ALBERT

Description: Basophils are the rarest granulocytes circulating in the peripheral blood. They play critical roles in anti-parasite Th2-type immune responses and chronic allergic disorders. The developmental pathway for basophils has been recently demonstrated; myeloid progenitors pass through common myeloid progenitors, granulocyte-monocyte progenitors, granulocyte-committed progenitors (GPs), and basophil-committed progenitors (BaPs) in the bone marrow. BaPs then give rise to mature basophils. However, our understanding of how this pathway is regulated remains still elusive. Interferon Regulatory Factor-8 (IRF8), a hematopoietic cell-specific IRF transcription factor, is essential for the development of monocytes, dendritic cells, and eosinophils, while it inhibits neutrophil differentiation. Its role in the development of basophils has yet to be analyzed. In this study, we investigated whether IRF8 has any role in the development of the basophil lineage. We found that Irf8–/– mice displayed a severe reduction of basophil counts in the bone marrow, peripheral blood and spleen compared to wild-type (WT) mice. Irf8–/– mice retained GPs but lacked BaPs. Cell transfer experiments revealed that the defect of basophil development in Irf8–/– mice resides in bone marrow cells. We utilized IRF8-GFP chimera knock-in mice to examine IRF8 protein expression in the basophil lineage at a single cell level. We found that GPs, but not BaPs and mature basophils, expressed IRF8. Furthermore, purified Irf8–/– GPs failed to efficiently give rise to basophils in vitro. These results indicate that IRF8 acts at the stage of GPs in a cell-intrinsic manner. To understand the mechanism by which IRF8 promotes basophil development, we performed transcriptome analysis of purified GPs from WT and Irf8–/– mice by microarray. Because IRF8 is no more expressed in BaPs, we envisaged that IRF8 acts by inducing downstream transcription factors in GPs. The expression of several transcription factor genes such as Gata2 and Spib was reduced in Irf8–/– GPs compared to WT GPs. Analysis of DNA motifs in the promoter regions of genes downregulated in Irf8–/– GPs predicted that GATA transcription factor(s) may act downstream of IRF8. Indeed, retroviral transduction of GATA2, known to be essential for basophil development, into Irf8–/– hematopoietic progenitor cells rescued basophil differentiation in vitro. On the other hand, Spib–/– mice showed no obvious defects in basophil development. Taken together, these results suggest that the IRF8-GATA2 axis in GPs critically regulates basophil development. Disclosures: No relevant conflicts of interest to declare.

Description: Background TET2 is known as an enzyme which converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Loss-of-function mutations in TET2 are frequent in angioimmunoblastic T-cell lymphoma (AITL) and peripheral T-cell lymphoma, not otherwise specified (PTCL-NOS). Normal counterpart of AITL is thought to be follicular helper T cells (Tfh). PTCL-NOS is likely to consist of heterogeneous groups. Some of the PTCL-NOS cases also have features of Tfh. In the last annual meeting, we reported that aged homozygous Tet2 gene-trap mice (Tet2gt/gt), which showed 80% reduction in the Tet2 mRNA level in various hematopoietic cells, developed T-cell lymphoma. To further investigate the mechanism of T-lymphomagenesis, we analyzed methylome, hydroxymethylome, and transcriptome in the lymphoma cells. Material and Method Tet2 gt mice have a trapping vector inserted into the second intron of the Tet2 locus. In all the analyses, we used CD4+ T cells prepared from lymphoma cells developed in these mice, as well as CD4+ T cells prepared from spleen of wild-type mice as a control. To investigate genome-wide methylation and hydroxymethylation statuses, we performed MeDIP and hMeDIP sequencing, and bisulfite sequencing. To examine comprehensive gene expression, we performed microarray-based analysis, followed by Gene Set Enrichment Analysis (GSEA). Result After observation over a year (median, 67 weeks), 5 out of 7 Tet2gt/gt mice developed T-cell lymphomas with Tfh-like immunostaining pattern. No differences were found in the average levels of 5mC between lymphoma and control CD4+ cells throughout the regions around transcription start sites (TSS) +/- 5 kb. In contrast, when the same regions were analyzed for 5hmC levels, those in the lymphoma cells were significantly lower at the regions around TSS +/- 1 kb. When focused on regions having high 5mC contents (MACS score〉5.0), lymphoma cells demonstrated a significant enrichment at regions around TSS +/- 1 kb, intragenic regions, and CpG islands (p=0.013, 0.006, 0.022 respectively). On the other hand, 5hmC was significantly decreased at regions around TSS +/- 1 kb in lymphoma cells than control cells (p=0.018). In a set of genes whose expression was higher in lymphoma cells than control cells, 5hmC levels were significantly lower in lymphoma cells. GSEA analysis revealed upregulation of Tfh-associated genes such as Bcl6 and cMaf (FDR q value=0.0004), key transcription factors for Tfh differentiation, in lymphoma cells compared with control cells. The expression of upregulated Tfh-associated genes was validated by real-time PCR. We focused on the epigenetic change of Bcl6 because it is among the most important transcription factors for Tfh development. It was reported that hypermethylation at intron 1 of Bcl6 upregulated its transcriptional activity in B cell lymphomas. Bisulfite sequencing revealed that the CpG sites in the intron 1 of Bcl6 were massively methylated/hydroxymathylated in lymphoma cells, whereas those in control cells were mostly at an unmodified status. MeDIP sequencing indicated that intron 1 of Bcl6 had more methylated status in lymphoma cells than control cells. We also found that CpG sites in the same region were densely methylated/hydroxymathylated in EL4, mouse T-cell lymphoma cell line, and that the decitabine treatment converted them into unmodified CpG along with the decrease in the Bcl6 expression levels. Discussion and Conclusion Tet2 gt/gt mice developed T-cell lymphoma with both Tfh-like immunohistological character and gene expression pattern. We found distinct changes in methylome, hydroxymethylome, and transcriptome. We also found a tight linkage between the increased methylation of intron 1 of Bcl6 and increased expression of its mRNA level in lymphoma cells developed in Tet2 knockdown mice. The same scenario is indicated in a T-cell lymphoma cell line. These observations imply that, in normal CD4+ T cells, reduced Tet2 function might increase the methylation status of the CpG sites in intron 1 of Bcl6, which may result in upregulation of Bcl6 expression and deviated Tfh generation. These processes might be an initiating event for the development of T-cell lymphoma with the Tfh features. Because of the long latency before lymphoma development in Tet2gt/gtmice, it is likely that additional hits are necessary. Disclosures: No relevant conflicts of interest to declare.

Description: Myelodysplastic syndromes (MDS) and related disorders are a heterogeneous group of chronic myeloid neoplasms with a high propensity to acute myeloid leukemia. A cardinal feature of MDS, as revealed by the recent genetic studies, is a high frequency of mutations and copy number variations (CNVs) affecting epigenetic regulators, such as TET2, IDH1/2, DNMT3A, ASXL1, EZH2, and other genes, underscoring a major role of deregulated epigenetic regulation in MDS pathogenesis. Meanwhile, these mutations/deletions have different impacts on the phenotype and the clinical outcome of MDS, suggesting that it should be important to understand the underlying mechanism for abnormal epigenetic regulation for better classification and management of MDS. SETD2 and ASH1L are structurally related proteins that belong to the histone methyltransferase family of proteins commonly engaged in methylation of histone H3K36. Both genes have been reported to undergo frequent somatic mutations and copy number alterations, and also show abnormal gene expression in a variety of non-hematological cancers. Moreover, germline mutation of SETD2 has been implicated in overgrowth syndromes susceptible to various cancers. However, the role of alterations in these genes has not been examined in hematological malignancies including myelodysplasia. In this study, we interrogated somatic mutations and copy number variations, among a total of 1116 cases with MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), who had been analyzed by target deep sequencing (n=944), and single nucleotide polymorphism-array karyotyping (SNP-A) (n=222). Gene expression was analyzed in MDS cases and healthy controls, using publically available gene expression datasets. SETD2 mutations were found in 6 cases, including 2 with nonsense and 4 with missense mutations, and an additional 10 cases had gene deletions spanning 1.8-176 Mb regions commonly affecting the SETD2 locus in chromosome 3p21.31, where SETD2 represented the most frequently deleted gene within the commonly deleted region. SETD2 deletion significantly correlated with reduced SETD2 expression. Moreover, MDS cases showed a significantly higher SETD2 expression than healthy controls. In total, 16 cases had either mutations or deletions of the SETD2 gene, of which 70% (7 out of 10 cases with detailed diagnostic information) were RAEB-1/2 cases. SETD2 -mutated/deleted cases had frequent mutations in TP53 (n=4), SRSF2 (n=3), and ASXL1 (n=3) and showed a significantly poor prognosis compared to those without mutations/deletions (HR=3.82, 95%CI; 1.42-10.32, P=0.004). ASH1L, on the other hand, was mutated and amplified in 7 and 13 cases, respectively, of which a single case carried both mutation and amplification with the mutated allele being selectively amplified. All the mutations were missense variants, of which 3 were clustered between S1201 and S1209. MDS cases showed significantly higher expression of ASH1L compared to healthy controls, suggesting the role of ASH1L overexpression in MDS development. Frequent mutations in TET2 (n=8) and SF3B1 (n=6) were noted among the 19 cases with ASH1L lesions. RAEB-1/2 cases were less frequent (n=11) compared to SETD2-mutated/deleted cases. ASH1L mutations did not significantly affect overall survival compared to ASH1L-intact cases. Gene Set Expression Analysis (Broad Institute) on suppressed SETD2 and accelerated ASH1L demonstrated 2 distinct expression signatures most likely due to the differentially methylated H3K36. We described recurrent mutations and CNVs affecting two histone methyltransferase genes, which are thought to represent novel driver genes in MDS involved in epigenetic regulations. Given that SETD2 overexpression and reduced ASH1L expression are found in as many as 89% of MDS cases, deregulation of both genes might play a more role than expected from the incidence of mutations and CNVs alone. Although commonly involved in histone H3K36 methylation, both methyltransferases have distinct impacts on the pathogenesis and clinical outcome of MDS in terms of the mode of genetic alterations and their functional consequences: SETD2 was frequently affected by truncating mutations and gene deletions, whereas ASH1L underwent gene amplification without no truncating mutations, suggesting different gene targets for both methyltransferases, which should be further clarified through functional studies. Disclosures Alpermann: MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Shih:Novartis: Research Funding.

Description: Induced pluripotent stem cells (iPSCs) can be generated by the expression of defined transcription factors not only from normal tissue, but also from malignant cells. Cancer-derived iPSCs are expected to provide a novel experimental opportunity to establish the disease model. We generated iPSCs from imatinib-sensitive chronic myelogenous leukemia (CML) patient samples. Remarkably, the CML-iPSCs were resistant to imatinib although they consistently expressed BCR-ABL oncoprotein. In CML-iPSCs, the phosphorylation of ERK1/2, AKT, and JNK, which are essential for the maintenance of both BCR-ABL (+) leukemia cells and iPSCs, were unchanged after imatinib treatment, whereas the phosphorylation of signal transducer and activator of transcription (STAT)5 and CRKL was significantly decreased. These results suggest that the signaling for iPSCs maintenance compensates for the inhibition of BCR-ABL. CML-iPSC–derived hematopoietic cells recovered the sensitivity to imatinib although CD34+38−90+45+ immature cells were resistant to imatinib, which recapitulated the pathophysiologic feature of the initial CML. CML-iPSCs provide us with a novel platform to investigate CML pathogenesis on the basis of patient-derived samples.

Description: Hypoxia induces erythropoiesis by stimulating erythropoietin secretion from kidney in vivo. Hypoxia also stimulates hemoglobin production in cultured murine erythroleukemia cells, but its mechanism is still unknown. We have found that hypoxia induces hemoglobin production in human erythroleukemia cell line, YN-1, and have analyzed the mRNA expression profile of YN-1 cells under hypoxic conditions (1% O2, 5% CO2 and 94% N2) using DNA microarrays, and compared it with that of normoxic conditions (5% CO2 and 95% room air). For DNA microarray analysis, total RNA was prepared from YN-1 cells, which were incubated under hypoxic or normoxic conditions for 6 hours. We thus found the increased mRNA expression for several genes involved in erythroid differentiation, including transforming growth factor β1 (TGF-β1) and mitoferrin (mitochondrial iron importer, previously known as SLC25A37). Among these candidates, we have focused on TGF-β1, since TGF-β1 is able to induce erythroid differentiation of several erythroleukemia cell lines, including YN-1 cells and K562 cells. We initially confirmed the increased mRNA expression of TGF-β1 under hypoxic conditions by Northern blotting analysis in YN-1 cells and YN-1-0A cells, which is a daughter cell line of YN-1 cells and is maintained in serum-free medium. Likewise, the hypoxic induction of mitoferrin mRNA in YN-1 cells was confirmed by Real Time PCR analysis. Then, using ELISA system, we measured the TGF-β1 concentration in the culture medium of YN-1-0A cells, and have found that hypoxia stimulates TGF-β1 secretion from the cells. Notably, the hypoxia-mediated increases in hemoglobin production and γ-globin mRNA expression were significantly suppressed in YN-1-0A cells, when TGF-β1 activity in the culture medium was neutralized with anti-TGF-β1 antibody. These results suggest that hypoxia stimulates TGF-β1 secretion from YN-1 cells and YN-1-0A cells, which in turn induces the terminal differentiation of erythroid cells in an autocrine manner. Interestingly, in contrast to the hemoglobin production, addition of TGF-β1 into the culture medium did not influence mitoferrin expression, suggesting that hypoxia induces mitoferrin expression by a mechanism independent of TGF-β1. Since several putative hypoxia responsive elements are present in the promoter region of the human mitoferrin gene, hypoxia inducible factor 1 (HIF-1) may be involved in inducible expression of mitoferrin under hypoxic conditions. The hypoxia-mediated induction of mitoferrin expression is important for effective supply of iron for heme biosynthesis in erythroid cells, as several missense mutations of the zebrafish mitoferrin gene are associated with hypochromic anemia and erythroid maturation arrest (Shaw G.C. et al., Nature, vol.440, p96–100, 2006). Taken together, we suggest that hypoxia stimulates hemoglobin production in erythroid cells by coordinately increasing the iron supply into mitochondria and the γ-globin expression, which may be achieved in part by the induction of mitoferrin and the enhanced secretion of TGF-β1, respectively.

Description: Adult T-cell leukemia/lymphoma (ATL) is a distinct form of peripheral T-cell lymphoma, which is etiologically associated with human T-cell leukemia virus type 1 (HTLV-1) infection during early infancy. Although HTLV-1 can effectively immortalize human T cells, there is a long latency period of ~50 years before the onset of ATL, suggesting that HTLV-1 infection alone may be insufficient for the development of ATL, but additional acquired genetic events that accumulate during the later life are essential for the development of ATL. However, such somatic alterations underlying the pathogenesis of ATL have not been fully elucidated. To obtain a complete registry of genetic alterations in ATL, we performed an integrated genetic study, in which whole-genome/exome and RNA sequencing (RNA-seq) was performed together with array-based methylation and genomic copy number analysis among a cohort of 50 paired ATL samples, followed by extensive validation using targeted deep sequencing of detected mutations in 〉 400 follow-up samples. Compared with other lymphoid malignancies, ATL cells carried higher numbers of mutations, copy number alterations, and rearrangements than in other lymphoid malignancies, suggesting the presence of global genomic instability in ATL. In addition to previously reported mutational targets in ATL (TP53,TCF8, and FAS) and known targets frequently mutated in other lymphoid malignancies (CARD11, GATA3, IRF4, POT1, and RHOA), we identified a variety of highly recurrent mutations affecting previously unknown mutational targets, many of which are involved in T-cell development, activation and migration, immunosurveillance, and transcriptional regulation. Molecular and functional analysis using human T-cell leukemia cell lines showed that some of these novel mutations actually augment T-cell receptor signaling, validating their biological significance in ATL. A comparison of mutations among disease subtypes revealed that several subtype-specific mutations, including TP53, CD58, IRF4 and TBL1XR1 mutations in acute and lymphoma types, and STAT3mutation in chronic and smoldering types, suggesting that different oncogenic mechanisms underlie different ATL subtypes. Furthermore, ATL cells had a distinct pattern of copy number changes and genomic rearrangements. Interestingly, their gene targets showed a significant overlap to mutational targets. Surprisingly, somatic focal deletions involving the 14q31.1 locus were observed in all the cases examined by whole-genome sequencing and therefore are thought to uniquely characterize ATL genomes, although their gene targets remained to be identified. Like other regions also frequently deleted in ATL, such as 7q31.1 and 1p21.3 loci, these deletions were thought to reflect high levels of genetic instability. Finally and conspicuously, pathway analysis revealed that multiple genes involved in the Tax interactome were systematically altered in ATL, although Tax itself underwent gene silencing in most cases. These data suggested that ATL cells can escape from cytotoxic T-lymphocytes by silencing immunogenic Tax expression, while developing alternative oncogenic mechanisms through acquiring somatic mutations or copy number alterations in the Tax-related pathway. Our findings suggest that deregulated T-cell functionalities caused by genetic alterations, especially those associated with HTLV-1 Tax oncoprotein, are central to ATL pathogenesis, and provide a novel clue to contrive new diagnostics and therapeutics for this intractable disease. Disclosures No relevant conflicts of interest to declare.

Description: MDS is a heterogeneous group of myeloid neoplasms caused by genetic and epigenetic alterations. During the past decade, the major driver mutations in MDS have been fully investigated. However, the role of epigenetic alterations, particularly those of DNA methylation, has less intensively been studied, even though abnormal DNA methylation has long been implicated in the pathogenesis of MDS. In this study, we analyzed DNA methylation status of bone marrow mononuclear cells from 320 cases with MDS-SLD (n = 7), MDS-RS (n = 63), MDS-MLD (n = 51), MDS-EB (n = 186), MDS-U (n = 1), and MDS with isolated del(5q) (n = 12), using Illumina 450K methylation array. Mutations in major driver genes (51 genes) and abnormal genomic copy numbers were also interrogated using targeted-capture sequencing. Using unsupervised consensus clustering, we identified 3 subgroups showing unique DNA methylation profiles. Subsequently, we assessed differentially methylated positions (DMPs) associated with each subgroup. Differentially hypermethylated positions (hyper-DMPs) were significantly more enriched in Group 3 (n = 82) (P 〈 0.001), while differentially hypomethylated positions (hypo-DMPs) were more prominent in Group 1 (n = 125). Group 1 was significantly enriched for SF3B1 (46%) mutations (q 〈 0.01), while Group 2 (n = 131) was characterized by the enrichment of ASXL1 (38%), RUNX1 (30%), TP53 (26%), STAG2 (15%), and SETBP1 (6.7%) mutations (q 〈 0.01). In contrast, Group 3 (n = 64) was significantly enriched for TET2 (67%) and IDH1/2 (12% and 15%, respectively) mutations (q 〈 0.01), suggesting a strong association between DNA methylation and gene mutations. To further elucidate mutation-specific DNA methylation patterns, supervised analysis was performed for each mutation. As expected from their enrichment in Group 3 (q 〈 0.01), TET2 and IDH1/2 mutations were significantly associated with hyper-DMPs (P 〈 0.001) involving 1891 and 8330 promotor sites, respectively. Conspicuously, among these hypermethylated promoter sites, 〉1616 were commonly hypermethylated, strongly supporting the common impact of TET2 and IDH1/2 mutations on deregulated DNA methylation. To clarify prognostic impact of abnormal DNA methylation, we first interrogated the correlation between unique methylation subgroups and revised IPSS. Patients with very low or low risk were significantly dominant (74%) in Group 1 (q 〈 0.01), and very high or high risk cases were significantly enriched (68%) in Group 2 (q 〈 0.01). In accordance with this finding, patients in Group 3 showed significantly shorter overall survival (OS) compared to Group 1 (HR: 1.94, 95%CI: 1.11-3.4, P 〈 0.05) and OS was even worse in Group 2 patients (vs. Group 1: HR: 5.18, 95%CI: 3.21-8.36, P 〈 0.001). Strong correlations between epigenetic and genetic profiles were further interrogated using a Bayesian statistical model; on the basis of DNA methylation and gene mutations, the original 3 clusters were re-classified into 5 discrete clusters, clusters A, B, C, D, and E (n = 124, 17, 74, 46, and 59, respectively); patients in Group 1 and 3 largely clustered into Cluster A and E, respectively, while Group 2 was further subclassified into clusters B, C, and D. Clusters B and D were characterized by a conspicuos enrichment of DNMT3A (88%) and TP53 (69%) mutations (q 〈 0.001), while Cluster C was characterized by higher frequency of ASXL1 (71%), RUNX1 (54%), STAG2 (27%), and EZH2 (21%) mutations (q 〈 0.001). In contrast to significant associations between epigenetic regulators and unique methylation clusters, splice factor mutations tended to be clustered into multiple clusters, depending on type of co-occurring mutations. For example, combined SF3B1 and TET2 mutations (n = 20) were enriched in Cluster A, where highly associated with MDS-RS, while patients with SF3B1 and RUNX1 mutations (n = 9) were more grouped in Cluster C, mostly showing MDS-EB phenotype (89%). Similarly SRSF2 mutations with RUNX1 and/or ASXL1 mutations (n = 36) were enriched in Cluster C, largely associated with MDS-EB phenotype (80%), while those with TET2 or IDH1/2 (n = 39) were mainly grouped into Cluster C, many of which showed MDS-EB phenotype (74%). These findings highlight differential roles of mutated epigenetic regulators and splicing factors in abnormal DNA methylation. In conclusion, we elucidated the collaborative impact of DNA methylation profiles and mutation status on heterogeneous pathogenesis and prognosis in MDS. Figure. Figure. Disclosures Nadarajah: MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Nakagawa:Sumitomo Dainippon Pharma Co., Ltd.: Research Funding. Inagaki:Sumitomo Dainippon Pharma Co., Ltd.: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Description: In megakaryocytes, the maturation process and oxidative stress response appear to be closely related. It has been suggested that increased oxygen tension and reactive oxygen species (ROS) promote megakaryopoiesis and that the expression of stress-responsive genes responsible for ROS elimination declines during megakaryocytic maturation. NF-E2 p45 is an essential regulator of megakaryopoiesis, whereas Nrf2 is a key activator of stress-responsive genes. Because p45 and Nrf2 have similar DNA-binding specificities, we hypothesized that p45 competes with Nrf2 to repress stress-responsive genes and achieves favorable intracellular conditions to allow ROS to be efficiently used as signaling molecules. We conducted comprehensive gene expression profiling with wild-type and p45-null megakaryocytes and examined the functional relationship between p45 and Nrf2. We found that 2 characteristic gene clusters are defined within p45 target genes: platelet genes and cytoprotective genes. The former are unique targets activated by p45, whereas the latter are common targets of p45 and Nrf2. Further analysis suggested that, as a less efficacious activator, p45 maintains moderate expression of cytoprotective genes through competing with Nrf2 and promotes ROS accumulation. Increased ROS enhanced platelet gene expression. These results suggest that p45 dominates over Nrf2 to enhance megakaryocytic maturation by promoting ROS accumulation.

Description: Background Myelodysplastic syndromes (MDS) are a heterogeneous group of myeloid neoplasms characterized by varying degrees of cytopenias and a predisposition to acute myeloid leukemia (AML). With conspicuous clinical and biological heterogeneity in MDS, an optimized choice of treatment based on accurate diagnosis and risk stratification in individual patients is central to the current therapeutic strategy. Diagnosis and prognostication in patients with myelodysplastic syndromes (MDS) may be improved by high-throughput mutation/copy number profiling. Methods A total of 944 patients with various MDS subtypes were screened for gene mutations and deletions in 104 known/putative genes relevant to MDS using targeted deep-sequencing and/or array-based genomic hybridization. Impact of genetic lesions on overall survival (OS) was investigated by univariate analysis and a conventional Cox regression, in which the Least Absolute Shrinkage and Selection Operator (lasso) was used for selecting variables. The linear predictor from the Cox regression was then used to assign the patients into discrete risk groups. Prognostic models were constructed in a training set (n=611) and confirmed using an independent validation cohort (n=175). Results After excluding sequencing/mapping errors and known or possible polymorphisms, a total of 2,764 single nucleotide variants (SNVs) and insertions/deletions (indels) were called in 96 genes as high-probability somatic changes. A total of 47 genes were considered as statistically significantly mutated (p10% of the cases. Less common mutations (2−10%) involved U2AF1, ZRSR2, STAG2, TP53, EZH2, CBL, JAK2, BCOR, IDH2, NRAS, MPL, NF1, ATM, IDH1, KRAS, PHF6, BRCC3, ETV6, and LAMB4. Intratumoral heterogeneity was evident in as many as 456 cases (48.3%), even though the small number of gene mutations available for evaluation was thought substantially to underestimate the real frequency. The number of observed intratumoral subpopulations tended to correlate with the number of detected mutations and therefore, advanced WHO subtypes and risk groups with poorer prognosis. Mean variant allele frequencies (VAFs) showed significant variations among major gene targets, suggesting the presence of clonogenic hierarchy among these common mutations during clonal evolution in MDS. The impact of these genetic lesions on clinical outcomes was initially investigated in 875 patients. In univariate analysis, 25 out of 48 genes tested significantly affected overall survival negatively (P

Description: Chronic myelomonocytic leukemia (CMML), the most common entity among myelodysplastic syndrome/myeloproliferative neoplasm, is characterized by monocytosis, morphologic dysplasia, and progression to acute myeloid leukemia. Despite its relatively high incidence, the pathogenesis of CMML remains elusive, mainly due to the paucity of suitable animal models and the difficulties in the establishment of CMML cell lines. As reprogramming technology has been established as a new powerful tool for disease modeling, here we developed induced pluripotent stem cells (iPSC) from CMML leukemic cells. By introducing episomal vectors expressing OCT3/4, SOX2, KLF4, L-MYC, LIN28 combined with shRNA for p53, several lines of iPSC were generated from CD34+cells of a healthy donor (wild-type: WT) and a CMML patient with der (1; 7) (q10; p10) translocation. All iPSC expressed pluripotent surface markers (TRA1-60 and SSEA4) and stemness-related genes (NANOG, OCT3/4, SOX2, KLF4, C-MYC, and REL). The der (1; 7) (q10; p10) translocation were detected in all CMML iPSC. When co-cultured with 10T1/2 stromal cells in the presence of VEGF, CMML and WT iPSC generated a comparable frequency of CD34+ CD43+ hematopoietic progenitor cells (HPC). However, when cultured in cytokine-supplemented semisolid medium, CMML iPSC-derived CD34+ CD43+ HPC yielded an increased number of hematopoietic colonies with larger sizes, especially CFU-GM and CFU-GEMM, compared with WT iPSC-derived cells. Importantly, CMML iPSC-derived hematopoietic colonies mainly consisted of monoblasts with a high nucleus/cytoplasm ratio, while those of WT iPSC predominantly composed of macrophages. Flow cytometric analysis showed marked increases of CD34+ hematopoietic progenitors and CD13+ myeloid cells in CMML iPSC-derived hematopoietic colonies. Of note, among myeloid lineages, there were remarkable increases in CD14+ monocytic cells and CD24+ CD14- immature granulocytes, which were unique characteristics of human CMML. In addition, CMML iPSC-derived CD13+myeloid cells exhibited a weak expression of CD56, which was never detected in WT iPSC-derived cells, suggesting CMML iPSC-derived hematopoietic cells recapitulate the original phenotype of CMML. Notably, we found that CMML iPSC-derived HPC retained the ability to serially replate and generate colonies even after the fourth plating, although essentially no WT iPSC-derived colonies were detected after the second plating, suggesting enhanced self-renewal capacity of CMML iPSC-derived HPC. In addition, when cultured in methylcellulose without cytokines, CMML iPSC-derived HPC were able to form spontaneous hematopoietic colonies, in contrast to WT iPSC-derived cells that gave rise to almost no detectable colonies. Taken together, these results suggest CMML iPSC-derived HPC possess the multiple biologic properties of CMML leukemic blasts. Then, we conducted a comprehensive gene expression and DNA methylation profiling of WT and CMML parental CD34+ cells, iPSC, and iPSC-derived CD34+ CD43+ HPC. Strikingly, although gene expression and DNA methylation status were quite different between WT and CMML parental CD34+ cells (R2 = 0.72 for gene expression and 0.90 for DNA methylation), WT and CMML iPSC-derived HPC exhibited similar gene expression and DNA methylation pattern (R2= 0.92 and 0.96), indicating reprogramming followed by redifferentiation may enable to obtain more homogenous population of normal and CMML cells that reside in almost the same differentiation stage. Using these multi-omics platforms, we searched differentially expressed and methylated genes between WT and CMML iPSC-derived HPC to identify molecular abnormalities that contribute to the pathophysiology of CMML. Indeed, gene set enrichment analysis revealed that embryonic stem cell (ESC)-related gene sets were enriched in CMML iPSC-derived HPC, which is consistent with a previous report that showed murine MLL leukemia stem cells employ a transcriptional program shared with ESC. Functional analysis of candidate genes is underway to further define the molecular aberrations involved in the CMML pathogenesis. In summary, we have established a novel CMML model of patient-derived iPSC and revealed their derived HPC recapitulate the disease’s primary features. These findings highlight the iPSC as an attractive platform to investigate the cellular and molecular pathophysiology of CMML. Disclosures: Kurokawa: Novartis: Consultancy, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Research Funding.