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The transcriptional repressor BCL11A is involved in hematological malignancies, B-cell development, and fetal-to-adult hemoglobin switching. However, the molecular mechanism by which it promotes the development of myeloid leukemia remains largely unknown. We find that Bcl11a cooperates with the pseudokinase Trib1 in the development of acute myeloid leukemia (AML). Bcl11a promotes the proliferation and engraftment of Trib1-expressing AML cells in vitro and in vivo. Chromatin immunoprecipitation sequencing analysis showed that, upon DNA binding, Bcl11a is significantly associated with PU.1, an inducer of myeloid differentiation, and that Bcl11a represses several PU.1 target genes, such as Asb2, Clec5a, and Fcgr3. Asb2, as a Bcl11a target gene that modulates cytoskeleton and cell-cell interaction, plays a key role in Bcl11a-induced malignant progression. The repression of PU.1 target genes by Bcl11a is achieved by sequence-specific DNA-binding activity and recruitment of corepressors by Bcl11a. Suppression of the corepressor components HDAC and LSD1 reverses the repressive activity. Moreover, treatment of AML cells with the HDAC inhibitor pracinostat and the LSD1 inhibitor GSK2879552 resulted in growth inhibition in vitro and in vivo. High BCL11A expression is associated with worse prognosis in humans with AML. Blocking of BCL11A expression upregulates the expression of PU.1 target genes and inhibits the growth of HL-60 cells and their engraftment to the bone marrow, suggesting that BCL11A is involved in human myeloid malignancies via the suppression of PU.1 transcriptional activity.

Introduction Myelodysplastic syndromes (MDS), a heterogeneous group of acquired clonal hematopoietic stem cell disorders, is characterized by peripheral cytopenia and has a high tendency to progress to acute myeloid leukemia (AML). Serum ferritin (SF) level is a well-known indicator of iron stores and inflammation. Elevated SF levels is common in patients with MDS and appear to be largely related to regular red blood cell (RBC) transfusions. However, some patients with MDS showed high SF levels at the time of diagnosis, even before receiving RBC transfusions, although the exact mechanisms is unclear. The impact of SF elevation without RBC transfusion has not been well characterized. In this study, we assessed cytogenetic, molecular, and clinical characteristics of patients with MDS and SF elevation. Methods Patients with newly diagnosed MDS who visited our hospital between Jan 2011 and Jan 2020 were included in this study. Patients were defined as high-SF-MDS when SF values were >350 ng/mL (patients with Results Among the 112 patients with MDS, SF value were available for 87 patients. This study analyzed 28 women and 59 men with a median age of 69 years (range: 28-87 years). The median follow-up period was 540 days (range: 3-3618 days). A total of 17 patients died during the observational period (until Apr 2021). Patients were diagnosed with MDS with single lineage dysplasia (MDS-SLD, n = 9), MDS with multilineage dysplasia (MDS-MLD, n = 26), MDS with ring sideroblasts with SLD (MDS-RS-SLD, n = 5), MDS-RS-MLD (n = 6), MDS with excess blasts 1 (MDS-EB1, n = 25), MDS-EB2 (n = 13), MDS with isolated del(5q) (n=1), and MDS, unclassifiable (MDS-U, n = 2). The median SF value was 263 ng/mL (range: 3-1,245 ng/mL); 31 patients were classified as high-SF-MDS and 56 were classified as low-SF-MDS. The Proportion of MDS subtypes differed significantly between the groups (P=0.004, Table1). Compared to low-SF-MDS, MDS-EB1/2 (51.6% vs. 39.3%) and MDS-RS-SLD/MLD (22.6% vs. 1.8%) was more common in high-SF-MDS. In the laboratory data analysis, high-SF-MDS patients showed low Hb levels, high RBC distribution width values, and high bone marrow RS percentages. In patients with high-SF-MDS, as well as MDS-RS, the presence of bone marrow RS was also observed in seven out of 11 patients with MDS-EB1/2 (median 8%, range 2-19%). Complex karyotype ( was more common in patients with high-SF-MDS (46.4% vs. 16.0%, P=0.007) than those with low-SF-MDS. Trisomy 8 and del (5q) were also frequently detected among those with high-SF-MDS. Patients with high-SF-MDS had worse 2-year overall survival (OS) than those with low-SF-MDS. To clarify the molecular characteristics of high-SF-MDS in more detail, NGS analysis was performed on 11 patients with high-SF-MDS whose samples were available. Ten gene mutations were detected in eight patients. Interestingly, five among 11 patients harbored TP53 mutation; the median variant allele frequency was 53.9% (range, 39.7-64.1%). Subsequently, SF3B1 gene mutation was detected in three patients; and two of them also harbored TP53 mutation. Other gene mutations, RUNX1, STAG2, SRSF2, ASXL1, ATM, DNMT3A, BCOR, and DDX41 were detected in one patient each. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Internal tandem duplications in the juxtamembrane domain of FMS-like tyrosine kinase 3 gene (FLT3-ITD) and missense mutations in the gene's tyrosine-kinase domain (FLT3-TKD) play critical roles in the pathophysiology of acute myeloid leukemia (AML). Recent study revealed that AML cells resistant to quizartinib, a type II TKI, consist of heterogeneous clonal populations harboring wild-type FLT3 as well as FLT3-ITD, FLT3-TKD and FLT3-ITD/TKD mutations, and on- and off-target mechanisms may contribute to the resistance to TKIs. To overcome the heterogeneous resistance mechanisms of FLT3-ITD and TKD mutations, various combinatorial therapies have been investigated. For example, BCL-2 inhibition in the presence of TKIs increased survival a murine FLT3-ITD AML model, and a phase Ib/II clinical trial of a combination of quizartinib with venetoclax, a BCL2 inhibitor is ongoing in relapsed/refractory FLT3-mutant AML patients (NCT03735875). To dissect underlying mechanisms of drug-resistance and exploring new targets in refractory AML with FLT3-ITD and TKD mutations, we investigated alterations of transcriptome signatures by analyzing AML samples with FLT3-ITD/D835 dual mutations. Previously, we reported BCL2A1 transcriptomes were upregulated in primary AML cells with FLT3-ITD/D835 dual mutations compared to cells with FLT3-ITD mutations only. This was recapitulated in the MV4-11 cells harboring FLT3-ITD/D835 dual mutations after 6 month-exposure to quizartinib. The MV4-11 cells with the FLT3-ITD/D835 dual mutations became resistant to quizartinib, and the cells also became resistant to venetoclax, a BCL2 inhibitor (Yamatani et al. ASH 2019). In this study, we further investigated BCL2A1 as new target in refractory AML with FLT3-ITD/D835 dual mutations. First, we examined whether overexpression of BCL2A1 induces drug-resistance in MV4-11 and Molm13 cell lines with FLT3-ITD. While parental MV4-11 and Molm13 cells are sensitive to venetoclax and quizartinib, MV4-11 and Molm13 cells transfected with lentivirus carrying BCL2A1 became resistant to venetoclax (IC50: MV4-11 with BCL2A1 over-expression >1000 nM vs. mock vector 0.71 nM; Molm13 with over-expression >1000 nM vs. mock vector 0.38 nM, 72h). In contrast, the sensitivity to quizartinib was retained in the BCL2A1 overexpressing MV4-11 and Molm13 cells. These findings indicate that the overexpression of BCL2A1 could play a role in the acquired resistance to venetoclax, but not to quizartinib. Bromodomain-containing protein 4 (BRD4), a family member of bromodomain and extra-terminal motif (BET) is known to transcriptionally modulate BCL2A1 gene expression. Next, we examined effects of CPI-0610, a BET inhibitor, on MV4-11 cells with FLT3-ITD or the FLT3-ITD/D835 dual mutation. CPI-0610 inhibited cell growth of MV4-11 cells by inducing apoptosis irrespective of co-existing FLT3 mutations (IC50: FLT3-ITD/D835, 255 nM vs. FLT3-ITD, 191 nM, 72h). Immunoblotting analyses confirmed that BET inhibition by CPT-0610 decreased the expression of BCL2A1 in MV4-11 cells FLT3-ITD/D835. WIn conclusion, transcriptome analysis and molecular pharmacological approaches identified alterations in the anti-apoptotic BCL2 family proteins in double-mutant FLT3 leukemias. BCL2A1 upregulation might be involved in drug resistance of FLT3-ITD/D835 dual mutant AML cells, and could be a promising new target in refractory AML with FLT3-ITD/D835 dual mutations. Disclosures Konopleva: Cellectis: Research Funding; Eli Lilly: Research Funding; Calithera: Research Funding; Sanofi: Research Funding; Ascentage: Research Funding; AstraZeneca: Research Funding; Agios: Research Funding; Stemline Therapeutics: Consultancy, Research Funding; Kisoji: Consultancy; Genentech: Consultancy, Research Funding; F. Hoffmann La-Roche: Consultancy, Research Funding; Forty-Seven: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Reata Pharmaceutical Inc.;: Patents & Royalties: patents and royalties with patent US 7,795,305 B2 on CDDO-compounds and combination therapies, licensed to Reata Pharmaceutical; Rafael Pharmaceutical: Research Funding; Ablynx: Research Funding; Amgen: Consultancy. Carter:Syndax: Research Funding; AstraZeneca: Research Funding; Amgen: Research Funding; Ascentage: Research Funding. Andreeff:Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Amgen: Research Funding; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy.

Key Points RUNX1a, but not RUNX1b, is overexpressed in CD34+ cells from patients with myelodysplastic/myeloproliferative neoplasms. SRSF2P95H mutation induces RUNX1a overexpression and a monocytic phenotype in TF-1 cells.

Genetic mutations in FLT3 (fms-like tyrosine kinase-3) play an important role in the pathogenesis of acute myeloid leukemia (AML). FLT3 internal tandem duplications (FLT3-ITD) occur in approximately 25% of all AML cases and various tyrosine kinase inhibitors (TKIs) targeting FLT3-ITD such as quizartinib, crenolanib, and gilteritinib have been developed. Although these selective FLT3 inhibitors were thought to be promising, their effects turned out to be temporary due to the rapid development of resistance associated with clonal switching. Acquired FLT3 point mutations at D835 in the activation loop of tyrosine kinase domain are often accountable for clonal switching at least for Type II TKIs. In addition, adjunct mutations in other genes are also found to be associated with TKI resistance. To investigate the underlying molecular mechanism of this secondary, mutation-driven acquired resistance, we first analyzed co-occurring mutations in the leukemia cells obtained from 26 AML patients with FLT3-ITD (n=14) or FLT3-ITD/D835 dual (n=12) mutations, and performed cap analysis of gene expression (CAGE) sequencing, which identifies and quantifies the 5' ends of capped mRNA transcripts (transcription start sites) and allows investigating promoter structures necessary for gene expression. Patients with FLT3-ITD/D835 harbored a higher number of co-mutations such as ASXL1 and RUNX1 compared to AML with FLT3-ITD (FLT3-ITD/D835: 2.83 ± 0.52, FLT3-ITD: 0.49 ± 0.13, p To recapitulate the observations obtained with primary human AML samples, we generated MV4;11 cells with acquired FLT3-ITD/D835 mutations (MV4;11-QR cells) by culturing FLT3-ITD MV4;11 leukemia cells in the presence of quizartinib (1.5 nM), a selective FLT3 inhibitor, for 6 months. While quizartinib (0.2 nM) suppressed the proliferation of 50% of the parental MV4;11 at 72 hours, much higher concentrations of quizartinib (10 nM) was required to suppress the proliferation of MV4;11-QR cells. Quantitative RT-PCR and immunoblot analysis revealed that MV4;11-QR cells expressed higher transcript and protein levels of BCL2A1 than MV4;11 parental cells, while BCL2 levels were similar in both cells and MCL1 and BCLxL expression were lower in the MV4;11-QR than in the parental cells. Next, to investigate the molecular properties of AML cells bearing FLT3-ITD or FLT3-ITD/D835 without other co-mutations, we created Ba/F3 cells stably expressing FLT3-ITD or FLT3-ITD/D835. Of notes, the FLT3-ITD/D835 Ba/F3 cells expressed markedly higher BCL2 transcript and protein levels with lower expression of BCLxL than in FLT3-ITD Ba/F3 cells. No significant difference of MCL1 expression was observed. The sensitivity to quizartinib was massively decreased in the FLT3-ITD/D835 Ba/F3 cells (IC50: FLT3-ITD/D835 >1000nM vs. FLT3-ITD, 0.8nM, at 48h). Finally, we examined the efficacy of the BCL-2 specific inhibitor venetoclax in FLT3-ITD/D835 dual mutated cells with or without upregulation of BCL2 or BCL2A1, the latter shown to confer resistance to venetoclax by sequestering released BIM (Esteve-Arenys, Oncogene. 2018). As expected, venetoclax caused more profound cell growth inhibition and apoptosis induction in BCL2 upregulated FLT3-ITD/D835 Ba/F3 compared to FLT3-ITD Ba/F3 cells (IC50: FLT3-ITD/D835 301nM vs. FLT3-ITD >1000 nM, 96 h). However, FLT3-ITD/D835 bearing MV4;11-QR cells with upregulated BCL2A1 were less sensitive to venetoclax than MV4;11 parental cells (IC50: MV4;11-QR, 149nM vs. MV4;11, 33 nM, 72 h). In conclusion, these results demonstrate that acquisition of D835 mutation in FLT3-ITD mutated AML is often accompanied with multiple co-occurring genetic mutations, and depends on anti-apoptotic BCL-2 associated pro-survival mechanisms. BCL2A1 upregulation might be involved in pathogenesis of acquired drug resistance of FLT3-ITD/D835 dual mutant AML cells, and is a promising new target in FLT3-ITD/D835 refractory AML with complex mutations. Disclosures Carter: Amgen: Research Funding; AstraZeneca: Research Funding; Ascentage: Research Funding. Shah:Bristol-Myers Squibb: Research Funding. Konopleva:Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ablynx: Research Funding; Astra Zeneca: Research Funding; Agios: Research Funding; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding. Andreeff:Daiichi Sankyo, Inc.: Consultancy, Patents & Royalties: Patents licensed, royalty bearing, Research Funding; Jazz Pharmaceuticals: Consultancy; Celgene: Consultancy; Amgen: Consultancy; AstaZeneca: Consultancy; 6 Dimensions Capital: Consultancy; Reata: Equity Ownership; Aptose: Equity Ownership; Eutropics: Equity Ownership; Senti Bio: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Oncoceutics: Equity Ownership; Oncolyze: Equity Ownership; Breast Cancer Research Foundation: Research Funding; CPRIT: Research Funding; NIH/NCI: Research Funding; Center for Drug Research & Development: Membership on an entity's Board of Directors or advisory committees; Cancer UK: Membership on an entity's Board of Directors or advisory committees; NCI-CTEP: Membership on an entity's Board of Directors or advisory committees; German Research Council: Membership on an entity's Board of Directors or advisory committees; Leukemia Lymphoma Society: Membership on an entity's Board of Directors or advisory committees; NCI-RDCRN (Rare Disease Cliln Network): Membership on an entity's Board of Directors or advisory committees; CLL Foundation: Membership on an entity's Board of Directors or advisory committees; BiolineRx: Membership on an entity's Board of Directors or advisory committees.

Background Myelodysplastic syndromes (MDS), commonly seen in elderly patients, represent a heterogeneous group of clonal hematopoietic stem cell disorders caused by the accumulation of gene mutations. By contrast, congenital bone marrow failure syndromes and genetic predispositions associated with MDS are known in pediatric patients. However, little is known about the pathogenesis of MDS in adolescent and young adult (AYA) patients. Previous reports showed the patients with MDS aged under 40 or 41.5 years at allo-HSCT were associated with good survival compared to those among the older population (N Engl J Med. 2017;376:536-547, Blood. 2017;129:2347-2358). However, AYA-MDS is rare, and its clinical features and genetic abnormalities have not been analyzed enough. It is suspected that the clinical and genetic features of AYA-MDS patients might be different from those of elderly patients or pediatric patients. Therefore, we investigated the gene abnormalities of AYA-MDS patients and aimed to elucidate the genetic characteristics associated with the good outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT). We analyzed the patients younger than 50 years of age in order to reduce the variation of patient-related factors. Methods We analyzed the outcomes of all consecutive patients aged under 50 years who were diagnosed with MDS or acute myeloid leukemia evolving from MDS in our hospital between January 2005 and July 2018. The study was approved by the institutional review board, and patients gave written informed consent for the study, according to the Declaration of Helsinki. Cytogenetic analysis and genomic DNA extraction were carried out using diagnostic bone marrow samples. We performed targeted next-generation sequencing to identify mutations in 68 driver genes using AmpliSeq for Illumina Myeloid Panel and On-Demand Panel on the MiniSeq system (Illumina). Gene variants were detected by in-house analysis pipeline. Overall survival (OS) was analyzed for all patients, and the Kaplan-Meier survival curve was used to assess OS using the log-rank test. Additionally, the cumulative incidence of relapse (CIR) was analyzed for patients who underwent allo-HSCT. Gray's test was used to evaluate the CIR. Results A total of 85 patients with MDS aged under 50 years (U40 between 15 and 39 years old: N=37, 40s between 40 and 49 years old: N=48) were analyzed. The median follow-up time of survivors was 2,041 days (range 176-5,085). There were no significant differences in patient characteristics between U40 and 40s. The 3-year OS of U40 were superior to 40s (79.9% vs. 58.1%, P=0.018), especially lower risk IPSS categories (3-year OS, 95.5% vs. 50.8%, P=0.002). In total, 69 of 85 patients (U40: N=31, 40s: N=38) had undergone allo-HSCT. U40 patients had lower percentage of bone marrow blasts at just before HSCT than 40s patients (over 10%, 12.9% vs. 36.8%, P=0.048), and better 3-year OS from HSCT in lower-IPSS (88.8% vs. 53.8%, P=0.024); but not in higher-IPSS (45.0% vs. 43.2%, P=0.834). In this cohort, at least one driver mutation was detected in 61% of allo-HSCT recipients. Frequently mutated genes (more than 10%) were ASXL1 and RUNX1; however, both of the genes did not have significant impact on the outcomes. While, only one patient in 40s had TP53 mutation. We detected 0.8 (range 0-3) and 1.8 (range 0-6) mutations at average in U40 and 40s, respectively (P=0.06). The proportions of the patients without any gene mutations were 52% in U40 and 30% in 40s. Transplanted patients with 0 or 1 mutation showed lower relapse rate than those with 2 or more mutations (3-year CIR, 23.3% vs. 45.2%, P=0.049). Conclusions The clinical outcomes of U40 patients with MDS were favorable than those in the 40s, especially in lower disease risk. The number of driver mutations in U40 tended to be lower than that in 40s. MDS in adult is regarded as a stem-cell aging disease with gene mutations; however, MDS-associated mutations were not detected in the half of U40. Moreover, TP53 mutation that is associated with extremely poor posttransplant survival was not detected in U40 patients. MDS patients with less than 2 mutations showed lower relapse rate, which maybe indicate genetic mutations have a great impact on transplant outcomes between 15 and 49 years old. Disclosures No relevant conflicts of interest to declare.

Background Constitutional partial trisomy 8 mosaicism (CT8M) is a congenital chromosomal abnormality with an estimated occurrence rate as one out of 25,000-50,000 pregnancies. CT8M has a wide variability in physical manifestation ranging from apparently normal to severe disablement. However, diagnosis of CT8M in adult without physical abnormality is difficult . Acquired trisomy 8, which is restricted to the malignant cells, is one of the most common chromosomal abnormalities in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), and clinical implication of carrying isolated trisomy 8 is considered as intermediate cytogenetic risk in MDS. However, isolated trisomy 8 without morphological dysplastic features is not definitive evidence for MDS. 15 to 20% of trisomy 8 in MDS are supposed to be derived from CT8M. We therefore diagnosed CT8M patients among patients with cytopenia and analyzed clinical and genetic features to uncover the association with MDS. Methods . Clinical features including cytogenetic analysis were analyzed regularly. Genomic DNA was extracted from whole PB cells or BM mononuclear cells. We performed targeted next-generation sequencing to identify mutations in 68 driver genes of myeloid neoplasms using AmpliSeq for Illumina Myeloid Panel and On-Demand Panel on the MiniSeq system (Illumina). Gene variants were detected by in-house analysis pipeline. The study was approved by the institutional review board and patients gave written informed consent for the study. Results We identified nine CT8M patients with cytopenia.They comprised 3 males and 6 females at a median age of 56 years (range 24-84 years) (Table). All the patients carried no physical abnormality nor conspicuous phenotypic features. Four patients (Patient #3, #4, #6 and #7) did not show apparent morphological dysplasia at the initial BM examination, and they were not diagnosed as MDS. Their cytopenia has not been exacerbated until now without any treatment, and the duration of stable cytopenia was from 2 to 12 years in these patients. By contrast, five patients with CT8M were diagnosed as MDS . Two patients (#5 and #8) were diagnosed as MDS-single lineage dysplasia (SLD), and their cytopenia has not become worse without any treatment for about 4 years. Other three patients diagnosed as MDS-multilineage dysplasia (MLD) showed various clinical courses. Patient #1 was treated with azacitidine and maintains complete hematological improvement after 34 courses of the treatment. Patient #2 was treated with erythropoietin stimulating agent and azacytidine but developed to AML 3 years after initial diagnosis but leukemic blasts has del(20), not +8. Patient #9 developed advanced pancytopenia in 3 months from initial diagnosis and received red blood cell transfusion regularly. Gene mutations were detected in five out of nine patients with CT8M. In three patients, gene mutations were detected at high (20 to 50%) variant allele frequency (VAF). Patient #2 who was analyzed at the AML phase had gene mutations of SRSF2, SF3B1, STAG2 and NOTCH1. BM sample from patient #9 showed ASXL1 mutation and two TET2 mutations. Patient #4 who did not show apparent myelodysplasia had a high VAF ASXL1 mutation, indicating clonal idiopathic cytopenias of undetermined significance. Two patients had low (2 to 5%) VAF mutations; patient #1 was analyzed after 34 courses of azacitidine had a TET2 mutation; patient #5 with MDS-SLD had a WT1 mutation and two PHF6 mutations. Four patients (#3, #6, #7 and #8) did not have any mutations. The clinical and genetic features showed that CT8M with cytopenia without MDS-related mutations were under 56 years old and did not develop to MDS or stayed at MDS-SLD. Patients with low VAF mutations were also stable. By contrast, patients with advanced diseases gained multiple MDS-related gene mutations with high VAF. One patient without dysplasia had a high VAF ASXL1 mutation. All the patients with gene mutations were age of 56 to 84 years. Conclusion Our results indicated that isolated trisomy 8 may cause cytopenia, but the cytopenia is not exacerbated without MDS-related driver gene mutations. CT8M patients with cytopenia might get gene mutations gradually with age, which leads to MDS or AML. Disclosures No relevant conflicts of interest to declare.

Background Azacitidine (AZA) is one of the hypomethylating agents (HMAs) proven to significantly prolong overall survival (OS) for patients with higher-risk myelodysplastic syndromes (h-MDS) via a prospective phase 3 trial. However, the optimal treatment schedule of AZA for h-MDS has not yet been prospectively determined. As a 7-day treatment schedule includes weekends, a 5-day administration has also been applied with h-MDS, though solid evidence has not been established. Based on this background, we planned to compare AZA treatment on 7-day and 5-day schedules. Patients Patients diagnosed with de novo or treatment-related MDS (FAB-defined RAEB and RAEB-t), aged 16 years or older were eligible for this study, if they showed adequate performance status (ECOG PS 0-2) and no history of HMA treatment or chemotherapy. Candidates for allogeneic stem-cell transplantation were excluded. Study design This was a multicenter, randomized, open-label, phase 3 trial to compare the efficacy of AZA treatment for 7-days (AZA-7) to 5-days (AZA-5). The primary endpoint was 2-year OS rate (2y OS). AZA was given subcutaneously at 75 mg/m² every 28 days. To test the non-inferiority of AZA-5 to AZA-7, 2y OS of AZA-7 was estimated at 30%, and the delta was defined as 11%, for which 410 patients were needed to complete the trial. However, because of the poor recruitment, this study closed prematurely, and the protocol-planned interim analysis (when 200 patients were registered) was performed. This protocol was approved by the IRB at each trial center. All patients provided written informed consent, and the trial was conducted in accordance with the Declaration of Helsinki. The efficacy of AZA was evaluated using IWG2006 criteria, and treatment was continued until study completion, or relapse, unacceptable toxicity, or disease progression was observed. Efficacy was analyzed by intention-to-treat. Secondary endpoints were the hematological response rate, 2-year leukemia-free survival (2y LFS) rate, the cytogenetic response, and occurrence of adverse events. Results Between January 2013 and Jun 2018, 201 patients were randomly assigned to AZA-7 (n=99) or AZA-5 group (n=102). The median age of all patients at the enrollment was 73.5 years old (range, 48 to 91). Between the two groups, there was no significant difference in the baseline characteristics, such as sex, type of MDS (de novo or treatment-related), percent of BM blasts, FAB and WHO classification, and IPSS and IPSS-R risk. After excluding 5 and 6 patients from AZA-7 and AZA-5, respectively (poor data entry, change in diagnosis), at the time of last follow-up, 59 and 67 patients died in AZA-7 and AZA-5, respectively. The Kaplan-Meier estimates of the 2y OS and 2y LFS were 35.1% (95% CI, 23.8 to 46.7) and 27.3% (95% CI, 17.3 to 38.3) for AZA-7, and 22.4% (95% CI, 13.1 to 33.3) and 20.5% (95% CI, 11.6 to 31.2) for AZA-5, respectively. There was no significant difference in the frequency of erythroid, platelet or neutrophil improvement, or hematological response (complete or partial response, or any hematological improvement) between the two groups. The most common non-hematological adverse event of grade 3 to 4 (in ≥20% of the patients in either group) was febrile neutropenia (33.7% for AZA-7, and 30.5% for AZA-5). Conclusion and Discussion Because of the premature termination of this trial, statistical analysis for the primary endpoint (2y OS of 35.1% for AZA-7, and 22.4% for AZA-5) could not provide any solid evidence. However, considering the difference of more than 10% in 2y OS, and no major differences in the safety profiles, we favor AZA-7 to AZA-5 for MDS patients with RAEB and RAEB-t. Figure Disclosures Kiguchi: Tejin Co., Ltd.: Research Funding; Taiho Pharmaceutical Co., Ltd.: Research Funding; SymBio Pharmaceutical Co., Ltd.: Research Funding; Celgene Co., Ltd.: Research Funding; Janssen Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharmaceutical Co., Ltd.: Research Funding; Novartis Pharmaceutical Co., Ltd.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Astellas Pharmaceutical Co., Ltd.: Research Funding; MSD CO., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myeres Squibb Co., Ltd.: Research Funding; Sanofi K.K., Ltd.: Research Funding; Celltrion, Inc.: Research Funding. Usuki:Daiichi Sankyo Co., Ltd.: Research Funding, Speakers Bureau; Astellas Pharma Inc: Research Funding, Speakers Bureau. Suzuki:kyowa Hakko Kirin: Consultancy, Honoraria; MSD k.k.: Research Funding; Nippon Shinyaku: Honoraria; Novartis: Honoraria; takeda: Research Funding; Incyte and Pfizer: Research Funding; astellas: Research Funding; Celgene: Honoraria; Chugai: Honoraria, Research Funding; daiichi sankyo: Research Funding; eisai: Research Funding. Tomita:Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Kyowa Kirin: Research Funding; Taiho Pharma: Research Funding. Handa:Ono: Research Funding. Maeda:Janssen Pharmaceutical K.K..: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria. Iyama:Otsuka Pharmaceutical Co., Ltd.: Honoraria; Otsuka Pharmaceutical Factory: Honoraria; Astellas Pharma: Honoraria; Daiichi Sankyo: Honoraria; Allexion Pharma: Honoraria; CSL Behring: Honoraria. Yamauchi:Astellas, AbbVie: Consultancy; Pfizer, Chugai, Teijin, Solasia: Research Funding. Kiyoi:Daiichi Sankyo Co., Ltd: Research Funding; Bristol-Myers Squibb: Research Funding; Pfizer Japan Inc.: Honoraria; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Perseus Proteomics Inc.: Research Funding; FUJIFILM Corporation: Research Funding; Astellas Pharma Inc.: Honoraria, Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Eisai Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding. Miyazaki:Chugai: Research Funding; Otsuka: Honoraria; Novartis: Honoraria; Nippon-Shinyaku: Honoraria; Dainippon-Sumitomo: Honoraria; Kyowa-Kirin: Honoraria.

High Mobility Group AT-hook 2 (HMGA2) is a chromatin modifier and its overexpression has been found in a subset of patients with myelodysplastic syndrome (MDS). The high level of HMGA2 expression appears to predict poor prognosis in various tumors; however, it remains unclear how HMGA2 dysregulates expression of target genes to facilitate the transformation. To elucidate the mechanisms by which the overexpression of Hmga2 promotes the development of MDS, we generated an Hmga2-expressing Tet2-deficient (Hmga2-Tet2Δ/Δ) mouse model showing the progressive phenotype of MDS. We found that Hmga2-Tet2Δ/Δ mice had neutropenia and anemia, but variable platelet counts, accompanied by elevated frequencies of mutant cells in myeloid cells. Hmga2-Tet2Δ/Δ mice showed a similar median survival to Tet2Δ/Δ mice (274 days vs 290 days), but shorter survival than Hmga2-Tet2wt/wt mice (274 days vs undetermined). Moribund Hmga2-Tet2Δ/Δ mice showed progressive leukopenia and anemia, accompanied by the emergence of dysplastic neutrophils, myeloblasts and anisocytosis in the PB and BM and dysplastic megakaryocytes in the BM. Hmga2-Tet2Δ/Δ mice had mildly increased spleen weights, and expanded myeloid cells and HSPCs in the spleen without the deposition of fibrosis. During a 12-month observation, we found that Hmga2-Tet2Δ/Δ mice developed lethal MDS/MPN overlap disease (47%), MDS (33%), MPN (13%), and AML (7%), while 6 out of 11 Tet2Δ/Δ mice developed MPN (55%). Hmga2-Tet2wt/wt mice subsequently showed similar blood counts in PB and died without the expansion of leukemic or dysplastic blood cells. Therefore, Hmga2 overexpression did not transform wild-type HSCs but promoted the development of MDS in the absence of Tet2 in vivo. In order to elucidate the molecular mechanisms underlying the transformation of Hmga2-Tet2Δ/Δ cells, we initially performed gene expression profiling by a RNA sequencing analysis in LSK HSPCs isolated from WT, Hmga2-Tet2wt/wt, Tet2Δ/Δ, and Hmga2-Tet2Δ/Δ mice at a pre-disease stage and those isolated from two Hmga2-Tet2Δ/Δ MDS/MPN and AML mice. Hmga2-Tet2Δ/Δ leukemic cells were placed closer to one out of two Hmga2-Tet2Δ/Δ cells at the pre-disease stage, but clearly apart from the other genotype cells, indicating that Hmga2 overexpression and Tet2 loss result in the accumulation of alterations in the transcriptional program during the development of MDS.In order to clarify the mechanisms by which the overexpression of Hmga2 alters the transcriptional program in Tet2-deficient cells, we performed the ChIP-sequencing of FLAG-tagged Hmga2 in bone marrow progenitor cells isolated from WT, Hmga2-Tet2wt/wt, and Hmga2-Tet2Δ/Δ mice. The numbers of Hmga2-binding peaks were markedly lower in Tet2-deficient cells than in Hmga2-Tet2wt/wt cells (2227 peaks versus 11500 peaks). Furthermore, annotated genes adjacent to Hmga2-binding sites partially overlapped in both genotype cells, whereas 2965 out of 3843 genes identified in Tet2 wild-type cells lost the binding peaks of Hmga2 upon the deletion of Tet2. Based on the DNA-binding capacity of Hmga2, the loss of Tet2 remodeled the binding sites of Hmga2 via change in DNA methylation in Hmga2-binding flanking regions, which were not observed in the presence of Tet2, leading to significant enrichments in genes involved in cell-to-cell adhesion and cell morphogenesis in Hmga2-Tet2Δ/Δ cells. Furthermore, we found that the overexpression of Hmga2 and loss of Tet2 resulted in the activation of oncogenic pathways (e.g. TGF-b, TNF-a), but suppressed the expression of genes in the unfolded protein response. Notably, the inhibition of bile acid metabolism to reactivate the unfolded protein response markedly attenuated the proliferation of Hmga2-Tet2Δ/Δ cells. These combinatory effects on the transcriptional program and cellular functions were not redundant to those in either single mutant cell, supporting Hmga2 being a proto-oncogene because its overexpression alone was not sufficient to develop MDS in vivo. Thus, Hmga2 overexpression exerts synergistic, but also gain-of-function effects with the loss of Tet2 to target these key biological pathways and promotes the transformation of Tet2-deficient stem cells. This study also provides a new rationale for targeting the unfolded protein response in MDS cells expressing HMGA2. Disclosures No relevant conflicts of interest to declare.

RUNX1/AML1 mutations have been identified in myelodysplastic syndromes (MDSs). In a mouse bone marrow transplantation model, a RUNX1 mutant, D171N, was shown to collaborate with Evi1 in the development of MDSs; however, this is rare in humans. Using enforced expression in human CD34(+) cells, we showed that the D171N mutant, the most frequent target of mutation in the RUNX1 gene, had an increased self-renewal capacity, blocked differentiation, dysplasia in all 3 lineages, and tendency for immaturity, but no proliferation ability. BMI1 overexpression was observed in CD34(+) cells from the majority of MDS patients with RUNX1 mutations, but not in D171N-transduced human CD34(+) cells. Cotransduction of D171N and BMI1 demonstrated that BMI1 overexpression conferred proliferation ability to D171N-transduced cells in both human CD34(+) cells and a mouse bone marrow transplantation model. Stepwise transduction of D171N followed by BMI1 in human CD34(+) cells resulted in long-term proliferation with a retained CD34(+) cell fraction, which is quite similar to the phenotype in patients with higher-risk MDSs. Our results indicate that BMI1 overexpression is one of the second hit partner genes of RUNX1 mutations that contribute to the development of MDSs.

Two types of mutations of a transcription factor CCAAT-enhancer binding protein α (C/EBPα) are found in leukemic cells of 5%-14% of acute myeloid leukemia (AML) patients: N-terminal mutations expressing dominant negative p30 and C-terminal mutations in the basic leucine zipper domain. Our results showed that a mutation of C/EBPα in one allele was observed in AML after myelodysplastic syndrome, while the 2 alleles are mutated in de novo AML. Unlike an N-terminal frame-shift mutant (C/EBPα-Nm)–transduced cells, a C-terminal mutant (C/EBPα-Cm)–transduced cells alone induced AML with leukopenia in mice 4-12 months after bone marrow transplantation. Coexpression of both mutants induced AML with marked leukocytosis with shorter latencies. Interestingly, C/EBPα-Cm collaborated with an Flt3-activating mutant Flt3-ITD in inducing AML. Moreover, C/EBPα-Cm strongly blocked myeloid differentiation of 32Dcl3 cells, suggesting its class II mutation-like role in leukemogenesis. Although C/EBPα-Cm failed to inhibit transcriptional activity of wild-type C/EBPα, it suppressed the synergistic effect between C/EBPα and PU.1. On the other hand, C/EBPα-Nm inhibited C/EBPα activation in the absence of PU.1, despite low expression levels of p30 protein generated by C/EBPα-Nm. Thus, 2 types of C/EBPα mutations are implicated in leukemo-genesis, involving different and cooperating molecular mechanisms.

Internal tandem duplications (ITD) in the juxtamembrane domain of FMS-like tyrosine kinase 3 (FLT3) cause ligand-independent constitutive activation of FLT3 kinase and its downstream signaling. FLT3-ITD mutations confer poor prognosis with high relapse rates in AML patients. FLT3-targeted therapies using tyrosine kinase inhibitors (TKIs) often induce additional point mutations within the tyrosine kinase domains (FLT3-TKD mutations), most commonly found at D835 activation loop. Additional FLT3-TKD mutations that cause secondary resistance emerge in at least 20% of patients with TKI treatment. To elucidate the alterations of transcriptome signatures of FLT3-ITD and TKD double mutations in AML, we performed cap analysis of gene expression (CAGE) sequencing for 26 primary AML samples (14 with FLT3-ITD, 12 with FLT3-ITD/D835). CAGE detects and quantifies the specific transcriptional start site (TSS) transcripts, which enables high-throughput gene expression profiling and promoter usage analysis. Altered transcription of TSS in FLT3-ITD/D835 AML samples were detected by comparison with TSS in FLT3-ITD samples, and upregulation of 310 TSS and downregulation of 22 TSS were mapped (FDR < 0.05, EdgeR). Based on Gene Ontology (GO) analysis, up-regulated genes were enriched in "apoptotic process", "intracellular signal transduction" and "immune system development", including pro-survival BCL2A1 and drug resistance related S100A8 and PRKCH. To validate these transcriptional changes, we utilized isogenic paired Ba/F3 cells transfected with FLT3-ITD or FLT3-ITD/D835. CAGE detected upregulation of 1945 TSS and downregulation of 1470 TSS in FLT3-ITD/D835 compared to FLT3-ITD cells (FDR < 0.05). TSS transcriptions of Bcl-2, Prkca, NF-κB1, Myc, and Cdkn1a (p21) were upregulated in FLT3-ITD/D835 cells. GO analysis consistently highlighted higher activation of NF-κB signaling and its downstream Bcl-2 in FLT3-ITD/D835 than in FLT3-ITD cells both for primary AML samples and Ba/F3 cells. To determine a correlation between activated promoters and transcription factors in FLT3-ITD/D835 cells, we performed an unbiased search for enriched sequence motifs using HOMER software. HOMER revealed that the promoter 4 of BCL2A1 contained a common motif of transcription factor STAT6, known to associate with NF-κB and cooperatively bound to their respective promoter elements. Ingenuity Pathway Analysis also highlighted higher activation of STAT6 in FLT3-ITD/D835 AML cells compared to FLT3-ITD. Immunoblot analysis confirmed higher expression of Bcl-2, c-Myc, p27, and lower expression of Bcl-xL and Mcl-1 in FLT3-ITD/D835 Ba/F3 compared to FLT3-ITD Ba/F3 cells. FLT3-ITD and FLT3-ITD/D835 cells showed similar expression level of Bax and Bid. We found that FLT3-ITD/D835 Ba/F3 cells proliferated slower than FLT3-ITD cells (growth rate; FLT3-ITD/D835 9.8±2.4 fold, FLT3-ITD 19.8±1.2 fold, p=0.003, 48 h) with G0/G1 accumulation (FLT3-ITD/D835 61.5±10.1%, FLT3-ITD 31.3±11.2%, p Finally, we hypothesized that targeting Bcl-2 may effectively overcome the acquired resistance of FLT3-ITD/D835 cells. Venetoclax (ABT-199/GDC-0199), a clinically available Bcl-2 selective inhibitor, as single agent was not capable of eliminating FLT3-ITD AML due to altered activation of FLT3 downstream signaling and Mcl-1 upregulation. FLT3-ITD/D835 cells showed higher expression of the Bcl-2 gene and/protein and were less dependent on FLT3 signaling compared to FLT3-ITD cells (Tabe, ASH 2017). As expected, venetoclax caused more profound cell growth inhibition and apoptosis induction in FLT3-ITD/D835 Ba/F3 than in FLT3-ITD Ba/F3 cells (IC50 2.98 μM vs 13.9 μM, ED50 28.8 μM vs 173 μM, 48 h). In conclusion, we identified Bcl-2 transcriptional activation as a novel mechanism by which the acquired D835 mutation in FLT3-ITD AML cells facilitates anti-apoptotic network activation and confers TKI resistance. Bcl-2 inhibition by venetoclax represents a putative therapeutic strategy in FLT3-ITD/TDK double-mutated AML cells. Disclosures Shah: Bristol-Myers Squibb: Research Funding; ARIAD: Research Funding. Konopleva:Stemline Therapeutics: Research Funding. Andreeff:AstraZeneca: Research Funding.

Background Although the outcome after allogeneic hematopoietic stem cell transplantation (Allo-HSCT) for the patients with acute myeloid leukemia (AML) in complete remission has been improved, the prognosis of patients with active disease is still dismal, generally with 20 - 30% of overall survival (OS) at 2 years. Prognostic value of gene mutations detected by the next generation sequencing (NGS) for this extremely poor group remains to be evaluated. Methods A total of 120 patients with AML not in hematological remission who received the first allo-HSCT at our institute between April 2005 and December 2017 were enrolled. For each patient, genomic DNA was extracted from the frozen bone marrow sample harboring leukemic blasts which was preserved at the nearest available date before the initiation of conditioning regimen. Sequencing was performed using TruSight Myeloid Sequencing Panel® on the MiniSeq system (Illumina). Gene variants were detected by in-house analysis pipeline. Charts were retrospectively reviewed on survival, relapse, and non-relapse mortality (NRM). The Kaplan-Meier method was used to assess OS using the log-rank test. Univariate and multivariate analysis were performed to identify potential prognostic factors. The Cox proportional hazards method was used for the multivariate analysis to assess OS. Gray's test and the Fine-Gray test were used to assess the cumulative incidence of relapse (CIR). Competing risks were relapse and NRM. Results Median follow-up of survivors was 1345 days (235 - 4888 days). Median age at transplant was 51 (range 21 - 71). Grafts were from bone marrow (n = 67, 55.8%), peripheral blood (n=42, 35.0%) and cord blood (n=11, 9.2%). Refined disease risk index (Blood. 2014;123:3664-71) scored high (n=61, 51.3%) and very high (n=58, 48.7%). OS at 2 years of the whole cohort was 27.3% (95% confidence interval [CI], 19.4% - 35.7%). There was no significant difference in OS between patients in primary induction failure and in relapse (OS at 2 years: 26.5% [n=50] vs 28.7% [n=70], p= 0.293). NGS analysis revealed TP53 loss-of-function mutation in 23 (19.2%) patients. Among all detected gene mutations, TP53 mutation was the most powerful predictor of poor OS after allo-HSCT (OS at 2 years: 13.5% vs 30.5% for TP53+ [n=23] vs TP53- [n=97], p= 0.0184). Consistent with previous reports, monosomal karyotype (MK, J Clin Ocnol 2010:26;4791-7) was significantly associated with positive TP53 mutation (13.3% of non-MK vs 37.9% of MK, p=0.006). Of note, all the patients (n=11) positive for both prognostic factors died within 1 year after allo-HSCT, whereas OS of the patients without either factor (n=78) was 33.6% at 2 years. Multivariate analysis on OS revealed MK, TP53 mutation, de novo AML (no prior history of MDS), ECOG performance status score 2 or more, C reactive protein 1.0 mg/dL or more, peripheral blood blast frequency of 1% or more at the initiation of conditioning regimen were independent prognostic factors for poor OS. Among them, to determine the prognostic factors critical for deciding the indication of allo-HSCT, we chose pre-transplant factors which were available around one months before transplant. From this point of view, multivariate analysis revealed independent prognostic factors for poor OS after allo-HSCT including MK (Hazard ratio [HR] 2.05; 95% confidence interval [CI] 1.30 - 3.25, p=0.00217), TP53 mutation (HR 1.72; 95% CI 1.04 - 2.86, p=0.035), and de novo AML (HR 1.67; 95% CI 1.04 - 2.86, p=0.036). Because the HR of these three factors were comparable, a score of 1 was assigned to each factor. OS at 2 years was 49.7%, 26.5% and 13.1% for patients with low (score 0, n=23), intermediate (score 1, n=63) and high (score 2 or 3, n=34) risk, respectively (p Conclusions NGS-analysis revealed the importance of TP53 mutation on transplant outcome of AML in non-remission. The combined score with the information on MK, de novo AML, and TP53 mutation stratified the patients into three groups, including low risk with 50% survival rate, and high risk with shorter survival after allo-HSCT for whom the other strategy might be optimal. Our results would require further validation in larger cohorts. Disclosures Harada: Celgene: Research Funding.

Introduction: PcG proteins form two main multiprotein complexes, Polycomb repressive complex 1 (PRC1) and PRC2. They repress the transcription of target genes. Polycomb group ring finger protein1 (PCGF1) is a component of PRC1.1, a non-canonical PRC1.1 that monoubiquitylates H2A at lysine 119 in a manner independent of H3K27me3. Several groups including ours showed that the loss of Ezh2, a component of PRC2, promotes the development of JAK2 V617F-induced Myelofibrosis (MF) in mice. However, the role of PRC1.1 in hematologic malignancies is still not fully understood. We found that the deletion of PCGF1 in mice promotes myeloid commitment of hematopoietic stem and progenitor cells (HSPCs), and eventually induces a lethal myeloproliferative neoplasm (MPN)-like disease in mice (Nakajima-Takagi Y, unpublished data). Based on these findings, we investigated the role of PCGF1 in a mouse model of JAK2V617F-induced myelofibrosis. Methods: We transplanted BM cells from Cre-ERT2, PCGF1flox/flox;Cre-ERT2, JAK2V617F;Cre-ERT2, and JAK2V617F;PCGF1flox/flox;Cre-ERT2 mice into lethally irradiated recipient mice. We deleted PCGF1 by tamoxifen administration 4 weeks after transplantation. Results: JAK2/PCGF1 KO mice developed lethal MF significantly earlier than the other genotypes (p Conclusions: Our findings suggest that dysregulated PRC1.1 function promotes JAK2V617F-induced MF with mechanisms distinct from MF associated with PRC2 dysfunction. Disclosures Harada: Celgene: Research Funding.

FMS-like tyrosine kinase 3 with internal tandem duplication (FLT3-ITD), the most frequent mutation in acute myeloid leukemia (AML), results in the constitutive ligand-independent activation of FLT3 receptor downstream signaling. FLT3-targeted therapies using tyrosine kinase inhibitors (TKIs) demonstrate single agent activity in these patients but long-term efficacy is limited by emerging resistance in part due to acquired point mutations in the tyrosine kinase domains (TKD) of the FLT3 gene, most commonly found at D835 within the activation loop. Exportin 1 (XPO1) mediates the nucleo-cytoplasmic transport, and is overexpressed in AML cells with FLT3-ITD mutations (Kojima, Blood, 2013). We have reported that the clinically available XPO1 inhibitor selinexor effectively inhibits cell proliferation in both FLT3-ITD and FLT3-ITD/D835 mutated cells through upregulation of TP53 and blockade of c-Myc signaling (Tabe, ASH, 2017). In this study, we aimed at developing novel mechanism-based combination therapies for TKI-resistant FLT3-ITD cells with acquired TKD mutation. First we investigated the transcriptional changes associated with XPO1 and FLT3 inhibition in FLT3-ITD and FLT3-ITD/D835 mutated cells. We utilized paired isogenic FLT3-ITD or FLT3-ITD/D835Y transfected Ba/F3 cell lines and performed the cap analysis of gene expression (CAGE) that identifies and quantifies gene expression at the transcription start sites (TSS). FLT3-ITD cells are sensitive and FLT3-ITD/D835 cells are resistant to FLT3 inhibitor quizartinib. CAGE detected the shared upregulation of 3833 TSS genes induced by selinexor in FLT3-ITD and FLT3-ITD/D835 cells (FDR Based on these findings, we focused on Bcl-2 as a novel therapeutic target in FLT3-ITD/D835 cells, and investigated the anti-leukemia efficacy of combined inhibition of Bcl-2 and XPO1. We utilized the clinically available selective Bcl-2 inhibitor venetoclax and observed synergistic anti-proliferative and pro-apoptotic effects of venetoclax and selinexor in FLT3-ITD/D835 (Combination Index, CI: 0.39 and 0.45). Simultaneous inhibition of Bcl-2 by venetoclax and FLT3 by quizartinib showed no synergistic or additive effects in FLT3-ITD/D835 cells. Western blot analysis showed that the combination of venetoclax / selinexor reduced Bcl-2 and Mcl-1 in FLT3-ITD/D835 cells, and activated cleaved caspase3. In FLT3-ITD cells, the venetoclax / selinexor combination induced only small decrease of Bcl-2, did not change Mcl-1 and Bcl-xL, and increased cleaved caspase3. These changes were not observed in wt-FLT3 transfected Ba/F3 cells. Collectively, CAGE analysis of transcription start sites identified the primary mechanism underlying the synergistic activity of concomitant inhibition of Bcl-2 and XPO1 in FLT3-ITD/D835 cells as related to the regulation of Bcl-2, Mcl-1, Bcl-xL and c-Myc. These findings suggest that Bcl-2 inhibition by venetoclax combined with XPO1 inhibition by selinexor could be developed into a promising therapeutic strategy for TKI-resistant AML patients with FLT3-ITD and secondary acquired TKD mutations. Disclosures Konopleva: Stemline Therapeutics: Research Funding. Andreeff:AstraZeneca: Research Funding.

Hairy enhancer of split 1 (Hes1) is a basic helix-loop-helix transcriptional repressor that affects differentiation and often helps maintain cells in an immature state in various tissues. Here we show that retroviral expression of Hes1 immortalizes common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) in the presence of interleukin-3, conferring permanent replating capability on these cells. Whereas these cells did not develop myeloproliferative neoplasms when intravenously administered to irradiated mice, the combination of Hes1 and BCR-ABL in CMPs and GMPs caused acute leukemia resembling blast crisis of chronic myelogenous leukemia (CML), resulting in rapid death of the recipient mice. On the other hand, BCR-ABL alone caused CML-like disease when expressed in c-Kit-positive, Sca-1-positive, and lineage-negative hematopoietic stem cells (KSLs), but not committed progenitors CMPs or GMPs, as previously reported. Leukemic cells derived from Hes1 and BCR-ABL-expressing CMPs and GMPs were more immature than those derived from BCR-ABL-expressing KSLs. Intriguingly, Hes1 was highly expressed in 8 of 20 patients with CML in blast crisis, but not in the chronic phase, and dominant negative Hes1 retarded the growth of some CML cell lines expressing Hes1. These results suggest that Hes1 is a key molecule in blast crisis transition in CML.

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic stem cell disorders characterized by proliferation of one or more myeloid cell lineages. Some patients exhibit leukemic transformation (LT) by unknown mechanisms, and chemotherapy may increase the risk of LT. To clarify the molecular mechanisms of LT, gene alterations involved in LT from patients in the chronic phase (CP) of MPNs were identified. Among 18 patients who progressed to leukemia, AML1/RUNX1 mutations were detected in 5 patients at the LT but in none at the CP. To investigate the leukemogenic effect of AML1/RUNX1 mutants, the AML1D171N mutant was transduced into CD34+ cells from patients in the CP of MPNs. The D171N transduction resulted in proliferation of immature myeloid cells, enhanced self-renewal capacity, and proliferation of primitive progenitors. Taken together, these results indicate that AML1/RUNX1 point mutations may have a leukemogenic potential in MPN stem cells, and they may promote leukemic transformation in MPN.

Mutations in the gene encoding Fms-like tyrosine kinase-3 (FLT3) are found in approximately 30% of acute myeloid leukemia (AML) patients, with two distinct groups of mutations; the internal tandem duplication mutations in juxtamembrane domain (FLT3-ITD) and the point mutations within the tyrosine kinase domains (FLT3-TKD). The second-generation tyrosine kinase inhibitor (TKI) quizartinib which primarily targets an inactive (type II) kinase conformation, has demonstrated responses in relapsed/refractory FLT3-ITD AML patients. However, duration of response to quizartinib is limited largely by secondary mutations in FLT3-TKD, most commonly at D835 activation loop, emerging in at least 20% of patients. To elucidate the alterations of transcript signatures of quizartinib-resistant FLT3-ITD/D835 mutants in AML, we performed cap analysis of gene expression (CAGE) sequencing utilizing 11 FLT3-ITD and 6 FLT3-ITD/D835 primary AML cells with wild type NPM1. CAGE identifies and quantifies the 5' ends of capped mRNA transcripts, which enables the identification of transcription start sites (TSS) and allows investigating promoter structures necessary for gene expression. The TSS of genes altered in AML cells with FLT3-ITD/D835 compared with FLT3-ITD were mapped, and CAGE detected upregulation of 124 genes and downregulation of 25 genes in FLT3-ITD/D835 cells (FDR < 0.05). Specifically, several genes closely linked with activation of MAPK signaling (MAPK6, TCF4), AKT/mTOR signaling (NBN) and both (TNFAIP6) were upregulated in the D835 acquired cells. Expression of inflammatory response cytokine genes IL1B and IL8 were also increased in FLT3-ITD/D835 cells. The FLT related functional pathway analysis by KEGG ontology indicated that Ras pathway with its downstream Raf/MAPK and PI3K/AKT/mTOR signaling including transcription factor NFKB are the most significantly activated pathways in FLT3-ITD/D835 cells compared to FLT3-ITD cells. No additional activation of STAT5 pathway, a direct downstream signaling of mutated FLT3, was found in FLT3-ITD/D835 cells. To assess the underlying molecular basis of transcription signature, we performed an unbiased search for enriched sequence motifs using HOMER software. HOMER revealed upregulation of 80 motifs and downregulation of 51 motifs in AML cells with FLT3-ITD/D835 compared with FLT3-ITD (q < 0.05). Motifs of AP-1, JUN, CEBP, ATF and NFKB were substantially enriched in FLT3-ITD/D835 compared to FLT3-ITD cells. ETS, SPI-1 and RUNX1 were the most common binding motifs altered (both increased and decreased) by D835 mutation acquisition. Of note, AP-1 modulates inflammatory response and is tightly regulated by MAPK. To validate the transcriptional changes and the highlighted gene ontology results of AML primary samples, we utilized paired isogenic FLT3-ITD or FLT3-ITD/D835Y transfected Ba/F3 cell lines. As predicted, type II kinase inhibitor quizartinib was active against FLT3-ITD Ba/F3 cells (IC50; 0.6nM) but not FLT3-ITD/D835Y Ba/F3 cells (IC50; > 5000nM). Type I kinase inhibitor crenolanib was highly active against both FLT3-ITD (IC50; 2.1nM) and FLT3-ITD/D835Y cells (IC50; 16.4nM). Focusing on FLT related Ras pathway, we first investigated MAPK activation in these cells. Immunoblot analysis demonstrated substantial reduction of phospho- (p-) ERK after quizartinib (5nM) or crenolanib (50nM) treatment both in FLT3-ITD and FLT3-ITD/D835Y cells, irrespective of their susceptibility. We next examined p-S6, the target of PI3K/AKT/mTOR and MAPK signaling, and observed that crenolanib but not quizartinib successfully diminished p-S6 expression in FLT3-ITD/D835Y cells. FLT3-ITD/D835 cells showed higher baseline expression of NFkB regulator p-IkB compared to FLT3-ITD cells, which was not repressed by crenolanib. In conclusion, our study showed that the acquired D835 mutation in FLT3-ITD AML cells facilitates transcriptional network activation to induce proinflammatory genes through Ras-MAPK-NF-κB/AP-1 pathway and Ras-PI3K-AKT-mTOR signaling. This transcriptional modulation promotes oncogenic pro-survival and proliferative signaling in AML cells and defines differential sensitivity against Type I and Type II kinase inhibitors. Disclosures Shah: ARIAD: Research Funding; BMS: Research Funding; Daiichi-Sankyo: Research Funding; Pfizer: Research Funding; Plexxikon: Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership; Abbvie: Consultancy, Research Funding; Genentech: Consultancy, Research Funding; Stemline: Consultancy, Research Funding; Eli Lilly: Research Funding; Cellectis: Research Funding; Calithera: Research Funding.

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.

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.

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.

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