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TET proteins convert 5-methylcytosine to 5-hydroxymethylcytosine, an emerging dynamic epigenetic state of DNA that can influence transcription. Evidence has linked TET1 function to epigenetic repression complexes, yet mechanistic information, especially for the TET2 and TET3 proteins, remains limited. Here, we show a direct interaction of TET2 and TET3 with O-GlcNAc transferase (OGT). OGT does not appear to influence hmC activity, rather TET2 and TET3 promote OGT activity. TET2/3-OGT co-localize on chromatin at active promoters enriched for H3K4me3 and reduction of either TET2/3 or OGT activity results in a direct decrease in H3K4me3 and concomitant decreased transcription. Further, we show that Host Cell Factor 1 (HCF1), a component of the H3K4 methyltransferase SET1/COMPASS complex, is a specific GlcNAcylation target of TET2/3-OGT, and modification of HCF1 is important for the integrity of SET1/COMPASS. Additionally, we find both TET proteins and OGT activity promote binding of the SET1/COMPASS H3K4 methyltransferase, SETD1A, to chromatin. Finally, studies in Tet2 knockout mouse bone marrow tissue extend and support the data as decreases are observed of global GlcNAcylation and also of H3K4me3, notably at several key regulators of haematopoiesis. Together, our results unveil a step-wise model, involving TET-OGT interactions, promotion of GlcNAcylation, and influence on H3K4me3 via SET1/COMPASS, highlighting a novel means by which TETs may induce transcriptional activation., JOURNAL ARTICLE, FLWOA, SCOPUS: re.j, info:eu-repo/semantics/published

The first two murine models of IDH1(R132H) mutation provide mechanistic insights into transformation. In hematopoietic cells, inhibition of TET2 and histone demethylases leads to epigenetic alterations and accumulation of hematopoietic precursors. In the central nervous system, inhibition of collagen and prolyl hydroxylases lead to altered microenvironment and defective angiogenesis.

t(8;21) is the most frequent chromosomal abnormality in acute myeloid leukemia (AML), occurring in 4-12% of adult and 12-30% of pediatric patients. This translocation fuses the N-terminus of AML1 to nearly the entire coding region of ETO, resulting in expression of the fusion protein AML1-ETO. Observations that mice expressing AML1-ETO develop AML only if treated with mutagenic agents have suggested that AML1-ETO requires cooperating disease alleles for leukemogenesis. Consistent with this, t(8;21)+ AML patients harbor multiple genetic abnormalities. Recent exome/genome sequencing studies have expanded the number of known mutations in t(8;21)+ AML patients; however, efforts to distinguish driver from passenger mutations have yielded few cooperative events and the requirements for AML1-ETO leukemogenesis remain largely unknown. To better define the genetic landscape in AML and distinguish driver from passenger mutations, we compared the mutational profiles of two specific AML1-ETO driven mouse models of leukemia to the mutational profiles of human AML patients. We found that the mouse models of AML1-ETO driven AML were phenotypically similar in terms of their extensive latency, myeloid progenitor immunophenotype, and the acquired secondary disease alleles. The first model relies upon the expression of AML1-ETO in transplanted p21 null cells, while the second model relies upon the expression of AML1-ETO9a, a splice variant of AML1-ETO, in transplanted wild type cells. p21 is neither disrupted, nor methylated in t(8;21)+ AML. Because loss of p21 prevents the repair of damaged DNA, leukemogenesis may occur in this model once a cooperating disease allele has been naturally acquired in an AML1-ETO positive hematopoietic progenitor. AML1-ETO9a itself deregulates the expression of several DNA repair genes, suggesting that AML1-ETO9a could similarly facilitate the acquisition of a cooperating disease allele. When we compared the mutational landscape of these murine leukemias to AML patients, we found that the murine leukemias enrich for disease alleles present in human AML (hypergeometric p ≤ 4.26x10-20) and that there is a significant tendency for disease alleles mutated in both species to possess mutations in the same protein domain (hypergeometric p ≤ 4.23x10-3). Furthermore, domains mutated in both species were affected by recurrent mutations (Spearman correlation of domain p-values r = 0.53, p ≤ 2.73x10-8). While the frequency with which various protein classes were affected by mutations was significantly different in MLL-AF9 and AML1-ETO/AML1-ETO9a positive murine AML compared to MLL-fusion and t(8;21)+ positive human AML (p = 0.049), the protein classes targeted in AML1-ETO/AML1-ETO9a murine AML vs. human t(8;21)+ AML were not significantly different (p = 0.327). To identify disease alleles capable of cooperating with AML1-ETO, we determined that of the 424 genes mutated in both species, 38 of those genes were significantly mutated in human AML (Genome MuSiC SMG FDR ≤ 30%). These 38 genes represented 45 mouse orthologues, 38 of which were significantly mutated in AML1-ETO driven murine leukemias (FDR ≤ 10%). These 38 orthologues corresponded to 32 human orthologues, 3 of which were annotated in COSMIC as cancer-related genes: TET2, PTPN11, and THRAP3. Using retroviral transduction and transplantation experiments, we demonstrated that the expression of AML1-ETO in transplanted Tet2 null cells or PTPN11 D61Y cells was sufficient for leukemogenesis. At euthanasia, mice exhibited leukocytosis, anemia, thrombocytopenia, splenomegaly, and an expansion in the myeloid progenitor compartment. Our identification of Tet2 loss as a cooperating allele implicates mutations in epigenetic regulators as potential driving events in t(8;21)+ AML, while the discovery of PTPN11 D61Y solidifies the role of constitutive MAPK signaling in t(8;21)+ AML. This integrative genetic profiling approach allowed us to accurately predict cooperating events in t(8;21)+ AML in a robust and unbiased manner, while also revealing functional convergence in mouse and human AML. Collectively, these findings illustrate the power of integrating murine and human genomic profiling to identify functionally relevant disease alleles in AML. Disclosures Levine: CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy.

Myelodysplastic syndromes (MDS) are a group of clonal hematopoetic disorders marked by ineffective hematopoiesis, peripheral cytopenias, and an increased risk of transformation to acute myeloid leukemia. Multiple processes govern hematopoietic progenitor proliferation and natural differentiation into mature myeloid elements. Molecular events that disrupt any of these processes have the potential to lead to ineffective hematopoiesis and an MDS phenotype. Recent advances in genomic analysis have identified a number of new genes that may be involved. The molecular description of MDS will lead to better understanding, classification, and treatment of this disease.

Therapy-related Myeloid Neoplasms (tMN) comprise a poor risk subset of myelodysplastic syndromes and acute myelogenous leukemia, are increasing in incidence, and represent a serious complication following treatment for primary malignancies. In our previous study of 11 genes in 38 tMN patient samples, the data suggested that the mutational spectrum of tMN was distinct from de novo myeloid malignancies. To confirm this finding and to refine the tMN mutation profile, we investigated the mutation profile in samples from 88 patients and 28 genes using Sanger and next-generation sequencing approaches. We performed amplification using RainDance microfluidic PCR, followed by HiSeq next-generation sequencing. Mutations were identified using a modified pipeline for SNP calling employing variant detection software programs. Our study cohort included 88 patients, 71 of whom had complete clinical data for analyses. Patients had a history of epithelial and hematologic malignancies (³2 malignancies n=11; breast n=9; colorectal n=5; head and neck n=4; genital-urinary n=6; lung n=1; lymphoma n=25; melanoma n=2; ovarian n=1; sarcoma n=2; other, n=5). Treatment of primary cancers included chemotherapy alone (n=27), radiation alone (n=8), autologous stem cell transplant (n=11), or chemotherapy plus radiation (n=25). The median latency time between primary malignancy treatment and tMN diagnosis was 5.7yrs (range, 0.7 - 30.9 yrs). Median age at tMN diagnosis was 64yrs (range, 26 - 85 yrs). International Prognostic Scoring System (IPSS) risk group for MDS at tMN diagnosis were Low risk (n=8), Int-1 (n=11), Int-2 (n=30), High risk (n=9). We identified somatic mutations in 56 of 88 (64%) patients (83 patients were evaluated by next-generation sequencing and 5 by Sanger sequencing only). Mutations in TP53 were most common and were detected in 27/88 patients (30.7%), followed by mutations in TET2 in 12/88 (13.6%), DNMT3A in 9/88 (10.2%), NRAS in 8/83 (9.6%), KRAS in 5/83 (6.0%), and KIT in 5/83 (6.0%). Gene mutations detected at lower frequencies included those in ASXL1 in 5/88 (5.7%), RUNX1 in 2/83 (2.4%), EZH2 in 1/88 (1.1%), and SF3B1 in 1/88 (1.1%). Of the 58 patients with complete sequencing and FISH data, 4 patients exhibited biallelic somatic TP53 mutations and 3 patients had TP53 mutation combined with del 17p TP53 loss, demonstrating that 7 of 58 evaluable patients (12.1%) experienced biallelic loss of TP53. We also identified biallelic mutations in TET2 and DNMT3A in 2 separate patients. 25 patients had 2 or more concurrent somatic mutations. The highest number of co-occurring mutations in one patient was 5 mutations; 12 patients had 2 somatic mutations. The most common co-occurrence was TP53 and TET2, which was observed in 5 patients. All 5 ASXL1 mutations co-occurred with additional mutations. By analyzing variant allele frequencies (VAFs) in patients with multiple mutations, we observed that some tMN patients harbored multiple clones with distinct VAFs. This observation was also supported by the co-occurrence of typical class I driver mutations in the same patient, (e.g. KRAS 6% and NRAS 21% VAF; NRAS 9% and KIT 34%; NRAS 26% and KIT 9% in individual patients). The allele frequency data also suggested that ASXL1 is likely an early occurring mutation as the VAF was higher than for other co-occurring mutations (mean VAF ASXL1 50%, other co-occurring genes 23.5%, p Because of previous reports on the prognostic significance of point mutations in myeloid malignancies (e.g. TP53 in MDS and TET2 in AML), we tested the impact of individual mutations on prognosis. TP53 mutation or loss was associated with worse prognosis in tMN (OS 17.6 vs 25.2 mos, n=72, p Our data reveal that tMNs display distinct mutation profile compared to de novo disease (Fig B). TP53 mutations and loss are the most common abnormalities and predict for adverse outcome. Epigenetic modifier mutations also occur in tMNs and can serve as disease-initiating mutations. Collectively our results demonstrate that characterizing these mutation profiles can enhance our understanding of disease mechanisms in tMNs and may guide the development of future therapies for these difficult to treat disorders. Figure 1 Figure 1. Disclosures Sekeres: Celgene Corp.: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Boehringer Ingelheim: Membership on an entity's Board of Directors or advisory committees.

Somatic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) are observed in patients with acute myeloid leukemia (AML). Leukemia-associated IDH1/2 mutations result in aberrant accumulation of the oncometabolite 2-hydroxyglutarate (2-HG). The observation that IDH1/2 mutations are mutually exclusive with TET2 mutations led to the finding that IDH1/2-mutant production of 2-HG inhibits TET2 function and induces changes in DNA methylation. These data suggested that small molecule inhibition of mutant IDH enzymes might reverse the aberrant epigenetic remodeling of IDH-mutant leukemia cells and restore normal hematopoietic differentiation. We therefore investigated the in vivo efficacy of AG-221, a potent and selective mutant IDH2 inhibitor in early-phase clinical trials, in murine models of IDH2-mutant leukemia. We first assessed the impact of AG-221 on 2-HG production in hematopoietic cells expressing mutant IDH2-R140Q. AG-221 treatment (10mg/kg or 100mg/kg bid) led to a reduction in 2-HG in vivo (96.7% below pre-treatment levels). Moreover, AG-221 treatment restored megakaryocyte-erythroid progenitor (MEP) differentiation that is suppressed by mutant IDH2 expression in vivo (mean MEP% mean, 39% Veh vs 50% AG-221). We next investigated the impact of mutant IDH2 inhibition with AG-221 on DNA methylation in vivo. We used eRRBS, a bisulfite-based next-generation sequencing platform, to assess the effect of AG-221 therapy on DNA methylation. AG-221 or vehicle therapy treated LSK stem cells (lin- Sca+ c-Kit+) were sorted from mice expressing IDH2-R140Q and evaluated by eRRBS. AG-221 therapy reversed the effects of mutant IDH2; we observed a significant reduction in DNA methylation, including 180 genes that had 20 or more hypomethylated differentially methylated cytosines (DMCs) following treatment. 84 of these genes had reduced methylation at 10 or more DMCs in the gene promoter with AG-221 therapy compared to vehicle. Mutant IDH2 inhibition with AG-221 reversed aberrant methylation at many genes with a known role in hematopoietic proliferation and differentiation, including the master transcriptional factor RUNX1. We next assessed in vivo effects of the small-molecule IDH2-R140Q inhibitor in a mouse model of IDH2-mutant leukemia. We generated mice that simultaneously expressed a constitutive Flt3ITD knock-in allele and a conditional mutant IDH2R140Q knock-in allele. As reported recently using retroviral/transgenic models, Mx1-Cre IDH2R140QFlt3ITD developed fully penetrant, transplantable AML with expansion of c-Kit+ positive blasts in the peripheral blood, and widespread leukemic infiltration. AG-221 inhibited the serial replating capacity of IDH2R140QFlt3ITD expressing cells in vitro. We competitively transplanted IDH2R140QFlt3ITD AML cells and normal bone marrow cells into secondary recipients, and then assessed the effect of AG-221 therapy on leukemia in vivo and on disease burden. AG-221 (100mg/kg bid) treatment of mice engrafted with Mx1-Cre IDH2R140QFlt3ITD AML cells markedly reduced 2HG levels consistent with on target inhibition in vivo. AG-221 therapy induced differentiation of leukemic cells, with an increase in the CD11b+ population and a decrease in the c-Kit+ population in the peripheral blood at 2wks. We next assessed the impact of treatment with both AG-221 therapy with AC220, a potent, specific Flt3 inhibitor in late phase clinical trials. Combined IDH2R140Q and Flt3ITD inhibition resulted in a marked decrease in leukemic burden to vehicle-treated mice, with a significant reduction in leukemic cell chimerism in vivo in the setting of combined inhibition at 2 wks, (mean 45.2 fraction 88% veh, 73% AG-221, p These data demonstrate that AG-221 inhibits mutant IDH2-mediated 2-HG production in vivo and reverses the effects of mutant IDH2 on DNA methylation in mutant stem/progenitor cells. AG-221 induces differentiation and impairs self-renewal of IDH2-mutant leukemia cells, effects that are further enhanced by simultaneous inhibition of Flt3ITD. Clinical trials combining IDH2 inhibitors with other targeted AML therapies are warranted in order to increase therapeutic efficacy. Disclosures Intlekofer: Foundation Medicine, Inc: Consultancy. Thompson:Agios: Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Travins:Agios Pharmaceuticals: Employment, Stockholder Other. Straley:Agios: Employment, Equity Ownership. Gliser:Agios Pharmaceuticals: Employment, Stockholder Other. Yen:Agios: Employment, Equity Ownership. Levine:Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Our knowledge of the causes of brain tumors has steadily increased and is leading to a refined understanding of the signaling pathways that may be essential for tumor formation. At the same time, we are gaining insights into the developmental processes that regulate the formation of the diverse range of cell types in the normal brain. Interestingly, many of these pathways seem to overlap and suggest common mechanisms regulating tumor formation and cellular development. This overlap may also inform us about the nature of the cell of origin for different types of brain tumors. By appreciating the inter-relationship between tumor formation and development, we may be able to design new therapeutics targeting tumors for new modes of treatment.

Platelet-derived growth factor (PDGF) is expressed in many different tumors, but its precise roles in tumorigenesis remain to be fully defined. Here, we report on a mouse model that demonstrates dose-dependent effects of PDGF-B on glial tumorigenesis. By removing inhibitory regulatory elements in the PDGFB mRNA, we are able to substantially elevate its expression in tumor cells using a retroviral delivery system. This elevation in PDGF-B production results in tumors with shortened latency, increased cellularity, regions of necrosis, and general high-grade character. In addition, elevated PDGF-B in these tumors also mediates vascular smooth muscle cell recruitment that supports tumor angiogenesis. PDGF receptor (PDGFR) signaling appears to be required for the maintenance of these high-grade characteristics, because treatment of high-grade tumors with a small molecule inhibitor of PDGFR results in reversion to a lower grade tumor histology. Our data show that PDGFR signaling quantitatively regulates tumor grade and is required to sustain high-grade oligodendrogliomas.

In this study we identified and characterized the receptor related to the modulation of growth of human gastric cancer by gastrin. By performing receptor binding assays on human AGS gastric cancer cells with the selective CCK-B/gastrin receptor antagonist [3H]L-365,260, specific and saturable binding were determined. Binding was dependent on protein concentration, time, temperature, and the presence of protease inhibitors, and was located in the membrane fraction. Gastrin, as well as CCK, stimulated gastric cancer cell growth in a receptor-mediated fashion at a concentration consistent with the binding affinity. Receptor gene expression for the CCK-B/gastrin receptor, but not for the CCK-A receptor, was found by reverse transcription polymerase chain reaction. Receptor binding assays as well as transcriptional and growth studies provide evidence that gastrin-stimulated growth of human gastric cancer is mediated by CCK-B/gastrin-like receptors.

Although DNA methyltransferase Inhibitor (DNMTI) therapy demonstrates efficacy in patients (pts) with Myelodysplastic Syndromes (MDS), to date there are no clinical or genomic markers to select patients most likely to benefit from DNMTI therapy. Two recent retrospective studies have suggested TET2 mutations (muts) are associated with a higher response rate to 5-azacytidine (5-aza) and decitabine (DAC). We investigated the efficacy of DAC (20 mg/m2/day x 5 days) plus tretinoin (ATRA) given on days 10-19 of a 28-day schedule in MDS pts in a phase I/II study. We comprehensively analyzed pretreatment samples from 23 pts (15M,8F; median age 67, range, 44-78) treated on this study for alterations in known oncogenes to identify potential correlations between mut profile and clinical outcomes. The median follow-up was 41 mos (range, 10-66). IPSS cytogenetics (CG) risk categories included Good (n=8), Intermediate (n=5), and High risk (n=8) pts (2 karyotype failures). The overall IPSS risk categories were Int-1 (n=3), Int-2 (n=16), and High (n=4). Of 23 pts, 16 responded, including 2 CRs, 9 mCR+/-HI, 1 PR, and 4 HI. Genomic DNA and total RNA were isolated from all pt samples. Adaptor ligated sequencing libraries were captured by solution hybridization using two custom baitsets targeting 374 cancer-related genes and 24 genes frequently rearranged for DNA-seq, and 258 genes frequently rearranged for RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging >584X for DNA and >20,000,000 total pairs for RNA, to enable the sensitive and specific detection of genomic alterations. The median number of muts detected was 2 (range, 0-6), with 22/23 (96%) pts having at least 1 mut. The number of muts detected for 14 PB samples with ≤ 1% blasts was 2 (range, 0-6) compared to 4 muts (range, 1-5) for 8 pts with > 1% PB or BM blasts (unknown source for 1 sample). The most common mut identified in this cohort was TP53 (35%). Previous studies have shown that complex CGs and mut TP53 are strong poor prognostic indicators. Six pts with complex CGs and either mut or deleted TP53 had a median of 0 (range 0-2) additional muts, whereas pts with non-complex CGs who were wild-type (WT) TP53 (n=12) had a median of 3 (range 1-6) muts in other disease alleles (p=0.006). We identified mutually exclusive recurrent muts in RNA splicing factors including SRSF2 (26%), SF3B1 (17%), and ZRSR2 (9%), and recurrent muts in RUNX1 (22%), IDH1/2 (22%), TET2 (17%), ASXL1 (17%), PTPN11 (13%), CEBPA (13%), STAG2 (9%), and KRAS (9%). Thirteen additional muts were detected in single pts, and we identified novel candidate muts in DNA repair pathways (ATM, ATR, FANCA BRCA2), and in the recently described tumor suppressor FAT3. We also hypothesized that prognostication of pts with normal karyotype (NK) MDS may be aided by molecular stratification, as we and others have shown in AML. Of note, all 5 pts with IDH1/2 muts occurred in pts with NK, and in 4/5 cases they were associated with concomitant SRSF2 muts that were previously shown to be associated with adverse outcomes. We identified TET2 muts in 4 pts who were also ASXL1 WT. Notably, all 4 TET2 mutated/ASXL1 WT pts responded to DAC/ATRA therapy. The other 12 responders included 7 pts with TP53 mut/loss +/- other muts. No other muts were associated with a differential response to DAC/ATRA in this trial cohort. The strong association between TET2 muts and DAC/ATRA response, and more detailed mutational profiling of the 12 TET2 WT pts who responded to DAC/ATRA therapy is being investigated in additional pt samples and will be presented in more detail. Pts with TP53 muts showed a trend toward a worse survival which was not statistically significant; however, no other individual mut was associated with a differential survival in this small cohort. Conclusions The use of comprehensive mutational profiling employed in this study allowed us to identify muts in 25 genes in MDS pts, and allowed us to show that the overall mut frequency was higher in pts with a NK and without TP53 muts, thereby providing evidence for alternate disease initiation events and pathogenesis in these two genomically defined MDS subsets. All pts with mutated TET2 responded to DAC/ATRA, suggesting that pts with muts in TET2 are the best candidates for novel therapeutic trials which include DNMTI therapy. Disclosures: Wang: Foundation Medicine, Inc: Employment. Sanford:Foundation Medicine, Inc: Employment. Brennan:Foundation Medicine, Inc: Employment. Nahas:Foundation Medicine, Inc.: Employment. Otto:Foundation Medicine, Inc: Employment. Lipson:Foundation Medicine, Inc: Employment. Stephens:Foundation Medicine, Inc: Employment. Levine:Foundation Medicine, Inc: Consultancy.

Specific combinations of Acute Myeloid Leukemia (AML) somatic mutations are associated with distinct clinical and biologic features. However, in vivo models do not exist for the majority of common, poor-prognosis genotypes. Concurrent mutations in FLT3 and TET2 are associated with adverse outcome. We hypothesized that activating mutations in FLT3 would cooperate with inactivating mutations in TET2to induce AML in vivo, and that we could investigate AML pathogenesis and therapeutic response using a genetic model of this poor-risk AML genotype. To understand how these genes cooperate to induce AML, we generated Vav+Tet2fl/flFlt3-ITD mice, which resulted in fully penetrant, lethal disease in all recipient mice. Flow cytometric analysis revealed expansion of mac1+ cells in the peripheral blood, with progressive expansion of a c-Kit+, blast population which was apparent in the blood and bone marrow at 28 days, leading to lethal AML in all Vav+Tet2fl/flFlt3-ITD mice with a median survival of 12 months. Consistent with genetic data demonstrating most AML patients have monoallelic TET2 mutations, Vav+Tet2fl/+Flt3-ITD mice also develop AML, suggesting haploinsufficiency for Tet2 is sufficient to cooperate with the Flt3-ITD mutation to induce AML. All mice developed leukocytosis (median 85K/uL), splenomegaly (median 554mg) and hepatomegaly (median 2900mg) with evidence of extramedullary disease cell infiltration by leukemic blasts. Flow cytometric analysis of stem/progenitor populations revealed expansion of the granulocyte-macrophage progenitor (GMP) population and the lin- sca+ kit+ (LSK) stem cell population. Detailed analysis of the LSK population revealed a decrease in the LT-HSC population (LSK CD150+ CD48-) that was replaced by a monomorphic CD48+ CD150- multipotent progenitor population. Given previous studies have shown that LSK and GMP cells can contain leukemia stem cells (LSC) in other models of AML, we performed secondary transplant studies with LSK and GMP populations. LSK (CD48+ CD150-) cells, but not GMP cells, were able to induce disease in secondary and tertiary recipients in vivo. In order to assess the sensitivity of Tet2/Flt3-mutant AML and specifically the LSCs, to targeted therapies, we treated primary and transplanted mice with chronic administration of AC220, a FLT3 inhibitor in late-stage clinical trials. AC220 treatment inhibited FLT3 signaling in vivo, and reduced peripheral blood counts/splenomegaly. However, FLT3 inhibition did not reduce the proportion of AML cells in the bone marrow and peripheral blood. AC220 therapy in vivo reduced the proportion of GMP cells, but not LSK cells, demonstrating LSCs in this model are resistant to FLT3-targeted anti-leukemic therapy. We hypothesized that Tet2/Flt3-mutant LSCs possess a distinct epigenetic/transcriptional signature that contributes to leukemic cell self-renewal and therapeutic resistance. We performed RNA-seq using the Lifetech Proton sequencer to profile the expression landscape of Vav+Tet2fl/flFlt3-ITD mutant LSKs compared to normal stem cells. We were able to obtain an average of 62 million reads per sample. We identified over 400 genes differentially expressed in LSCs relative to normal hematopoietic stem cells (FC>2.5, padj Our data demonstrate that TET and FLT3 mutations can cooperate to induce AML in vivo, with a defined LSC population that is resistant to targeted therapies and characterized by site-specific changes in DNA methylation and gene expression. Current studies are aimed to assess the functional role of specific gene targets in LSC survival, and at defining therapeutic liabilities that can be translated to the clinical context. Disclosures: No relevant conflicts of interest to declare.

Abstract 308 Loss-of-function somatic mutations in Addition of Sex Combs Like 1 (ASXL1) occur in a subset of patients with myeloid malignancies, most commonly in myelodysplastic syndrome (MDS). In addition, germline mutations in ASXL1 are observed in patients with Bohring-Opitz Syndrome, a developmental disorder characterized by neurologic and skeletal abnormalities. Previous work characterizing a constitutive gene-trap Asxl1 model was notable for significant perinatal lethality of Asxl1−/− mice with evidence of an overt hematopoietic phenotype in surviving mice. Given the lack of detailed data on the hematopoietic phenotype of Asxl1 loss in vivo, we created a murine model for conditional knockout of Asxl1 for tissue- and temporal-specific deletion with 4 different Cre recombinase alleles. We crossed our conditional Asxl1 allele to EIIa-cre to generate mice with germline loss of Asxl1; this revealed embryonic lethality of mice with homozygous germline Asxl1 loss. Mice with heterozygous germline deletion of Asxl1 were viable and fertile although a significant proportion had cranio-facial abnormalities. Timed-sacrifice of pregnant mothers from heterozygous EIIa-cre Asxl1+/− crosses revealed that homozygous Asxl1−/− pups survived to 18–20.5 days post-coitus, with all embryos characterized by craniofacial abnormalities including anopthalmia, microcephaly, cleft palates, and mandibular malformations similar to those seen with Bohring-Opitz syndrome. We then generated mice with conditional Asxl1 deletion in the hematopoietic compartment by crossing floxed mice with Vav-cre recombinase mice for hematopoietic deletion at birth, and with Mx1-cre mice for inducible deletion of Asxl1. Deletion of Asxl1 in both systems resulted in complete loss of Asxl1 as assessed by Western blot. In both models we noted progressive development of leukopenia and anemia in mice with homozygous loss of Asxl1 compared to age-matched controls (Figure). This was associated with extramedullary hematopoiesis and morphologic evidence of myeloid, erythroid, and megakaryocytic dysplasia, similar to that observed in human MDS (Figure). Flow cytometry revealed a progressive increase in immunophenotypically-defined multipotent progenitors (Lineage-negative, Sca1+, cKIT+, CD150-, CD48+ cells) in bone marrow and spleen of knockout (KO) mice. Characterization of HSC's from 6-week old Vav-cre Asxl1−/− mice using serial competitive transplantation revealed a competitive disadvantage with transplantation of SLAM+ cells from 6-week old KO mice. A subset of Vav-cre Asxl1−/− mice developed a transplantable, monocytic-like leukemia beyond 6 months of age which was characterized by both proliferative and dysplastic features. Restriction of Asxl1 deletion to the megakaryocytic compartment using Pf4-cre revealed an age-specific decline in platelet production in Pf4-cre Asxl1−/− mice compared to age-matched controls, and a concomitant increase in bone marrow megakaryocytes in KO mice. Consistent with previous in vitro studies, hematopoietic-specific deletion of Asxl1 was associated with a marked decrease in H3K27me3 as assessed by histone Western blots of murine splenocytes and H3K27me3 ChIP-Seq of myeloid progenitors from 1 year-old Vav-cre Asxl1−/− mice compared with littermate controls. RNA-Seq from myeloid progenitors was integrated with H3K4me3/K27me3 ChIP-Seq to identify gene targets of Asxl1 loss associated with myelodysplasia. Given that MDS patients frequently present with concomitant ASXL1 and TET2 mutations, Vav-cre Asxl1fl/fl mice were also crossed with mice bearing floxed alleles of Tet2. Mice with combined hematopoietic-specific deletion of both Asxl1 and Tet2 developed bone marrow failure and hastened death compared with age-matched, single-gene deleted counterparts at 25 to 40 weeks of age. The findings here reveal that hematopoietic-specific deletion of Asxl1 results in progressive ineffective hematopoiesis, an increase in hematopoietic progenitors, a propensity for leukemic transformation with age, and morphologic features of human MDS (Figure). Combining Asxl1 deletion with loss of Tet2, a combined genotype present in at least 5% of patients with de novo MDS, resulted in shorter latency, progressive MDS. These data suggest that these two models represent novel, genetically accurate models of MDS amenable to epigenomic, functional and preclinical therapeutic studies. Disclosures: No relevant conflicts of interest to declare.

Abstract 2802 Background: Therapy-related myelodysplastic syndromes and acute myelogenous leukemia (tMDS/AML) comprise a poor-risk subset of MDS/AML and are associated with a higher rate of cytogenetic abnormalities and complex karyotypes. Large scale mutation profiling efforts in de novo MDS have identified mutations that correlate with clinical features but such mutations have not been investigated in tMDS/AML. Methods: Cryopreserved (mononuclear cell fractions) bone marrow and peripheral blood samples from tMDS/AML patients were analyzed. Lymphocytes were depleted from these fractions by either fluorescence-activated cell sorting or affinity column selection. Genomic DNA was subjected to high throughput PCR and sequenced for TP53, TET2, DNMT3a, ASXL1, IDH1, IDH2, SF3B1, EZH2, EED, SUZ12, and RBBP4. Somatic mutations were validated by comparison to lymphocyte DNA controls. Patient selection was based on sample availability for tMDS/AML patients with untreated or active, previously treated disease. Results were correlated with clinical outcomes, cytogenetic profiles, and response to therapy. Results: Samples from 38 patients (20 males, 18 females) with tMDS/AML were analyzed. For their primary malignancy (≥ 2 malignancies, n=3; AML, n=1; breast, n=4; colorectal, n=3; Hodgkins or composite lymphoma, n=3; gastric, n=1; melanoma, n=2; NHL, n=12; ovarian, n=1; prostate, n=3; sarcoma, n=2; thyroid, n=3), patients received chemotherapy alone (n=17), radiation alone (n=4), radioactive iodine alone (n=1), chemotherapy plus radiotherapy (n=13), radiotherapy plus radioactive iodine (n=1), chemotherapy plus radiotherapy plus radioactive iodine (n=2). Median latency time between primary malignancy treatment and MDS diagnosis date was 5.68yrs (range, 0.71–30.88). Median age at MDS diagnosis was 65yrs (range, 34–83). Not surprising was the finding that of the 35 MDS patients with complete IPSS parameter data most were IPSS Int-2 (45%) or High Risk (13%) compared to Low (11%) or Int-1 Risk (24%) disease, and in contrast to expected proportions at diagnosis for de novo MDS. WHO Classifications were: RA, n=3; RCMD, n=11; RAEB-1, n=7; RAEB-2, n=7; AML, n=3; CMML-1, n=1; MDS-U, n=3; Unknown, n=3. The median survival was 16.8mo. Median time between MDS diagnosis and sample procurement was 3.0mo (range, 0–57.2) during which 2 patients progressed to AML, 11 received a DNA methyltransferase inhibitor (DNMTI), 1 induction chemotherapy, and 2 DNMTI plus induction. We identified somatic mutations in 15/38 (39.5%) patients. Including cytogenetic abnormalities, we identified somatic alterations in 34/38 (88%) of the patients in this cohort. TP53 mutations were most common, detected in 8/38 (21%) patients, followed by TET2 in 4/38 (10.5%), DNMT3a in 3/38 (7.9%), ASXL1 in 1/38 (2.6%), IDH1 in 1/38 (2.6%), and EZH2 in 1/38 (2.6%). No IDH2, SF3B1, EED, SUZ12, or RBBP4 mutations were detected. Only 2 patients had concurrent point mutations (one patient with TP53/TET2/DNMT3 mutations and one patient with TET2/EZH2 mutations). 7/38 (18.4%) patients had TP53 loss by FISH analysis or exhibited loss of chromosome 17/17p; 2 of these patients showed concurrent TP53 point mutations consistent with biallelic TP53 loss. 12/13 patients with TP53 abnormalities (point mutations, loss of TP53 by FISH, or loss of chromosome 17) had IPSS Poor Risk cytogenetics and 11/13 patients had abnormalities in chromosome 5 (del 5q or monosomy 5). In patients without TP53 abnormalities, 9/25 had Poor Risk cytogenetics and 5/25 had abnormalities in chromosome 5. The median survival for patients with TP53 abnormalities was 9.7mo compared to 64.4mo for patients with no TP53 abnormalities (p=0.0043). Of the 13 patients with TP53 abnormalities, 12 received treatment for tMDS/AML. 3/12 were refractory to the first line of therapy, and 7 were unable to receive an adequate course of therapy with hypomethylating agents due to toxicity or progressive disease. Conclusions: TP53 point mutations are more common in tMDS/AML than in de novo MDS (7.5%, Bejar et al) while incidence of TET2 and ASXL1 mutations were lower in tMDS/AML compared to de novo MDS (20.5% and 14.4%, respectively). TP53 and TET2 point mutations were more strongly associated with exposure to prior chemotherapy, but not with exposure to radiation therapy. Taken together, these data demonstrate that TP53 mutations are common in tMDS/AML and are correlated with adverse clinical outcomes. Disclosures: No relevant conflicts of interest to declare.

Abstract 614 Myeloproliferative neoplasms (MPN) are clonal disorders of hematopoiesis but the mechanisms of clonal dominance in these diseases are poorly understood. The JAK2V617F mutation is found in the majority of patients with MPN and is sufficient to confer the MPN phenotype. We recently described a Jak2V617F knock-in MPN model in which the mutation was expressed from the endogenous murine Jak2 promoter and the disease phenotype closely recapitulated human polycythemia vera (PV). In the model we found that the MPN-initiating population is contained within the CD150+CD48− LineagelowSca1+cKithigh(LSK) long-term hematopoietic stem cell (HSC) compartment, and showed that the MPN is cell autonomous and serially transplantable. In long-term competitive transplantation experiments we found that the Jak2V617F CD150+CD48− LSK population demonstrates gradual clonal expansion over time and we are now investigating the regulation of this primitive HSC population in vivo. Erythropoietin (EPO) signaling is reported to be the fundamental defect in polycythemia vera (PV). The JAK2V617F mutation is present in 95% PV patients and can be detected in the HSC compartment. We evaluated the role of EPO signaling in the JAK2V617F mutant HSC compartment using a conditional Jak2V617F knock-in murine model. Floxed Jak2+/VF mice were crossed with Vav Cre or erythropoietin receptor GFP Cre (ErGFPcre) mice resulting in Jak2V617F expression in all hematopoietic lineages or in erythroid restricted Jak2V617F expression respectively. Jak2V617F-ErGFPcre mice demonstrated elevated hematocrit, expanded committed erythroid progenitors and suppressed EPO levels but had an attenuated MPN phenotype as compared with Jak2V617F-Vavcre mice. Notably, the hematopoietic stem and progenitor cell (HSPC) compartment was not expanded in Jak2V617F-ErGFPcre mice and HSCs from both Jak2V617F-Vavcre and Jak2V617F-ErGFPcre mice did not activate phosphoStat5 signaling in response to EPO stimulation. These results indicate that expression of Jak2V617F in the HSC compartment is required for development of a full MPN phenotype and suggest that cytokine receptors other than the EPO receptor are important in mediating clonal dominance within the HSC compartment in JAK2V617F mediated MPN. TET2 is one of three TET gene family members that appear to play a role in DNA demethylation. Acquired TET2 deletions and loss-of-function mutations have been found across a broad spectrum of myeloid malignancies indicating that TET2 may drive a common pathogenic step in myeloid cancers, such as the establishment and/or enhancement of clonal dominance. Tet2 null HSCs have recently been shown to have a competitive repopulating advantage over wild-type HSCs in murine transplantation assays. TET2 loss-of-function mutations are found in approximately 12% of MPN patients, are often found co-mutated with JAK2V617F and have been associated with leukemic transformation of MPN. To evaluate the effects of loss of Tet2 function on the self-renewal and differentiation of Jak2V617F mutant HSCs, we crossed Jak2V617F knock-in mice with Tet2 conditional knockout mice. At 6 weeks of age, Jak2V617F/Tet2 +/− Vav Cre mice do not demonstrate significant differences in MPN phenotype as compared with Jak2V617F/Tet2+/+ Vav Cre mice, in terms of peripheral blood counts, hematopoietic stem and progenitor cell numbers or in colony formation. Additional studies using older mice and Jak2V617F/Tet2−/− Vav Cre animals are underway to further investigate the impact of loss of Tet2 function on Jak2V617F mutant HSCs. Understanding the mechanisms that contribute to clonal dominance in MPN will help facilitate the development of strategies to selectively target MPN stem cells therapeutically, and thereby advance the treatment of MPN patients. Disclosures: No relevant conflicts of interest to declare.

Abstract 405 Somatic mutations in ASXL1 have been identified in patients with myeloid malignancies and are associated with worsened overall survival in AML and MDS patients. However the mechanisms of myeloid transformation of ASXL1 mutations had not been delineated. We therefore performed extensive in vitro and in vivo studies to assess the functional implications of ASXL1 mutations in the hematopoietic compartment. Transcriptional and Western blot analysis demonstrated loss of ASXL1 protein in primary leukemia samples with endogenous ASXL1 mutations indicating that these mutations are loss-of-function disease alleles. Further, ASXL1 depletion by shRNA in normal and malignant hematopoietic cells leads to robust upregulation of a set of genes including the posterior HOXA cluster (HoxA5-HoxA13). Increased HoxA gene expression was confirmed in human hematopoietic stem progenitor cells targeted with ASXL1 siRNA and in mice with conditional deletion of Asxl1 in the hematopoietic compartment. Previous studies in Drosophila had revealed that Asxl forms the polycomb-repressive deubiquitinase (PR-DUB) complex with BAP1, which normally opposes the function of polycomb repressive complex 1 (PRC1) by removing H2AK119 ubiquitination. We verified that wild-type, but not mutant ASXL1 associates with BAP1 in co-immunoprecipitation studies. However, BAP1 depletion in hematopoietic cells did not result in significant changes in HoxA gene expression, suggesting that ASXL1 regulates gene expression in hematopoietic cells independent of its role in the PR-DUB complex. We therefore performed CHIP sequencing for known activating and repressive chromatin marks and histone mass spectrometry to elucidate the genome-wide effects of ASXL1 loss on chromatin state in hematopoietic cells. This allowed us to show that ASXL1 loss resulted in genome-wide loss of the transcriptionally repressive mark H3K27me3 in hematopoietic cells and primary patient samples with ASXL1 mutations. These data were supported by western blot analysis and histone mass spectrometry demonstrating a significant loss of H3K27 trimethylation in ASXL1-mutant cells. Moreover, ASXL1 mutations in primary leukemia samples are characterized by loss of H3K27 trimethylation at the HoxA locus. These data led us to hypothesize that ASXL1 interacts with the PRC2 complex; co-immunoprecipitation studies revealed that ASXL1 associates with members of the PRC2 complex including EZH2 and SUZ12 but not with the PRC1 repressive complex. Importantly, ASXL1 downregulation resulted in loss of EZH2 recruitment to the HOXA locus indicating a role of ASXL1 in recruiting the PRC2 complex to known leukemogenic loci. We next assessed the effects of ASXL1 loss in vivo by generating a conditional knock-out model of ASXL1 and also by employing shRNA to deplete ASXL1 in hematopoietic cells expressing the NRASG12D oncogene. Consonant with the in vitro data, we observed HOXA9 overexpression with ASXL1 loss/depletion in vivo. Preliminary analysis reveals that conditional, hematopoietic specific ASXL1-knockout (ASXL1fl/fl Vav-Cre) mice are characterized by progressive expansion of LSK and myeloid progenitor cells in mice less than 6 months of age. After 6 months of age a significant proportion of ASXL1fl/fl Vav-Cre mice developed leukocytosis, anemia, thrombocytopenia, and splenomegaly; pathologic analysis of tissues revealed a phenotype consistent with myelodysplasia with myeloproliferative features. Moreover, loss of ASXL1 in cooperation with expression of NRasG12D resulted in impaired survival, increased myeloproliferation, and progressive anemia consistent with MPN/MDS in vivo. Taken together, these results reveal that ASXL1 mutations result in a loss-of-function and suggest a specific role for ASXL1 in epigenetic regulation of gene expression by facilitating PRC2-mediated transcriptional repression of known leukemic oncogenes. Moreover, our in vivo data validate the importance of ASXL1 mutations in the pathogenesis of myeloid malignancies and provide insight into how mutations that inhibit PRC2 function contribute to myeloid transformation through epigenetic dysregulation of specific target genes. Disclosures: Carroll: Agios Pharmaceuticals: Research Funding; TetraLogic Pharmaceuticals: Research Funding; Sanofi Aventis Corporation: Research Funding; Glaxo Smith Kline, Inc.: Research Funding.

SummaryRecurrent somatic ASXL1 mutations occur in patients with myelodysplastic syndrome, myeloproliferative neoplasms, and acute myeloid leukemia, and are associated with adverse outcome. Despite the genetic and clinical data implicating ASXL1 mutations in myeloid malignancies, the mechanisms of transformation by ASXL1 mutations are not understood. Here, we identify that ASXL1 mutations result in loss of polycomb repressive complex 2 (PRC2)-mediated histone H3 lysine 27 (H3K27) tri-methylation. Through integration of microarray data with genome-wide histone modification ChIP-Seq data, we identify targets of ASXL1 repression, including the posterior HOXA cluster that is known to contribute to myeloid transformation. We demonstrate that ASXL1 associates with the PRC2, and that loss of ASXL1 in vivo collaborates with NRASG12D to promote myeloid leukemogenesis.

SummaryHematopoietic stem cell differentiation involves the silencing of self-renewal genes and induction of a specific transcriptional program. Identification of multiple covalent cytosine modifications raises the question of how these derivatized bases influence stem cell commitment. Using a replicative primary human hematopoietic stem/progenitor cell differentiation system, we demonstrate dynamic changes of 5-hydroxymethylcytosine (5-hmC) during stem cell commitment and differentiation to the erythroid lineage. Genomic loci that maintain or gain 5-hmC density throughout erythroid differentiation contain binding sites for erythroid transcription factors and several factors not previously recognized as erythroid-specific factors. The functional importance of 5-hmC was demonstrated by impaired erythroid differentiation, with augmentation of myeloid potential, and disrupted 5-hmC patterning in leukemia patient-derived CD34+ stem/early progenitor cells with TET methylcytosine dioxygenase 2 (TET2) mutations. Thus, chemical conjugation and affinity purification of 5-hmC-enriched sequences followed by sequencing serve as resources for deciphering functional implications for gene expression during stem cell commitment and differentiation along a particular lineage.

Recent findings suggest that Notch signaling is active in brain tumors and stem cells, and that stem cells or cells with progenitor characteristics contribute to brain tumor formation. These stem cells are marked by expression of several markers, including nestin, an intermediate filament protein. We have studied how the Notch signaling pathway affects nestin expression in brain tumors. We find that Notch receptors and ligands are expressed in vitro and in human samples of glioblastomas, the highest grade of malignant gliomas. In culture, Notch activity activates the nestin promoter. Activation of the Notch pathway also occurs in a glioblastoma multiforme mouse model induced by Kras, with translational regulation playing a role in Notch expression. Combined activation of Notch and Kras in wild-type nestin-expressing cells leads to their expansion within the subventricular zone and retention of proliferation and nestin expression. However, activation of Notch alone is unable to induce this cellular expansion. These data suggest that Notch may have a contributing role in the stem-like character of glioma cells.