 74). Among AML1 mutations identified in this study, truncated-type mutants lost DNA binding potential and trans-activation activity. All missense mutations with one exception (Gly42Arg) lacked DNA binding ability and down-regulated the trans-activation potential of wild-type AML1 in a dominant-negative fashion. The Gly42Arg mutation that was shared by 2 patients bound DNA even more avidly than wild-type AML1 and enhanced thetrans-activation potential of normal AML1. These results suggest that AML1 point mutations are related to low-dose radiation or alkylating agents and play a role distinct from that of leukemogenic chimeras as a result of chromosomal translocations caused by sublethal radiation or topoisomerase II inhibitors.

Mutations in a variety of genes have been identified in MDS patients. Among them, mutations of additional sex combs-like 1 (ASXL1), found in 15-20% of MDS patients, have been identified as an independent poor prognostic factor. We previously demonstrated that C-terminal–truncating ASXL1 mutations (ASXL1-MT) inhibited myeloid differentiation and induced an MDS-like disease in mice after 1~2 years by inhibiting polycomb repressive complex 2–mediated methylation of histone H3K27 (Inoue et al. J Clin Invest. 2013). Given that ASXL1 mutations have been shown to be related to high-risk MDS or leukemic transformation, it is not clear how ASXL1-mutated MDS clones can transform into advanced MDS or AML. First, we examined genetic alterations in 368 WHO-defined MDS patients; ASXL1 mutations were detected in 64 of them (17.39%). Intriguingly, the patients with ASXL1 mutations had a significantly higher incidence of the concurrent SET binding protein 1 (SETBP1) mutation than those with the wild-type ASXL1 (6 out of 64, 9.38% vs. 2 out of 304, 0.66%, P=0.0005). Moreover, among ASXL1-mutated MDS patients, those harboring SETBP1 mutations had a higher incidence of leukemic transformation than those without (P=0.042), and MDS patients with both mutations had a significantly shorter overall survival compared to those without SETBP1 mutations (median, 10.5 vs. 22.5 months, P=0.046). In addition, we demonstrated that most SETBP1 mutations, such as D868N, occur in the PEST domain of the SKI homology region, preventing ubiquitination and subsequent proteasomal degradation. These results prompted us to investigate whether SETBP1 mutations play a critical role in the leukemic transformation of ASXL1-mutated MDS cells. In in vitro experiments, the expression of SETBP1-D868N enhanced myeloid colony formation of ASXL1-MT-transduced LSK cells, augmenting ASXL1-MT-induced differentiation blocking of 32Dcl3 cells. Of note, SETBP1-D868N collaborated with ASXL1-MT to induce AML after a short latency (median survival, 73 days) in a murine BMT model, while all mice expressing either ASXL1-MT or SETBP1-D868N survived for 6 months after transplantation (P To clarify the molecular mechanism leading to leukemic transformation, we first investigated the Pp2a-Akt pathway because SETBP1 protein has been shown to interact with SET oncoprotein, resulting in Pp2a phosphorylation and subsequent inhibition. Consistent with previous reports using overexpression systems of SETBP1 wild type protein (SETBP1-WT), BM cells of leukemic mice displayed phosphorylated Pp2a and Akt compared to those of the control mice. Administration of FTY720, a Pp2a activator, efficiently repressed the growth rate in vitro and slightly improved the survival of serially transplanted mice. Next, using RNA-seq and GSEA, we demonstrated that SETBP1-D868N enriched hematopoietic stem cell-related genes and posterior Hoxa genes. Chromatin immnoprecipitation assay showed that both SETBP1-WT and SETBP1-D868N interacted with the promoter regions of Hoxa9 and Hoxa10, raising the possibility that a gain-of-function mutant of SETBP1 enhances transcription of these genes, directly or indirectly. Moreover, GSEA indicated global repression of the TGF-β signaling pathway and reciprocal upregulation of the Myc pathway in leukemic mice. In conclusion, our data provide evidence for the role of SETBP1 mutations in leukemic transformation and suggest the resulting deregulated pathways as potential therapeutic targets to prevent disease progression in MDS. Disclosures Harada: Kyowa Hakko Kirin Co., Ltd.: Research Funding.

[Background] Erythropoiesis stimulating agents (ESAs) are used as standard treatment for anemia in patients with myelodysplastic syndromes (MDS) of low- or intermediate-1 (Int-1) risk according to the International Prognostic Scoring System (IPSS). However, no randomized study has been conducted to determine the optimal dose of ESAs. We conducted a randomized controlled trial of darbepoetin alfa (DA), a long-acting ESA, to find the optimal dose and evaluate efficacy and safety. [Methods] This multicenter randomized controlled trial was conducted in 41 sites in Japan and South Korea. Main inclusion criteria were red blood cell (RBC) transfusion-dependent, IPSS low/Int-1-risk MDS patients with hemoglobin (Hb) ≤ 9.0 g/dL and serum erythropoietin (EPO) ≤ 500 mIU/mL. Eligible patients were randomized to receive weekly subcutaneous doses of DA at a fixed dose of 60, 120 or 240 μg for 16 weeks (initial dose evaluation phase). DA was administered for up to 48 weeks, with dose adjustment allowed from week 17 onward. Dosing frequency was allowed either once every week (QW) or once every 2 weeks (Q2W), with the maximum single dose of 240 µg (extended treatment evaluation phase). The primary endpoint was modified IWG 2000 erythroid response defined as the proportion of patients who achieved an erythroid response [major erythroid response (MaR; RBC transfusion-free with an increase in Hb ≥ 1.0 g/dL above baseline) or a minor erythroid response (MiR; a ≥ 50% reduction in RBC transfusion as compared to baseline)] in the initial dose evaluation phase. Mortality and progression to AML were evaluated for one year. Changes in serum drug concentration and pharmacokinetic parameters were also evaluated. [Results] A total of 52 subjects were randomized. The subjects’ baseline demographics (mean±SD) included Hb of 7.9±0.9 g/dL, serum EPO concentration of 221.1±134.2 mIU/mL, and total amount of RBC transfusion given within 56 days prior to the first DA treatment of 1459±707.2 mL. Among 50 subjects evaluable for efficacy, the proportion of those who achieved an erythroid response during the initial dose evaluation phase in the 60, 120 and 240 μg group was: 64.7% (11/17 subjects) including 17.6% with MaR; 44.4% (8/18 subjects) including 16.7% with MaR; and 66.7% (10/15 subjects) including 33.3% with MaR, respectively. The proportion of subjects achieving erythroid response was similar across the three groups, but the highest proportion of MaR was noted in the 240 μg group. The mean baseline Hb was similar among the groups (7.7, 8.0, 8.0 g/dL in the 60, 120, 240 μg group, respectively). During the initial dose evaluation phase, the mean Hb level continued to rise over the first 2 weeks of DA treatment and remained between 8.6-9.1 g/dL thereafter in the 240 μg group, whereas the mean Hb was slightly lower in the 60 μg group (7.6-8.1 g/dL) and 120 μg group (8.1-8.4 g/dL). During the extended treatment evaluation phase (week 17-48), an increase in DA dose resulted in an improved response from "no response" to MiR and eventually to MaR in 1 subject in the 60 μg group, and from MiR to MaR in 2 subjects each in the 60 and 120 μg groups. There was no major difference among the groups with respect to time to erythroid response. No clinically significant safety concerns were identified. After 1 year of starting the treatment with DA, the survival rate and the proportion of subjects free from AML estimated by Kaplan-Meier method were 84.5% and 96.0%, respectively. The low baseline serum EPO concentration and low RBC transfusion needs were identified as strong predictors of efficacy. [Conclusion] These results demonstrated that treatment with DA is effective and safe in reducing or eliminating transfusion requirements in transfusion-dependent, IPSS low/Int-1-risk MDS patients. Given the highest proportion of subjects achieving MaR in the 240 μg group and the absence of dose-dependent adverse events, the optimal dose was determined to be 240 μg QW. No adverse effects were observed in terms of mortality and proportion of progression to AML. Table. Erythroid Response during the Initial-Dose Evaluation Phase Overall 60 ug 120 ug 240 ug N 50 17 18 15 Major or Minor Response n(%) 29(58.0%) 11(64.7%) 8(44.4%) 10(66.7%) Major Response n(%) 11(22.0%) 3(17.6%) 3(16.7%) 5(33.3%) Disclosures Harada: Kyowa Hakko Kirin Co., Ltd.: Research Funding. Shibayama:Kyowa Hakko Kirin Co., Ltd.: Research Funding. Jang:Kyowa Hakko Kirin Co., Ltd.: Research Funding. Shimazaki:Kyowa Hakko Kirin Co., Ltd.: Employment, Equity Ownership. Mitani:Kyowa Hakko Kirin Co., Ltd.: Consultancy, Research Funding. Sawada:Kyowa Hakko Kirin Co., Ltd.: Consultancy. Kim:Kyowa Hakko Kirin Co., Ltd.: Consultancy.

Myelodysplasitc syndromes (MDS) are associated with genetic and epigenetic defects in transcription factors and/or chromatin-modifying enzymes that regulate self-renewal, survival, and differentiation. Mixed lineage leukemia (MLL) is a transcription factor which regulates its downstream targets by an epigenetic manner and plays a critical role for regulation of normal hematopoiesis. Mutations in MLL have been identified in variety of hematopoietic malignancies. Internal partial tandem duplication (MLL-PTD) is a major aberration of MLL gene. MLL-PTD is often found in the patients with MDS, secondary AML (sAML), and de novo AML. We have previously shown that MLL-PTD knock-in (MLLPTD/WT) mice present increased self-renewal and apoptosis in hematopoietic stem/progenitor cells (HSPCs) and skewed myeloid differentiation, but never develop MDS or AML phenotypes. This suggests that additional genetic and/or epigenetic defects are necessary for the transformation. Interestingly, MLL-PTD is significantly associated with RUNX1 mutations in sAML and de novo AML. Thus, we hypothesized that combination of these commonly co-mutated genes might generate faithful mouse models for human diseases. To understand the impact of RUNX1 mutations in the MLL-PTD background, we retrovirally introduced MDS patient-derived RUNX1 mutants (RUNX1-D171N and RUNX1-291sX300) into BM cells obtained from 5-FU treated MLLPTD/WT mice. These transduced cells were transplanted into lethally irradiated recipient mice. Both MLLPTD/WT/D171N and MLLPTD/WT/291fsX300 BMT mice quickly developed macrocytic anemia and mild thrombocytopenia. Tri-lineage dysplasia was observed in BM from both groups. Anemia in MLLPTD/WT/291fsX300 BMT mice was more severe than that in MLLPTD/WT/D171N mice. MLLPTD/WT/D171N BMT mice presented hypo-cellular marrow with excess blasts (blasts< 5%), while MLLPTD/WT/291fsX300 BMT mice presented hyper-cellular marrow with higher percentage of blasts (blasts < 20%). Interestingly monocytes in PB from MLLPTD/WT/291fsX300 BMT mice were significantly increased compared to control and MLLPTD/WT/D171N BMT mice. MLLPTD/WT/291fsX300 BMT mice also developed BM fibrosis with slight splenomegaly. Interestingly, 20-30% of MDS patients, especially high-risk MDS with BM fibrosis, are also accompanied with splenomegaly. We have previously shown that MLL-PTD enhances self-renewal of HSPCs. To understand the mechanism of enhanced self-renewal, we performed gene expression array analysis and mRNA-sequencing, followed by KEGG pathway analysis. Interestingly, one of the top dysregulated pathways was Glycolysis/Gluconeogenesis pathway. HIF-1α is one well-known transcription factor for this pathway. Thus, we measured HIF-1α expression in HSPCs from MLLPTD/WT mice at both mRNA and protein levels. HIF-1α protein and mRNA of HIF-1α target genes in HSPCs from MLLPTD/WT mice were significantly increased compared with those from WT mice. To determine the direct interaction between MLL-PTD and HIF-1α, we introduced WT MLL or MLL-PTD together with HIF-1α cDNA into 293T cells and performed western blot (WB) and imuunoprecipitation-WB. We found the strong accumulation of HIF-1α in MLL-PTD co-transfectants and interaction between MLL-PTD and HIF-1α. To assess the significance of HIF-1α in MLL-PTD mediated pathogenesis, we performed CFU assay in the presence of a HIF-1α inhibitor, Echinomycin. MLLPTD/WT cells formed significantly fewer colonies than WT cells. RUNX1 mutants transduced MLLPTD/WT cells were hyper-sensitive to Echinomycin while WT HSPCs or AML cells derived from Cbfb/SMMHC knock-in mice showed less sensitivity. Echinomycin could induce myeloid differentiation and inhibit MDS-initiating cells. We further took a genetic approach and performed BMT assay. HIF-1α deletion significantly reduced reconstitution ability of MLLPTD/WT cells and MDS development in our mouse models. These results strongly suggest that HIF-1α is essential for MLLPTD/WT and MLLPTD/WT/RUNX1 mutant mediated self-renewal and MDS-like disease development. In conclusion, 1) we have established novel MDS-like mouse models which recapitulate human diseases by using genetic/epigenetic mutations (RUNX1/MLL-PTD); 2) we identified HIF-1α as a critical factor for MDS-like disease by using our robust MDS-like mouse models; 3) targeting HIF-1α could eradicate the MDS phenotypes and the MDS-initiation cells. Disclosures Harada: Kyowa Hakko Kirin Co., Ltd.: Research Funding.

Germline mutations in RUNX1 cause familial platelet disorder with predisposit ion to acute myelogenous leukemia (FPD/AML) which is characterized by platelet defects and hematological malignancies. The molecular pathogenesis is poorly understood because of the rarity of this disease, which has impeded the clinical improvement to refine cancer therapy and to identify patients at risk for the onset of malignancies in FPD/AML. Thus, we conducted a nationwide survey to identify patients with familial thrombocytopenia or hematological malignancies. After screening germline RUNX1 mutations in 73 index patients, 13 patients in 7 pedigrees were diagnosed as having FPD/AML, of which 7 had developed hematological malignancies. The mutational spectrum of FPD/AML was unveiled by whole-exome sequencing and targeted deep sequencing, and we identified CDC25C mutation as a recurrent target of FPD/AML, which was found in 7 of 13 (53 %) patients, including 4 FPD/AML patients who had not developed malignancies, although the variant allele frequencies were much lower than those found in patients with malignancies. Furthermore, GATA2 mutations were subclonal in 3 of 7 individuals with CDC25C abnormalities. Deep sequencing enabled an accurate estimation of allele frequencies for individual mutations and prediction of hierarchical architecture, which disclosed that CDC25C mutations form a founding pre-leukemic clone, followed by stepwise acquisition of subclonal mutations that contributed to leukemia progression. Single cell sequencing validated the estimation of the clonal evolution model. In all of the mutated forms of CDC25C, their binding capacity with c-TAK1 was reduced, resulting in decreased phosphorylation of CDC25C at Ser216. As a consequence, mutated CDC25C failed to bind 14-3-3 protein efficiently and stayed in the nucleus at G1/S phase. In accordance with these observations, CDC25C mutants enhanced mitosis entry, which was exaggerated by low dose radiation-induced DNA damage. These results suggest that CDC25C mutation results in disruption of the DNA checkpoint machinery, indicating that CDC25C is a novel target in human cancer. It is known that FPD/AML-associated RUNX1 mutations evokes DNA damage and activates the DNA checkpoint mechanism. We found, however, that introduction of CDC25C mutation results in the marked enhancement of mitotic entry in spite of co-expression of RUNX1 mutation in Ba/F3 cells. Thus, premature mitosis by loss of DNA checkpoint mechanisms in the presence of mutated CDC25C may contribute to malignant transformation of RUNX1-mutated cells. In conclusion, malignant transformation of FPD/AML is formed by stepwise acquisition of mutations and clonal selection, which is initiated by a CDC25C mutation in the pre-leukemic phase and further driven by mutations in GATA2 or other genes. These findings will facilitate the diagnosis and monitoring of patients with FPD/AML who are at an increased risk of developing hematological malignancy. Citation Format: Akihide Yoshimi, Takashi Toya, Masahito Kawazu, Toshihide Ueno, Ayato Tsukamoto, Hiromitsu Iizuka, Masahiro Nakagawa, Yasuhito Nannya, Shunya Arai, Motoshi Ichikawa, Hironori Harada, Kensuke Usuki, Yasuhide Hayashi, Etsuro Ito, Keita Kirito, Hideaki Nakajima, Hiroyuki Mano, Mineo Kurokawa. Recurrent CDC25C mutations drive malignant transformation in FPD/AML. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3439. doi:10.1158/1538-7445.AM2014-3439

In the previous paper, we described a series of 2-phenylethenesulfonamide derivatives, a novel class of ET(A)-selective endothelin (ET) receptor antagonists, including the 2-methoxyethoxy derivative 2a and the 2-fluoroethoxy derivative (2b). In this paper, we wish to report further details of structure-activity relationships (SARs) of the two regions of the molecule in compound 2b, which were the alkoxy region at the 6-position of the core pyrimidine ring and the 2-phenylethenesulfonamide region. In these modifications, replacement of the 2-fluoroethoxy group with a methoxy group (6e) and replacement of the 2-phenylethenesulfonamide group with a 2-(pyridin-3-yl)ethenesulfonamide group (6l) or 2-phenylethanesulfonamide group (6q) were well tolerated both in the ET(A) binding affinity and ET(A) selectivity. Among them, compound 6e showed further improvement in oral activity compared to 2b. After oral administration, compound 6e inhibited the big ET-1 induced pressor response in conscious rats at 0.3mg /kg with a duration of >6.5h. Compound 6e also exhibited a potent antagonistic activity in the pithed rats.

The functions of AML1 in hematopoietic differentiation are repressed by AML1-mutants including the AML1/ETO chimeric protein, which is seen in t(8;21) acute myeloid leukemia. Erythroid progenitors of the patients with t(8;21) AML expressed AML1/ETO. To investigate the effect of AML1/ETO in erythroid cells, we made a tetracycline-regulated AML1/ETO overexpression system in mouse erythroleukemic (MEL) cells. Enforced AML1/ETO repressed the terminal erythroid differentiation. Furthermore, we performed representational difference analysis using this MEL cell system to clone the downstream targets of AML1 in erythroid cell differentiation. We cloned a novel transmembrane protein, Art-1 (AML1-regulated transmembrane protein 1), which is a member of tetramembrane spanning superfamily. Art-1 expression was restricted in hematopoietic cells. It was upregulated by AML1 and downregulated by AML1/ETO in both erythroid and myeloid cells, and increased during erythroid cell differentiation. Art-1 may play an important role in the differentiation of erythroid cells, possibly as a direct downstream target of AML1.

The t(8;21)-encoded AML1-ETO chimeric product is believed to be causally involved in up to 15% of acute myelogenous leukemias through an as yet unknown mechanism. To directly investigate the role of AML1-ETO in leukemogenesis, we used gene targeting to create anAML1-ETO “knock-in” allele that mimics the t(8;21). Unexpectedly, embryos heterozygous for AML1-ETO(AML1-ETO/+) died around E13.5 from a complete absence of normal fetal liver–derived definitive hematopoiesis and lethal hemorrhages. This phenotype was similar to that seen following homozygous disruption of either AML1 orCBFβ. However, in contrast to AML1- or CBFβ-deficient embryos, fetal livers from AML1-ETO/+ embryos contained dysplastic multilineage hematopoietic progenitors that had an abnormally high self-renewal capacity in vitro. To further document the role of AML1-ETO in these growth abnormalities, we used retroviral transduction to express AML1-ETO in murine adult bone marrow–derived hematopoietic progenitors. AML1-ETO–expressing cells were again found to have an increased self-renewal capacity and could be readily established into immortalized cell lines in vitro. Taken together, these studies suggest that AML1-ETO not only neutralizes the normal biologic activity of AML1 but also directly induces aberrant hematopoietic cell proliferation.

Familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML) is an autosomal dominant disorder and is characterized by inherited thrombocytopenia and a lifelong risk of development of hematological malignancies. Although inherited RUNX1 mutation is the cause of thrombocytopenia, additional genetic events may be responsible for the tumor development as only 40 % of FPD/AML patients develop leukemia. Because of the rarity of this disorder, underlying mechanisms for malignant transformation in FPD/AML have not been elucidated. Thus we conducted a nationwide survey in Japan to collect samples and make a diagnosis of patients with familial thrombocytopenia or hematological malignancies. As a result, 56 pedigrees were extracted, and seven pedigrees with RUNX1 mutation were diagnosed as having FPD/AML, in which eight out of 14 patients had developed hematological malignancies. To systematically identify additional genetic alterations, we utilized whole exome sequencing in two patients with FPD/AML who had RUNX1_F303fsX566 mutation and developed MDS (Patient 1) or myelofibrosis (Patient 2) followed by AML. We identified 12 and 10 somatically acquired nonsynonymous mutations in these patients, respectively. Intriguingly, the two patients had common CDC25C mutation at codon 234 (D234G). Further genomic screening of other pedigrees revealed that four out of eight FPD/AML patients who developed hematological malignancy harbored somatic mutations in CDC25C (CDC25C_D234G in three patients and CDC25C_H437N in one patient). Recurrent CDC25C mutation in FPD/AML with subsequent hematological malignancy implies that it forms common genetic foundation of transformation in this disease. CDC25C is a phosphatase that prevents premature mitosis in response to DNA damage at the G2/M checkpoint, while it is constitutively phosphorylated at Ser216 throughout the interphase by c-TAK1. When phosphorylated at Ser216, CDC25C binds to 14-3-3 protein, which sequestrates CDC25C in the cytoplasm and inactivates it. In all of the mutated forms of CDC25C that we found in FPD/AML, their binding capacity with c-TAK1 and 14-3-3 protein was reduced, resulting in decreased phosphorylation status of CDC25C at Ser216. As a consequence, those CDC25C mutants led to enhanced mitosis entry, which was exaggerated by radiation-induced DNA damage. These results demonstrate that CDC25C mutation results in disruption of DNA checkpoint machinery. It is known that FPD/AML-associated RUNX1 mutations evokes DNA damage and induces cell cycle arrest in hematopoietic cells, suggesting that the DNA checkpoint mechanism is activated in the presence of those types of RUNX1 mutation. We found, however, that introduction of CDC25C mutation results in the marked enhancement of mitosis entry in spite of co-expression of RUNX1 mutation in Ba/F3 cells. Thus, premature mitosis by loss of DNA checkpoint mechanisms in the presence of mutated CDC25C may contribute to malignant transformation of RUNX1-mutated cells. Interestingly, analysis of clonal evolution during leukemic transformation revealed that a clone defined by CDC25C mutation was dominant in the early phase of disease progression in both patients, which supports the idea that CDC25C mutation is associated with establishment of the founder clone during the leukemic progression of FPD/AML. In Patient 1, the founder clone with CDC25C mutation acquired FAM22G and COL9A1 (group 1) mutations, followed by occurrence of GATA2 and LPP (group 2) mutations to become a dominant clone in the AML phase, whereas another subclone of the founder defined by CHEK2 and DTX2 (group 3) mutations regressed. Similar hierarchical progression was observed in Patient 2. An additional single cell genomic sequencing of bone marrow cells from Patient 1 in the AML phase revealed that group 1/2 mutations and group 3 mutations were mutually exclusive, which supports our predicted model. Collectively, these results indicate that somatic mutation in CDC25C is a recurrent event in the early phase of leukemic progression of FPD/AML, which induces premature mitosis and genetic instability in hematopoietic cells with germline RUNX1 mutation. Disclosures: Usuki: Alexion Pharmaceuticals, Inc.: Speakers Bureau. Kurokawa:Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Research Funding.

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.

Abstract 2820 RUNX1/AML1 mutations have been frequently detected in patients with myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). RUNX1 mutations are rarely detected in lower-risk MDS, whereas approximately 20% patients with higher-risk MDS (H-MDS) have the mutations. The mutations were distributed throughout the RUNX1 protein, and replacement of D171 amino acid in runt homology domain was the most frequent target of mutations in the RUNX1 gene. The D171N mutant showed a loss of normal RUNX1 trans-activation potential and dominant-negative suppression. In mouse transplantation systems D171N-transduced mice exhibited AML with multilineage dysplasia in collaboration with Evi1 overexpression. However, EVI1 overexpression was very rare in patients. Instead, most of H-MDS patients with RUNX1 mutations displayed a high expression of BMI1. RUNX1 D171N mutant showed an increased self-renewal capacity, differentiation block, dysplasia in all 3 lineages, slightly increased immature cells and no proliferation ability using enforced expression in human CD34+ cells, and the D171N-transduced cells showed low expression level of BMI1. Both D171N and BMI1 transduced cells displayed long-term proliferation ability. When BMI1 transduced later into D171N cells, the cells expanded with a retained CD34+ cell fraction, suggesting that BMI1 have a potential to boost the D171N cells to H-MDS. To confirm the collaboration of BMI1 overexpression with D171N mutant in vivo, we performed mouse BMT using BM cells transduced with both D171N and BMI1. Ly-5.1 murine BM mononuclear cells infected with retrovirus harboring D171N/BMI1 or control vectors were transplanted into sublethally irradiated syngeneic Ly-5.2 mice. Mice that received transplants of BMI1-transduced cells remained healthy over the observation period (n=12/12), as well as those that were transplanted with empty vectors-transduced cells (n=4/4). Most of the mice that received transplants of D171N -transduced cells developed MDS/AML mainly 6–8 months after transplantation (n=6/11, P BMI1 is well-known to be essential for self-renewal of HSCs, in part via repression of genes involved in senescence, and self-renewal of HSCs is enhanced by BMI1 expression. To address the mechanisms by which BMI1 would contribute to MDS/AML development, we analyzed gene expressions involved in BMI1 downstream signaling pathways. It is suggested that BMI1 overexpression may act as one of the partner abnormalities collaborating with master gene mutations for MDS-genesis. RUNX1 D171N mutant showed no proliferation ability using enforced expression in human CD34+ cells, and D171N-transduced mice exhibited MDS/AML in collaboration with Evi1 overexpression. In addition, co-transduction of D171N and BMI1 into BM cells resulted in faster induction of MDS/AML in BMT mice. Taken together, the RUNX1 mutant may require collaborating genes such as EVI1 and BMI1 to develop MDS/AML. We confirmed that BMI1 have a potential to boost the D171N cells to MDS/AML in vivo. Disclosures: No relevant conflicts of interest to declare.

Abstract 767 Somatic mutation of RUNX1 has been implicated in a variety of hematopoietic malignancies including myelodysplastic syndrome and acute myeloid leukemia, and previous studies using mouse models disclosed its critical roles in hematopoiesis. During embryonic development, Runx1 is absolutely essential in the emergence of hematopoietic stem and progenitor cells through hemogenic endothelium. In contrast, conditional disruption of Runx1 in adult hematopoietic system revealed that it was critical in the differentiation of megakaryocytes and lymphocytes as well as in the function of hematopoietic stem cells (HSCs). However, these results were derived from gene-disruption studies in mouse models, and the role of RUNX1 in human hematopoiesis has never been tested in experimental settings. Familial platelet disorder/ acute myeloid leukemia (FPD/AML) is a rare autosomal dominant disorder caused by germline mutation of RUNX1, marked by thrombocytopenia and propensity to acute leukemia. To investigate the physiological function of RUNX1 in human hematopoiesis and the pathophysiology of FPD/AML, we derived induced pluripotent stem cells (iPSCs) from three distinct FPD/AML pedigrees (FPD-iPSCs) and examined their defects in hematopoietic differentiation. These pedigrees have distinct heterozygous mutations in RUNX1 gene, two in the N-terminal RUNT domain affecting its DNA-binding activity and one in the C-terminal region affecting its transactivation capacity. After obtaining informed consent from the affected patients, we established iPSCs from their peripheral T cells by infecting Sendai viruses expressing four reprogramming factors (OCT3/4, SOX2, KLF4 and c-MYC). FPD-iPSCs could be established in comparable frequency as the one from normal individuals (WT-iPSCs). Initial characterization of FPD-iPSCs revealed that the established clones retained typical characteristics of pluripotent stem cells such as the expression of Nanog, Oct3/4, SSEA-3, SSEA-4, Tra-1-60 or Tra-1-81, and the teratoma formation in immunodeficient mice. Next we examined the hematopoietic differentiation capacity of FPD-iPSCs by co-culturing on AGMS-3 cells, a stromal cell line established from aorta-gonad-mesonephros (AGM) region. FPD-iPSCs and WT-iPSCs were dispersed and plated on inactivated AGM-S3 cells and were co-cultured in the presence of vascular endothelial growth factor. On day 10 through day 14 of co-culture, cells were collected and analyzed for the emergence of hematopoietic progenitors (HPCs) by flow cytometry. Interestingly, FPD-iPSCs generated CD34+ cells or CD45+ cells in significantly lower frequencies as compared to WT-iPSCs. To evaluate the differentiation capacity of HPCs generated from iPSCs, CD34+ cells were sorted by flow cytometry and subjected to colony forming assays. This revealed that CD34+ cells derived from FPD-iPSCs generated significantly fewer colonies as compared to those from WT-iPSCs in all colony types examined, showing that differentiation capacity of HPCs were impaired by RUNX1 mutation. Furthermore, CD34+ cells from FPD-iPSCs generated CD41a+CD42b+ megakaryocytes (MgK) in significantly lower frequencies as compared to WT in in vitro liquid culture with stem cell factor (SCF) and thrombopoietin (TPO). Of note, MgKs differentiated from FPD-iPSCs are smaller in size as evidenced by mean-FSC by flow cytometry. These results indicate that differentiation of MgKs is impaired both quantitatively and qualitatively. Importantly, all three FPD-iPSC lines share the same phenotype in the above-described assays, suggesting that N-terminal and C-terminal RUNX1 mutations impose similar defects in hematopoietic differentiation of FPD-iPSCs. Taken together, this study, for the first time, demonstrated that mutation of RUNX1 leads to the defective differentiation of hematopoietic cells in human settings. The phenotype observed in this study, at least in part, recapitulates the ones previously reported in Runx1-homozygously deficient mice, suggesting that the mutations of RUNX1 seen in FPD/AML indeed act in dominant negative manner. Disclosures: No relevant conflicts of interest to declare.

Abstract 1337 Erythroid hypoplasia or aplasia is a hematological condition observed in including idiopathic pure red cell aplasia (PRCA), thymoma-associated PRCA and aplastic anemia. Myelodysplastic syndrome (MDS) with erythroid hypoplasia/aplasia in bone marrow is a rare type of MDS that was not included in existing classifications of MDS. Patients with erythroid hypoplasia/aplasia have common characteristics; transfusion dependencies, immunologic abnormalities and successful immunosuppressive therapies with cyclosporin A (CsA). Thus, we may regard erythroid hypoplasia/aplasia as one of hematological disease entities. However, pathogenic mechanisms of erythroid hypoplasia/aplasia have not been fully elucidated, although T-lymphocyte-mediated inhibition of erythropoiesis is suspected to be the most possible mechanism of the pathogenesis. Recently, we reported that oligoclonal expansion of CD8+/perforin+ T cells was observed in patients with thymoma-associated PRCA and the oligoclonality was exclusively detected in CD8+ T cells, but not CD4+ T cells. To clarify the pathogenetic role of the T-cells, we analyzed the T-cell subsets and therapeutic responses in patients with erythroid hypoplasia/aplasia in bone marrow. Among 253 patients with MDS diagnosed at Hiroshima University Hospital between 2000 and June 2011, 12 patients (4.7%) showed erythroid hypoplasia/aplasia. A total of 22 patients with erythroid hypoplasia/aplasia, including 8 MDS with erythroid hypoplasia/aplasia, 3 idiopathic PRCA, 3 thymoma-associated PRCA and 8 aplastic anemia, were enrolled in this study. All patients were treated with CsA and improvement in anemia in this study followed the International Working Group (IWG) 2006 criteria. For T-cell subset analysis, mononuclear cells (MNCs) were purified from bone marrow (BM) or peripheral blood (PB) of the patients. MNCs were stained with fluorescent (FITC, PE, PerCP or APC)-conjugated antibodies for CD8, perforin, CCR7, CD62L, CD27, CD28 and CD45RO, CD45RA and were subjected to flow cytometric analysis. As controls, 30 patients with MDS without erythroid hypoplasia/aplasia and 30 patients without BM abnormalities were also analyzed. Among 22 patients with erythroid hypoplasia/aplasia, 10 patients (4 MDS with erythroid hypoplasia/aplasia, 1 idiopathic PRCA, 3 thymoma-associated PRCA and 2 aplastic anemia) responded to CsA therapy within 2 to 8 weeks. The median blood hemoglobin concentration increased from 6.5 g/dL at the baseline to 9.3 g/dL with treatment, with a median increase of hemoglobin of 2.8 g/dL from the baseline. We attempted to compare the T-cell subsets between CsA-responders and non-responders. All of 3 thymoma-associated PRCA showed good response to CsA therapy, suggesting that the oligoclonal expansion of a CD8+/perforin+ T-cell subset may be associated with the responses to immunosuppressive therapy. Thus, we focused on a T-cell subpopulation expressing CD8+/perforin+. Intriguingly, the CD8+/perforin+ T cells were significantly increased in the CsA-responders (44.3 ± 9.6%, n=10) compared to the non-responders (19.0 ± 9.3%, n=12, P Our study reveals that CD8+/perforin+ T cell subset is a large population in the patients with CsA-responsive erythroid hypoplasia/aplasia. It is suggested that CD8+/perforin+ T cell subset may have functions to reduce erythroid progenitors via immunological mechanisms. The mechanisms may be easily suppressed by immunosuppressive therapies. In conclusion, expansion of CD8+/perforin+ T cell subset predicts response to cyclosporin A therapy in patients with erythroid hypoplasia/aplasia. The disease entity of “erythroid hypoplasia/aplasia in bone marrow with expansion of CD8+/perforin+ T cell subset”, including MDS, PRCA with or without thymoma and aplastic anemia, may have common pathogenetic mechanisms. Disclosures: No relevant conflicts of interest to declare.

Abstract 275 RUNX1/AML1 gene has been investigated in the pathogenesis of hematopoietic diseases and point mutations of RUNX1 have been frequently detected in patients with MDS or AML. We have found RUNX1 mutations in patients with MDS and MDS-related AML, including therapy-related cases. The mutations were distributed throughout the full length of the RUNX1 protein, and replacement of D171 amino acid in runt homology domain was the most frequent target of mutations in the RUNX1 gene. The D171N mutant showed a loss of normal RUNX1 trans-activation potential and dominant-negative trans-activation suppression, suggesting that this mutant may have some oncogenic potential in addition to the loss of function. In mouse transplantation systems D171N-transduced mice exhibited hyper-proliferative AML with multilineage dysplasia in collaboration with Evi1 overexpression. This impressive result indicated that RUNX1 mutations may be a cause of MDS with a leukemogenic potential. However in human, most of D171-mutated patients were MDS refractory anemia with excess blasts, and EVI1 overexpression was observed in a patient with MDS rapidly progressed to AML. Instead, most of patients with RUNX1 mutations displayed a high expression level of BMI1, suggesting that mouse phenotypes were not always meets clinical features of the patients with the mutations, partly because gene circumstances in mouse are different from those in human. Thus, biological analysis using human hematopoietic stem cells was considered to be necessary to clarify the molecular mechanisms of the RUNX1 mutations in the pathogenesis of MDS/AML. To examine the effect of RUNX1 mutants, we transduced the D171N mutant into human CD34+ cells from cord blood cells. In colony forming cell (CFC) assay, the number of burst forming unit-erythroid colonies was significantly decreased, while the number of granulocyte-macrophage colony-forming unit colonies was increased. Most of the D171N-transduced cells expressed myeloid lineage markers. The D171N cells showed replating capacity for 3 replatings, suggesting that they have self-renewal advantage. The presence of progenitors with long-term self-renewal capabilities was confirmed by long-term culture-initiating cell assay. The D171N cells showed a drastic increase in the number of colonies. However, long-term culture in complete cytokine liquid medium showed that the D171N cells grew a little, exhibiting lower proliferation ability than control. The percentage of CD34+ cells increased slightly, but gradually decreased with a maximum around day 35. At this point, although the percentage of CD34+/CD38+ cells did not increase in comparison to control cells, the percentage of CD34+/CD38− cells increased to 4 %. On day 35, a vast majority of the control cells terminally differentiated into mature myeloid cells and monocytes, whereas the D171N cells contained a large number of immature cells and displayed morphological abnormalities in all 3 hematopoietic lineages. Cell cycle analysis revealed that most of the D171N cells accumulated in G1 phase on day 53 when the cells stopped proliferating. These results indicated that the D171N cells had no proliferation ability although the mutant probably gives rise to the multi-lineage dysplasia of hematopoietic cells with increase in the number of blasts that is the main characteristic of MDS. Because the D171N cells showed low expression level of BMI1, we next performed stepwise transduction of BMI1 following D171N. In CFC assay, stepwise vector-transduced D171N cells seemed to no longer have colony forming ability, whereas stepwise BMI1-transduced D171N cells displayed an increase in both colony forming ability and replating capacity. Moreover, CD34+ cell population remained in the stepwise BMI1-transduced D171N cells. Furthermore, the cells showed long-term proliferation with a retained CD34+ cell fraction. Morphological findings showed myeloid cell dysplasia with increased blast-like cells. Taken together, the results of the D171N forced expression demonstrate that the mutation requires collaborating genes for proliferation. EVI1 and BMI1 may add distinct proliferating forces to the D171N cells, reflected in clinical features of MDS patients with their overexpression. Our results support the concept that RUNX1 mutations may become one of the genetic classification categories of myeloid neoplasms. Disclosures: No relevant conflicts of interest to declare.

Recent immunophenotypic analysis has shown that the heterogeneous expression of the adhesion molecule VLA-5 classifies myeloma cells into VLA-5+ mature and VLA-5- immature subpopulations. To further clarify the two myeloma subpopulations, we generated a monoclonal antibody, MPC- 1, by immunizing mice with an adherent human myeloma cell line, KMS-5. The MPC-1 antibody recognized a 48-Kd surface antigen on KMS-5 but not on U-266, a nonadherent human myeloma cell line. Specificity characterization showed that MPC-1 antigen was expressed on mature myeloma cells, normal plasma cells, and mature B cells, whereas pre-B cells and germinal center B cells lacked its expression. Monocytes and a human bone marrow stromal cell line, KM102, also expressed this antigen. Two subclones of MPC-1+ VLA-5+ (KMS-5Ad) and MPC-1-VLA-5+ (KMS- 5NAd) were separated from the KMS-5 cell line. The KMS-5NAd adhered to KM102 more tightly than did the KMS-5NAd, and the U-266 (MPC-1-VLA-5-) displayed almost no adherence to the KM102. The adhesion of the KMS-5Ad was partially inhibited by the MPC-1 antibody. These results, taken together, suggest that the MPC-1 antigen serves as a differentiation marker for B-lineage cells, including plasma cells, and may function as an adhesion molecule involved in the interaction of mature myeloma cells with bone marrow stromal cells.

With regard to the expression of adhesion molecules, human myeloma cells freshly isolated from bone marrow were heterogeneous. By two- color analysis with anti-VLA-5 antibody (PE staining) and FITC-labeled anti-CD38 antibody, we found all myeloma cells located at CD38-strong positive (CD38++) fraction and identified two subpopulations among these myeloma cells: CD38++ VLA-5-(VLA-5-) myeloma cells and CD38++ VLA- 5+ (VLA-5+) myeloma cells. To clarify the biologic character of these two subpopulations, the morphology, in vitro proliferative activity and in vitro M-protein secretion were examined in each fraction isolated by the purification procedure or a cell sorter. Morphologic examination showed that VLA-5- myeloma cells were mostly immature or plasmablastic and VLA-5+ cells were mature myeloma cells. Furthermore, VLA-5- myeloma cells proliferated markedly in vitro and responded to interleukin 6 (IL- 6), a growth factor for myeloma cells, while VLA-5+ myeloma cells showed very low uptakes of 3H-thymidine and no responses to IL-6 but secreted higher amounts of M-protein (immunoglobulin) in vitro significantly. Therefore, we could clarify here heterogeneity of human myeloma cells in the bone marrow with regard to the expression of VLA- 5, one of integrin adhesion molecules; VLA-5- myeloma cells were proliferative immature cells and VLA-5+ cells were mature myeloma cells.

We have recently shown that two-color analysis with fluorescein isothiocyanate (FITC)-anti-CD38 antibody could clearly distinguish myeloma cells (plasma cells) from other hematopoietic cells in the bone marrow. Myeloma cells (plasma cells) alone were located at CD38strong positive (++) fractions. To further distinguish normal plasma cells from mature myeloma cells phenotypically, we examined immunophenotypes of normal plasma cells and myeloma cells by two-color flow cytometry with FITC-anti-CD38 antibody and phycoerythrin staining with antibody to VLA-4, MPC-1, CD44, CD56, CD19, CD20, CD24, or CD10. Normal plasma cells were all VLA-4+VLA-5+MPC-1+CD44+ CD19+CD56- in the bone marrows from seven healthy donors, tonsils from four patients with chronic tonsillitis, a spleen from one patient with idiopathic thrombocytopenic purpura, and lymph nodes from two patients with chronic lymphadenitis, respectively. On the other hand, mature myeloma cells (12 of 20 cases), VLA-4+VLA-5+MPC-1+, were all CD19- and most of them CD56+, and there were no myeloma cells with the CD19+CD56- phenotype in the 20 cases of myelomas we tested. Thus, as for the expression of CD19 and CD56, normal plasma cells from various tissues are all CD19+CD56-, whereas no myeloma cells have the CD19+CD56- phenotype. According to this finding, we investigated the expression of CD19 and CD56 on plasma cells (CD38++ fractions) in monoclonal gammopathy of undetermined significance (MGUS). Both CD19+CD56- and CD19-DC56+ plasma cells were found in all five cases of MGUS we tested, suggesting that MGUS consists of phenotypically normal plasma cells and myeloma cells. Therefore, it is reasoned that phenotypic analysis of plasma cells with anti-CD19 and anti-CD56 antibodies can distinguish normal plasma cells from malignant plasma cells (myeloma cells), and can detect malignant plasma cells even in MGUS or premyeloma states.

Abstract 183 RUNX1 transcription factor regulates hematopoietic ontogeny and is a frequent target of gene rearrangements in hematological malignancies. In addition to gene rearrangements, loss-of-function mutations of RUNX1 have been found in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). Mutations of RUNX1 have been detected in about 10–20% patients classified as MDS/AML (high-risk MDS and AML following MDS). Although loss-of-function mutations of RUNX1 cause leukemia together with additional cooperating events in mouse models, the mechanisms, by which impaired RUNX1 functions led to the subsequent genetic alterations, remain unclear. Because DNA damage-repair response has an important role for prevention of many types of tumors, including hematological malignancies, we analyzed the role for RUNX1 in DNA repair system. First, we stably expressed a dominant-negative mutant of RUNX1, RUNX1dC, in a murine myeloid cell line 32Dcl3. RUNX1dC lacks the C-terminal 225 amino acids, which was originally found in a patient with MDS and suppresses functions of wild-type (WT) RUNX1 by inhibiting its DNA binding activity. To analyze the roles for RUNX1 in the DNA repair system, we took advantage of in vitro DNA repair assays with DNA cross-linking agents in 32D-neo and 32D-RUNX1dC cells. Since the cells that recovered from DNA damage make colonies after cisplatin exposure, we can evaluate DNA repair ability of test cells with this assay. As a result, clonogenic ability of 32D-RUNX1dC was significantly decreased by the 2-hour exposure of cisplatin (10nM treatment: 93.4% reduction) compared to that of 32D-neo cell (10nM treatment: 58.6% reduction) (p=0.0006). In addition, 32D-RUNX1dC showed significantly lower clonogenic ability than 32D-neo after exposure to UV-B and gamma-ray, respectively. To evaluate DNA-damage accumulation in 32D-neo and 32D-RUNX1dC cells, we performed immunofluorescent microscopic analysis using monoclonal antibodies for (6–4) photoproducts (6–4 PPs) and cyclobutane pyrimidine dimers (CPDs), which are major products of DNA damage induced by UV-B. These types of DNA lesions are repaired by nucleotide excision repair (NER) system. After six hours from UV-B exposure, both 6–4 PPs and CPDs accumulated in 32D-RUNX1dC cells more abundantly than in 32D-neo cells. These results suggest that RUNX1dC attenuates NER in 32D cells, thereby leading to the sustained accumulations of DNA lesions after exposure to UV-B and cisplatin. To identify the molecule(s) involved in DNA-damage signaling, we profiled expression of 84 genes involved in DNA damage signaling by real-time RT-PCR array. The expression profiling revealed that RUNX1dC repressed Gadd45a, a regulator of NER system in 32D cells. Because genetic alteration of RUNX1 is supposed to occur at a HSC level in MDS and AML, we next evaluated whether RUNX1dC modifies Gadd45 expression in murine Lineage−Sca1+c-Kit+ (LSK) cells. As a result, RUNX1dC-transduced LSK cells showed significantly lower expression of Gadd45a and Gadd45b compared to Mock-transduced LSK cells. Luciferase reporter and chromatin immunoprecipitation assays showed that RUNX1 directly regulates Gadd45a expression via two RUNX1-binding sites neighboring to the p53-binding site in the intron 3 of the human Gadd45a gene. To confirm the roles for endogenous RUNX1 in NER system, we next performed RUNX1-knockdown experiments by short hairpin RNA (shRNA) -mediated gene silencing. RUNX1-shRNA-transduced 32D cells showed significantly lower expression of Gadd45a and Gadd45b than non-silensing-shRNA-transduced 32D cells. As expected, RUNX1-shRNA-transduced 32D cells showed significantly lower clonogenic ability after UV-B exposure than non-silensing-shRNA-transduced 32D cells (p=0.0008). These results suggest that endogenous RUNX1 regulates Gadd45 expression, thereby controlling NER system. Finally, we screened mRNA expression of Gadd45a in the samples from 23 MDS/AML patients, and found that its expression was significantly decreased in MDS/AML patients harboring RUNX1-C-terminal mutation compared to those with WT RUNX1 (p=0.0233). In summary, we here demonstrated that RUNX1 participates in the DNA damage-repair response through transcriptional regulation of Gadd45a. Our study suggests that the impaired RUNX1 function deteriorates NER system and may cause additional mutation(s), which are required for multi-step leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

Abstract 1902 Poster Board I-925 Myeloproliferative disorders (MPD), including polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF), are clonal hematopoietic stem cell disorders characterized by proliferation of one or more myeloid cell lineages; they are associated with a high frequency of the Janus kinase 2 (JAK2) mutation V617F (JAK2V617F). Some patients with MPD exhibit leukemic transformation (LT) after several years of disease, but the mechanism of LT has been a matter of some controversy due to insufficient insight into the underlying molecular pathogenesis. LT may be a natural sequela of these diseases, whereas treatment with alkylating agents, hydroxycarbamide or their combination may increase the risk of LT. Previous studies have reported that chromosomal abnormalities including -5/5q- and -7/7q- were frequently detected in patients with MPD at the time of LT, suggesting that the development of cytogenetic abnormalities may be associated with the LT of patients with MPD. However, the genes involved in LT still remain obscure. Therefore, we attempted to identify gene alterations involved in LT from patients in the chronic phase (CP) of MPD. Patients with MPD were diagnosed and classified according to the WHO criteria between 1985 and 2007. Patients with PV, ET, PMF, MPD unclassified (MPD-U) and LT of these diseases were examined as approved by the institutional review board at Hiroshima University. Patients gave written informed consent, according to the Declaration of Helsinki. Mutations of JAK2V617F, AML1, CEPBA, FLT3, N-RAS, c-KIT, PTPN11 and TP53 were screened and were identified. Among 417 patients with MPD, 18 (4.3 %) patients progressed to leukemia. At LT, 13 of the 18 patients showed additional cytogenetic abnormalities, including -7/7q- with trisomy 21, inv(3)(q21q26), i(17)(q10) and t(11;19)(q23;p13.3), which are known to be associated with therapy-related leukemia. JAK2V617F was detected in 10 of 14 patients at LT. No patients had a JAK2 mutation pattern that changed during CP to LT. We analyzed gene mutations that may play an important role in leukemogenesis and found five AML1 mutations, one PTPN11 mutation and one CEBPA mutation in patients at the LT, whereas no mutation was detected in patients at CP. The gene alterations were detected at LT in both JAK2V617F-positive and −negative MPD patients, raising the possibility that the hematopoietic stem cells (HSCs) may have been transformed into leukemic blasts as a result of gene abnormalities. Among these gene abnormalities in the MPN patients, we focused on AML1 mutations. To clarify the leukemogenic effect of AML1 mutants, the AML1D171N mutant was transduced into CD34+ cells from eight patients in the CP of MPD using retrovirus transduction methods. The effect of this mutant on cell differentiation/proliferation was assessed by CFC re-plating assays. The D171N plates contained fewer erythroid colonies and more myeloid colonies than the controls. After re-plating, new colonies were detected on all D171N plates but on only few control plates. The D171N-transduced cells contained more CD34+ cells and proliferated more strongly than the controls. These results indicate that the D171N mutant has the potential to increase the myeloid immature cells and to enhance their self-renewal capacity. Furthermore, D171N-transduced cells retained more CD34+ cells than the controls after long-term culture on MS5 stroma cells, and showed significantly more colonies in long-term culture-initiating cells experiments. In this study, AML1 point mutations were detected with high frequency in patients at the LT from both JAK2V617F-positive and -negative MPD. Furthermore, the AML1D171N mutant transduced into CD34+ cells from MPD patients promoted proliferation of primitive progenitors, i.e. leukemic stem cells. These results indicate that AML1 point mutations may have a leukemogenic potential in JAK2V617F-positive stem cells or in pre-JAK2 stem cells, and they may promote leukemic transformation in MPD. Disclosures: No relevant conflicts of interest to declare.

To understand myelodysplastic syndromes (MDS) at the molecular level, we used a mouse bone marrow transplant (BMT) model. Several leukemogenic fusion proteins were demonstrated to induce leukemia in mouse BMT models. However, analysis of molecular basis of MDS has been hampered by the lack of mouse MDS models. Mutations of a transcription factor AML-1/Runx-1 were identified in 15–40% of MDS-RAEB and MDS/AML and 5–10% of de novo AML. To test transforming abilities of the mutant AML-1, we transduced AML-1 with different mutations (#5 and #27) into mouse bone marrow cells using our highly efficient retrovirus-mediated gene transfer, transplanted the transduced cells to irradiated mice, and found that most mice developed leukemia within 4–13 months after the transplant (table1 and figure1). The leukemic mice exhibited marked hepato-splenomegaly, and morphological abnormalities of myeloid and erythroid lineages were frequently observed. Some mice developed to leukemia after a long latency with MDS-like symptoms, either with pancytopenia or leukocytosis with anemia, thus mimicking the human disease. To clarify the differences between the leukemic mice with early onsets and those that developed leukemia after a long period of MDS-like symptoms in the mutant AML-1-induced mouse diseases, the integration sites of the transduced retroviruses were investigated using a polymerase chain reaction (PCR)-based technique bubble PCR. Our preliminary results suggested that the integration sites of the retroviruses harboring the mutant AML-1 contribute to the early onset of leukemia. In conclusion, we have developed a useful mouse in vivo model of MDS/AML that should help understand the molecular basis of MDS and progression of MDS to leukemia. penetrance and latency mutation number position form penetrance latency #5 D171N point mutation in RHD 69.6 % 4–13 months #27 S291fsX300 truncation type 100 % 4–9 months Figure Figure

Cyclization of 3-benzoyl-2-cyanobutyronitrile to 2-amino-4-methyl-5-phenylfuran-3-carbonitrile was effected under acidic conditions, rather than the basic conditions previously reported. Since treatment with trifluoroacetic acid (TFA) at room temperature is very mild, 2-amino-4-methyl-5-phenylfuran-3-carbonitriles containing various functional groups can be accessed via this route.