Tet2 Loss Research Articles

TET2 mutations predict response to hypomethylating agents in myelodysplastic syndrome patients

AbstractOnly a minority of myelodysplastic syndrome (MDS) patients respond to hypomethylating agents (HMAs), but strong predictors of response are unknown. We sequenced 40 recurrently mutated myeloid malignancy genes in tumor DNA from 213 MDS patients collected before treatment with azacitidine (AZA) or decitabine (DEC). Mutations were examined for association with response and overall survival. The overall response rate of 47% was not different between agents. Clonal TET2 mutations predicted response (odds ratio [OR] 1.99, P = .036) when subclones unlikely to be detected by Sanger sequencing (allele fraction <10%) were treated as wild-type (WT). Response rates were highest in the subset of TET2 mutant patients without clonal ASXL1 mutations (OR 3.65, P = .009). Mutations of TP53 (hazard ratio [HR] 2.01, P = .002) and PTPN11 (HR 3.26, P = .006) were associated with shorter overall survival but not drug response. Murine-competitive bone marrow transplantation followed by treatment with AZA demonstrated that Tet2-null cells have an engraftment advantage over Tet2-WT cells. AZA significantly decreased this advantage for Tet2-null cells (P = .002) but not Tet2-WT cells (P = .212). Overall, Tet2 loss appears to sensitize cells to treatment with AZA in vivo, and TET2 mutations can identify patients more likely to respond to HMAs.

Abstract 460: Base resolution epigenomic analysis reveals a role for Tet2 in modulating enhancer activity

Abstract Cytosine methylation (5mC) is a well-established epigenetic mechanism essential for genomic imprinting, silencing of retrotransposons, and lineage-specific expression of developmental genes. This epigenetic mark is extensively remodeled during mammalian development through the action of several DNA methyltransferases (DNMT) and the Ten-Eleven-Translocation (TET) family of protein dioxygenases. TET proteins function in DNA de-methylation by catalyzing the oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) and other oxidized intermediates. Recurrent loss of function mutations of Tet2 are found in 27% of acute myeloid leukemia patients. In mouse models, TET2 loss is sufficient to cause myeloid malignancy through defects in hematopoietic stem cell differentiation. However, it remains unclear how TET2-dependent de-methylation regulates differentiation, a process often disrupted in tumorigenesis. Base-resolution methylomes have revealed that 5mC is dynamically regulated in a cell-type specific manner. Notably, DNA methylation is depleted at distal regulatory elements such as enhancers, where reduction of 5mC is correlated with the activity of these sequences. 5hmC is also significantly enriched at distal cis-regulatory sequences, suggesting that dynamic DNA methylation at these regions is likely mediated by interplays between DNMT-mediated methylation and TET-mediated de-methylation. Despite these discoveries, several open questions remain. First, are the different TET proteins functionally redundant? Does Tet2 uniquely modulate the methylome? If so, how? Second, what is the precise role of DNA hypomethylation and hydroxymethylation at enhancers? Do these modifications play active roles in gene regulation? We have employed genome-wide technologies to examine the effects of loss of Tet1 and Tet2 genes on DNA methylation, chromatin modification and gene expression landscapes in mouse embryonic stem cells. We generated base resolution DNA methylation and hydroxymethylation maps in wild type and Tet1 or Tet2 mutant mES cells, and determined the genomic regions where dynamic DNA methylation depends on either Tet1 or Tet2. Loss of Tet2 leads to dramatic reduction of DNA hydroxymethylation genome-wide, and local hypermethylation predominantly at enhancers. Furthermore, we observed reduced activities of re-methylated enhancers in Tet2-/- mES cells, supporting an active role for Tet2 and DNA hypomethylation at enhancers. Together, our results clarify the distinct functions of Tet1 and Tet2 in mammalian cells and highlight an active role of DNA hypomethylation at enhancers. Citation Format: Gary Hon, Chun-Xiao Song, Fulai Jin, Siddarth Selvaraj, Ah Young Lee, Chia-an Yen, Zhen Ye, Shi-Qing Mao, Bang-An Wang, Samantha Kuan, Lee Edsall, Boxuan Simen Zhao, Guo-Liang Xu, Chuan He, Bing Ren. Base resolution epigenomic analysis reveals a role for Tet2 in modulating enhancer activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 460. doi:10.1158/1538-7445.AM2014-460

Abstract 454: TET2 alterations facilitate progression of metastatic prostate cancer

Abstract Metastatic prostate cancer (mPCa) is a highly lethal disease and molecular markers identifying disease subtypes and/or therapeutic targets are needed. We sequenced the exomes of five metastatic tumors and healthy kidney tissue from an index patient with castration resistant mPCa to identify lesions associated with disease progression and metastasis. A somatic missense TET2 alteration predicted to be deleterious to the protein was observed at the metastatic stage of disease. We sequenced TET2 in additional mPCa tumors and PCa cell lines and detected somatic missense alterations, frequent LOH, and a frameshift truncation in PCa cell line, DU-145, derived from a mPCa brain tumor. We also observed complex combinations of frequent and rare germline missense SNPs, which may contribute to a previously described association of TET2 with PCa risk by GWAS. In this study, we confirm transcription factor binding to TET2 alleles associated with disease by GWAS. We examine TET2 binding partners in prostate cells by affinity purification, mass spectrometry, and forward and reverse immunoprecipitation. We confirmed a protein complex containing TET2, OGT, and two transcriptional co-activators/splicing factors implicated in receptor-mediated gene expression. Functional analysis shows TET2 loss alters expression of a unique set of genes, increases cell proliferation, and therefore is a driver of PCa. TET2 loss may facilitate progression to castration resistant mPCa and appears to define a subset of cases with metastatic disease. Citation Format: Michael L. Nickerson, Sudipto Das, Hong Lou, Hongchuan Li, Sevilay Turan, Kate Im, Stephen Anderson, Thorkell Andresson, Michael Dean. TET2 alterations facilitate progression of metastatic prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 454. doi:10.1158/1538-7445.AM2014-454

TET2 Inhibits Differentiation of Embryonic Stem Cells but Does Not Overcome Methylation-Induced Gene Silencing.

TET2 is a methylcytosine dioxygenase that is frequently mutated in myeloid malignancies, notably myelodysplasia and acute myeloid leukemia. TET2 catalyses the conversion of 5′-methylcytosine to 5′-hydroxymethylcytosine within DNA and has been implicated in the process of genomic demethylation. However, the mechanism by which TET2 loss of function results in hematopoietic dysplasia and leukemogenesis is poorly understood. Here, we show that TET2 is expressed in undifferentiated embryonic stem cells and that its knockdown results in reduction of 5′-hydroxymethylcytosine in genomic DNA. We also present DNA methylation data from bone marrow samples obtained from patients with TET2-mutated myelodysplasia. Based on these findings, we sought to identify the role of TET2 in regulating pluripotency and differentiation. We show that overexpression of TET2 in a stably integrated transgene leads to increased alkaline phosphatase expression in differentiating ES cells and impaired differentiation in methylcellulose culture. We speculate that this effect is due to TET2-mediated expression of stem cell genes in ES cells via hydroxylation of 5′-methylcytosines at key promoter sequences within genomic DNA. This leads to relative hypomethylation of gene promoters as 5′-hydroxymethylcytosine is not a substrate for DNMT1-mediated maintenance methylation. We sought to test this hypothesis by cotransfecting the TET2 gene with methylated reporter genes. The results of these experiments are presented.

TET1 exerts its tumor suppressor function by interacting with p53-EZH2 pathway in gastric cancer.

TET1 protein is reported to suppress cancer invasion and metastasis in prostate and breast cancer while EZH2, a polycomb group protein, has been identified as an oncogene in many types of cancers including gastric cancer. Here we report that there is an inverse relation of the expression pattern of TET1 and EZH2 in both normal gastric mucosa and gastric cancer. In gastric mucosa, EZH2 is selectively expressed in the proliferating neck cells while TET1 and 5-hydroxymethyl-cytosine (5-hmc) exhibit very low expression in the neck cells. In contrast, TET1 and 5-hmc expression is high in gastric glandular epithelium while EZH2 expression is absent in this cell population. On the other hand, in proliferating Ki67-positive gastric cancer cells, EZH2 is highly expressed while TET1 and 5-hmc expression is significantly down-regulated. When the mouse homologue of human TET1 protein Tet1 is overexpressed in a gastric cancer cell line MGC-803, we observed the dramatically down-regulation of EZH2 in one-third of the Tet1 overexpressed cells. In addition, Tet1 overexpressing cells also lost the H3K27 trimethylation mark and the cell proliferation protein Ki67. Furthermore, Tet1 overexpression induced p53 tumor suppressor protein. The increase of p53 protein level is accompanied by the phosphorylation of p53 by activated DNA-PK. Together, these results suggested a mechanism by which TET1 suppresses cancer formation by coupling DNA demethylation with DNA-PK activation of p53 and suppression of oncogenic protein EZH2. Conversely, loss of TET1 and 5-hmc expression might contribute to EZH2 up-regulation during gastric cancer development.

Loss of function of TET2 cooperates with constitutively active KIT in murine and human models of mastocytosis.

Systemic Mastocytosis (SM) is a clonal disease characterized by abnormal accumulation of mast cells in multiple organs. Clinical presentations of the disease vary widely from indolent to aggressive forms, and to the exceedingly rare mast cell leukemia. Current treatment of aggressive SM and mast cell leukemia is unsatisfactory. An imatinib-resistant activating mutation of the receptor tyrosine kinase KIT (KIT D816V) is most frequently present in transformed mast cells and is associated with all clinical forms of the disease. Thus the etiology of the variable clinical aggressiveness of abnormal mast cells in SM is unclear. TET2 appears to be mutated in primary human samples in aggressive types of SM, suggesting a possible role in disease modification. In this report, we demonstrate the cooperation between KIT D816V and loss of function of TET2 in mast cell transformation and demonstrate a more aggressive phenotype in a murine model of SM when both mutations are present in progenitor cells. We exploit these findings to validate a combination treatment strategy targeting the epigenetic deregulation caused by loss of TET2 and the constitutively active KIT receptor for the treatment of patients with aggressive SM.

A Global Increase in 5‐Hydroxymethylcytosine Levels Marks Osteoarthritic Chondrocytes

To investigate the role of the newly discovered epigenetic mark 5-hydroxymethylcytosine (5hmC) and its regulators in altered gene expression in osteoarthritis (OA). Cartilage was obtained from OA patients undergoing total knee arthroplasty and from control patients undergoing anterior cruciate ligament reconstruction. Global levels of 5hmC and 5-methylcytosine (5mC) were investigated using immunoblotting, enzyme-linked immunosorbent assays, and cellular staining. Gene expression changes were monitored by quantitative polymerase chain reaction (PCR) analysis. Levels of locus-specific 5hmC and 5mC at CpG sites in the matrix metalloproteinase 1 (MMP-1), MMP-3, ADAMTS-5, and hypoxanthine guanine phosphoribosyltransferase 1 (HPRT-1) promoters were quantified using a glucosylation and enzyme digestion-based method followed by quantitative PCR analysis. Global and locus-specific 5hmC levels and gene expression changes were monitored in normal chondrocytes stimulated with inflammatory cytokines to identify the effect of joint inflammation. A global 5-6-fold increase in 5hmC concomitant with a loss of TET1 was observed in human OA chondrocytes compared to normal chondrocytes. Enrichment of 5hmC was observed in promoters of enzymes critical to OA pathology, MMP-1 and MMP-3. Short-term treatment of normal chondrocytes with inflammatory cytokines induced a rapid decrease in TET1 expression but no global or locus-specific 5hmC enrichment. This study provides the first evidence of an epigenetic imbalance of the 5hmC homeostasis in OA leading to TET1 down-regulation and 5hmC accumulation. Our experiments identify 5hmC and its regulators as potential diagnostic and therapeutic targets in OA.

Concurrent Loss Of Ezh2 and Tet2 Cooperates In The Pathogenesis Of Myelodysplastic Disorders,

Inactivating somatic mutations in polycomb-group (PcG) genes such as EZH2 and ASXL1occur frequently in patients with myelodysplastic syndromes (MDS), myeloproliferative neoplasm (MPN) and MDS/MPN overlap disorders. While these mutations suggest a tumor suppressor function of polycomb repressive complex 2 (PRC2)-related genes in these diseases, both the impact of each PcG mutation and its interplay with coinciding mutations remain largely unknown. To understand the contribution of inactivating PcG mutations to the development of myeloid malignancies, genomic DNA from 119 patients with MDS and related neoplasms were analyzed for mutations in EZH2, ASXL1 and TET2 by high-throughput sequencing. Inactivating mutations in EZH2 and ASXL1 were detected in 8.4 and 16.8 % of patients, respectively. Moreover, 3.4 % of patients had deletion of EZH2 (located at 7q36) associated with -7 and 7q- chromosomal abnormalities. Notably, 57.1 % of these EZH2 mutations coexisted with TET2 mutations. Conversely, 34.8 % of patients with TET2 mutations had coexisting EZH2mutations. In order to understand the impact of inactivating EZH2 mutations and concurrent EZH2 and TET2 mutations on hematopoiesis, we crossed Cre-ERT;Ezh2fl/fl mice and Tet2 gene trap mice (Tet2KD/KD). Due to the early time of death in Tet2KD/KD mice and a necessity to exclude the influence of the loss of Tet2 and Ezh2 in BM niche cells, we transplanted E14.5 fetal liver cells from Cre-ERT control (WT), Cre-ERT;Tet2KD/KD, Cre-ERT;Ezh2fl/fl and Cre-ERT;Tet2KD/KDEzh2fl/fl CD45.2 mice into lethally irradiated CD45.1 recipient mice and deleted Ezh2 by intraperitoneal injection of tamoxifen at 4 weeks post-transplantation. During a long observation period, we found that Ezh2Δ/Δ mice developed MDS/MPN and half of the mice died by 10 months post-transplantation. They showed myeloproliferative features characterized by extramedullary hematopoiesis in the spleen as evident from splenomegaly with a marked increase in LSK cells. They were anemic and showed increased apoptosis in Ter119highCD71high erythroblasts in the BM, suggesting ineffective erythropoiesis, a feature compatible with myelodysplastic disorders. Ezh2Δ/Δ mice also showed dysplasia of myeloid cells, including a pseudo Pelger-Huët anomaly. To our surprise, concurrent deletion of Tet2 and Ezh2 significantly shortened the latency of disease development of not only MDS/MPN but also MDS, and all of the compound mice died of pneumonia by 10 months. Tet2KD/KDEzh2Δ/Δ MDS/MPN mice showed myeloproliferative features, including monocytosis and/or splenomegaly with extramedullary hematopoiesis. In contrast, Tet2KD/KDEzh2Δ/Δ MDS mice did not show obvious myeloproliferative features, but showed a trend of pancytopenia. The proportion of Annexin V+ cells in CD71highTer119high erythroblasts was significantly higher in both MDS/MPN and MDS mice compared to their WT counterparts, implicating enhanced apoptosis as a cause of anemia. Furthermore, myeloid dysplasia was more pronounced in these mice compared to Ezh2Δ/Δmice. Gene set enrichment analysis with microarray data showed that the Myc module was significantly enriched in Ezh2Δ/Δ LSK cells and became highly enriched in Tet2KD/KDEzh2Δ/Δ LSK cells during the development of MDS/MPN and MDS in Tet2KD/KDEzh2Δ/Δ mice. As expected, all of the PRC2 gene sets (Ezh2 targets and Ezh1 targets) showed a trend of positive enrichment in Ezh2Δ/Δ and Tet2KD/KDEzh2Δ/Δ LSK cells. Notably, however, Ezh1 targets became negatively enriched in Tet2KD/KDEzh2Δ/Δ LSK cells during the development of myelodysplastic disorders. ChIP-seq and microarray analysis data showed that upon deletion of Ezh2, a series of potential PcG related target oncogenes, such as Hmga2 and Pbx3, became derepressed in LSK cells. On the other hand, key developmental regulator genes, such as genes encoding homeobox, paired-box, T-box, forkhead and Gata family transcription factors and zinc finger DNA-binding proteins, were kept transcriptionally repressed by the compensatory action of Ezh1. Our findings provide the first evidence of the tumor suppressor function of EZH2 and demonstrate the cooperative effect of concurrent gene mutations in the pathogenesis of myelodysplastic disorders. These two models represent novel, genetically accurate models of myelodysplastic disorders amenable to epigenomic as well as preclinical therapeutic studies. Disclosures: No relevant conflicts of interest to declare.

Tet2 Loss Accelerates The Myeloproliferative Neoplasm (MPN) Phenotype Of Jak2V617F Knockin Mice But Is Insufficient To Cause Leukemic Transformation

TET2 mutations are early somatic events in the pathogenesis of acute myeloid leukemia (AML), myelodysplastic syndrome (MDS) and myeloproliferative neoplasms (MPN) and are one of the most common genetic lesions found in these diseases. In MPN, TET2 mutations are enriched within more advanced disease phenotypes such as myelofibrosis and leukemic transformation and often co-occur with the JAK2V617F mutation, which is present in the majority of MPN patients.We have developed and characterized a Jak2V617F conditional knockin mouse (Jak2VF/+), the phenotype of which closely recapitulates the features of human MPN. To determine the impact of Tet2 loss on Jak2V617F-mediated MPN, we crossed Tet2 conditional knockout mice with Jak2VF/+ knockin and Vav-Cre transgenic mice and backcrossed the compound mutant animals. We then characterized the effects of heterozygous and homozygous loss of Tet2 on the phenotype of Jak2VF/+ mice. We assessed peripheral blood counts, histopathology, hematopoietic differentiation using flow cytometry, colony formation and re-plating capacity. We also evaluated the effects of Tet2 loss on the transcriptome of the HSC compartment using gene expression microarrays and on HSC function using competitive bone marrow transplantation assays.Similar to Jak2VF/+/VavCre+ mice, Tet2+/-/Jak2VF/+/VavCre+ and Tet2-/-/Jak2VF/+/VavCre+ mice develop leukocytosis, elevated hematocrits (HCT) and thrombocytosis. Tet2-/-/Jak2VF/+/VavCre+ mice demonstrate enhanced leukocytosis and splenomegaly compared to the other groups. All groups demonstrate myeloid expansion, erythroid hyperplasia and megakaryocytic abnormalities consistent with MPN in the bone marrow and spleen, while more prominent myeloid expansion and megakaryocytic morphological abnormalities are observed in Tet2-/-/Jak2VF/+/VavCre+ mice as compared to the other groups. Notably, we do not see the development of acute myelogenous leukemia (AML) in Tet2-/-/Jak2VF/+/VavCre+ mice at 6 months. We see enhanced expansion of lineagelowSca1+cKithigh (LSK) cells (enriched for HSC) most prominently in the spleens of Tet2+/-/Jak2VF/+/VavCre+ and Tet2-/-/Jak2VF/+/VavCre+ mice as compared to Jak2VF/+/VavCre+ mice.In colony forming assays, we find that Tet2-/-/Jak2VF/+/VavCre+ LSK cells have enhanced re-plating activity compared to Jak2VF/+/VavCre+ LSK cells and that Tet2-/-/Jak2VF/+/VavCre+ LSK cells form more colonies that Tet2-/-/Jak2+/+/VavCre+ cells. Gene expression analysis demonstrates enrichment of a HSC self-renewal signature inTet2-/-/Jak2VF/+/VavCre+ LSK cells. Concordant with this, we find that Tet2-/-/Jak2VF/+/VavCre+ LSK cells have enhanced competitive repopulation at 16 weeks as compared to Jak2VF/+/VavCre+ and Tet2+/-/Jak2VF/+/VavCre+ LSK cells.In aggregate these findings demonstrate that Tet2 loss promotes disease progression in MPN but is insufficient to drive full leukemic transformation. Disclosures:No relevant conflicts of interest to declare.

Loss Of Tet2 Collaborates With AML1-ETO In Inducing Leukemogenesis

▪Disruption of the epigenome can contribute to both cancer initiation and tumor progression. The methylcytosine dioxygenase TET2, which is involved in the regulation DNA methylation patterns, is frequently mutated in Acute Myeloid Leukemia (AML) patients and plays a central role in normal hematopoiesis. However, Tet2–deficient mouse models do not readily develop acute leukemia suggesting that additional oncogenic events are necessary for full-blown transformation.The translocation t(8;21)(q22;q22) results in expression of the AML1-ETO (AE) fusion protein and is present in ∼5-10% of AML patients. Interestingly, the combination of t(8;21)(q22;q22) and TET2 mutations, although rare, have been observed in several studies in patients groups covering both pediatric and adult de novo AML. To confirm a direct role of TET2 mutations in the development of t(8;21)(q22;q22)-rearranged leukemias we expressed the AE fusion protein in hematopoietic stem and progenitor cells (HSPC) isolated from WT or Tet2-deficient animals. Serial replating assays in semisolid media as well as growth in liquid culture suggested a cooperative effect of AE and Tet2 deficiency as indicated by a significant increase in proliferation and colony formation efficiency. We then used a bone marrow transplantation model to assess the in vivo leukemic potential of AE;Tet2-/- HSPCs. The majority of recipient mice transplanted with AE;Tet2-/- (n=5/6) HSPCs succumbed to an aggressive and transplantable AML-like condition with a median latency of 5-6 months, whereas mice receiving AE HSPCs remained healthy over a 12 month observation period (n=6/6). The morbid mice showed many signs of disease including loss of weight, anemia, splenomegaly and large number of leukemic blasts (CD45neg) in circulation. Further analysis of the bone marrow and spleen revealed a massive expansion of a linneg (CD3e, Gr-1, Mac-1, B220, Ter119), c-Kitlow-high, CD16/32pos cell population indicating a block of differentiation and expansion of an immature myeloid progenitor population from AE:Tet2-/- HSPCs. To investigate how Tet2-deficiency contributes to transformation of t(8;21)(q22;q22)-rearranged cells we generated and cultured pre-leukemic AE-transduced cells in vitro from Tet2fl/fl HPSCs harboring the inducible Rosa26-CreERT2 allele. Genome-wide analysis of methylation patterns and gene expression showed that induction of Tet2-deficiency in pre-leukemic cells leads to progressive hypermethylation of gene-regulatory elements and altered expression of several genes implicated in tumorigenesis. Thus, this study illustrates how aberrant DNA methylation patterns can contribute to disease and confirm the role of Tet2 as a tumor suppressor preventing leukemogenesis. Disclosures:Helin:EpiTherapeutics : Consultancy, Equity Ownership.

Epigenetic Profiling Of Leukemia Stem Cells In a Model Of TET2/FLT3-Mutant AML

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&gt;2.5, padj &lt;0.05). Of note, we found that genes involved in normal myelo-erythroid differentiation, including GATA1, GATA2, and EPOR, were transcriptionally silenced in LSCs relative to normal stem cells, consistent with their the impaired differentiation and increased self-renewal observed in LSCs. Enhanced representation bisulfite sequencing revealed a subset of these genes were marked by increased promoter methylation. The number of hyper differentially methylated regions (HyperDMRs, 10% methylation difference, FDR&lt;0.2) was significantly greater in Vav+Tet2fl/flFlt3-ITD cells (787 HyperDMRs) compared to Vav+Tet2fl/fl cells (76 DMRs) suggesting FLT3 activation and TET2 loss cooperate to alter the epigenetic landscape in hematopoietic cells. 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.

Cooperation of KITD816V and TET2 loss of function in the pathogenesis of systemic mastocytosis and associated hematological malignancies

Systemic mastocytosis (SM) is a clonal disease of the hematopoietic stem cell compartment, frequently associated with the KITD816V activating mutation. This mutation alone cannot account for the variable clinical phenotypes of this disease, ranging from indolent forms to mast cell leukemia (MCL). Loss of function mutations in TET2 have been described in various myeloproliferative neoplasms including SM, where their coexistence with the KITD816V mutation at a frequency as high as 50% has an uncertain biological significance.

Abstract IA20: Epigenetic deregulation of gene expression

Abstract Epigenetic deregulation of gene expression through aberrant DNA methylation or histone modification plays an important role in the malignant transformation of hematopoietic cells. In particular, acute myeloid leukemias (AMLs) can be classified according to epigenetic signatures affecting DNA methylation or histone modifications affecting specific gene sets. Heterozygous somatic mutations in the loci encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in ~20% of AMLs and are accompanied by global DNA hypermethylation and hypermethylation and silencing of a number of specific gene promoters. IDH1/2 mutations are almost completely mutually exclusive with somatic loss of function mutations in TET2, which hydroxylates methylcytosine (mCpG). DNA hydroxymethylation can function as an intermediate step in mCPG demethylation. TET2 mutant de novo AMLs also display global and promoter specific hypermethylation partially overlapping with IDH1/2 mutant cases. Mutations in the IDH1/2 loci result in a neomorphic enzyme that generates the aberrant oncometabolite 2-hydroxyglutarate (2HG) using alpha-ketoglutarate (aKG) as a substrate. 2HG can disrupt the activity of enzymes that use aKG as a cofactor, including TET2 and the Jumonji family of histone demethylases. Expression of mutant IDH isoforms inhibit TET2 hydroxymethylation and Jumonji histone demethylase functions. IDH and TET2 mutant AMLs accordingly exhibit reduced levels of hydroxymethylcytosine and a trend towards reduced histone methylation. Mutant IDH or TET2 loss of function causes differentiation blockade and expansion of hematopoietic stem cells and TET2 knockout results in a myeloproliferative phenotype in mice. Hydroxymethylcytosine is abundance in hematopoietic stem cells, and displays specific distribution patterns, and yet the function of this covalent modification is not fully understood. Recent data link TET2 with the function of cytosine deaminases as a pathway towards DNA demethylation, which has implications as well for B-cell lymphomas and CML lymphoid blast crisis, which are linked with the actions of activation induced cytosine deaminase. Altogether, the available data implicate mutations in IDH1/2 and TET2 in promoting malignant transformation in several tissues, by disrupting epigenomics programming and altering gene expression patterning. Citation Format: Ari Melnick. Epigenetic deregulation of gene expression. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr IA20.

Notch pathway activation targets AML-initiating cell homeostasis and differentiation

Notch signaling pathway activation is known to contribute to the pathogenesis of a spectrum of human malignancies, including T cell leukemia. However, recent studies have implicated the Notch pathway as a tumor suppressor in myeloproliferative neoplasms and several solid tumors. Here we report a novel tumor suppressor role for Notch signaling in acute myeloid leukemia (AML) and demonstrate that Notch pathway activation could represent a therapeutic strategy in this disease. We show that Notch signaling is silenced in human AML samples, as well as in AML-initiating cells in an animal model of the disease. In vivo activation of Notch signaling using genetic Notch gain of function models or in vitro using synthetic Notch ligand induces rapid cell cycle arrest, differentiation, and apoptosis of AML-initiating cells. Moreover, we demonstrate that Notch inactivation cooperates in vivo with loss of the myeloid tumor suppressor Tet2 to induce AML-like disease. These data demonstrate a novel tumor suppressor role for Notch signaling in AML and elucidate the potential therapeutic use of Notch receptor agonists in the treatment of this devastating leukemia.

In aggressive forms of mastocytosis, TET2 loss cooperates with c-KITD816V to transform mast cells

AbstractAlthough a role for oncogenic KIT in driving mast cell disease is clear, the mechanisms driving the multiple phenotypic and clinical manifestations of this disorder are not well elucidated. We now show, using a large cohort of mastocytosis patients, including an almost equal number of aggressive and nonaggressive cases of systemic mastocytosis, that in contrast to the oncogenic KITD816V, TET2 mutation statistically associates with aggressive forms of the disease. By infecting primary murine bone marrow–derived mast cells with KITD816V, we also observe a significant and competitive growth advantage for KITD816V in Tet2-nullizygous compared with wild-type cells. TET2-deficient cells display increased proliferation and can survive in the absence of cytokines. Taken together, these data demonstrate a oncogenic cooperation in mast cells and reveal TET2 mutation as a potential marker to diagnose and predict severe forms of mastocytosis.

Conditional Deletion of Asxl1 Results in Myelodysplasia

AbstractAbstract 308Loss-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. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Tet2 Uniquely Regulates Self-Renewal and Long Term Repopulating Capacity of Fetal Liver and Bone Marrow Hematopoietic Stem Cells

AbstractAbstract 4739Ten-eleven-translocation 2 (TET2) gene is one of the frequent targets of mutation in various hematologic malignancies. These observations suggest critical roles of TET2 dysfunction in their molecular pathogenesis. To investigate physiological roles of TET2 in hematopoiesis, we previously analyzed fetal liver (FL) hematopoiesis of Tet2 gene-trap (Tet2gt) mice and showed that Tet2gt/gt FL cells displayed enhanced self-renewal and long term repopulating (LTR) capacity with expansion of Lineage(−)Sca-1(+)c-Kit(+) (LSK) and common myeloid progenitor (CMP) fractions. However, there remain several questions unanswered. First, self-renewal capacity was examined only by using bulk FL cells and therefore effects of Tet2 loss on purified cell populations such as hematopoietic stem cells (HSCs) or hematopoietic progenitor cells (HPCs) remain elusive. Second, because other groups have reported myeloid transformation in Tet2 conditional knockout mice, it is possible that Tet2 loss confers self-renewal capacity to non-self-renewing myeloid progenitors such as CMPs. Third, effects of Tet2 haploinsufficiency on adult hematopoiesis was not examined using purified HSCs or HPCs.To address these issues, we analyzed E14.5 FL and adult bone marrow (BM) cells from Tet2gt mice. We first performed serial replating assay of FL-LSK cells in methylcellulose containing interleukin (IL)-3, IL-6, stem cell factor (SCF) and erythropoietin (Epo). In this assay, Tet2gt/gt FL-LSK cells showed significantly higher replating capacity as compared to that of WT cells. Interestingly, Tet2gt/gt FL-LSK cells formed various types of colonies including granulocyte-macrophage (GM) and erythrocyte-megakaryocyte (EM) colonies, whereas WT FL-LSK cells generated only GM colonies at the second time of replating, showing that multipotent differentiation capacity was maintained in Tet2gt/gt cells even in the presence of lineage-acting cytokines.Next we examined the self-renewal capacity of highly purified FL-HSCs (CD34+LSK or CD150+LSK cells) by competitive repopulation assay. As expected, the recipients of Tet2gt/gt CD34+LSK cells showed significantly higher donor chimerism in peripheral blood as compared to those receiving WT cells. Furthermore, CD150+LSK cells from Tet2+/gt and Tet2gt/gt FLs demonstrated higher peripheral blood repopulation in the secondary and tertiary recipient mice as compared to that of WT recipients in serial transplantation assay. These results indicate that the enhanced self-renewal and LTR capacity of Tet2-mutant FL cells was uniquely associated with highly purified HSCs. This conclusion also applied to the BM LSK cells from adult mice, since Tet2+/gt BM LSK cells also showed significantly higher peripheral blood contribution compared to the WT cells in serial transplantation assays. This result demonstrates that Tet2 haploinsufficiency is sufficient to confer the enhanced self-renewal and LTR capacity to HSCs in adult hematopoiesis.Lastly, we examined self-renewal capacity of FL CMPs by serial replating assay. Interestingly, Tet2gt/gt FL CMP cells displayed increased replating capacity as compared to WT cells. However, in vivo repopulation assay using Tet2+/+, Tet2+/gt, and Tet2gt/gt FL CMP cells showed no significant difference in peripheral blood chimerism among these recipients.Taken together, enhanced self-renewal and LTR capacity by Tet2 ablation is uniquely associated with HSCs in FL and adult BM, but not with myeloid progenitors, indicating that Tet2 regulates self-renewal program intrinsic to HSCs. In addition, Tet2 haploinsufficiency is sufficient to enhance self-renewal and LTR capacity of HSCs, which explains pathological relation between high incidence of heterozygous TET2 mutations and hematologic malignancies. Disclosures:No relevant conflicts of interest to declare.

Long-Term Cell Autonomous Effect of Tet2 Loss in Hematopoietic Cells in Mice.

AbstractAbstract 2416TET2 mutations are frequently observed in myeloid malignancies including myelodysplastic syndrome (MDS), myeloproliferative neoplasm (MPN) and MDS/MPN. Several groups have already reported that deletion of Tet2 in mice leads to dysregulated hematopoietic stem cell self-renewal and subsequent development of myeloid malignancies. Of note, mice hypomorphic or heterozygous for the Tet2 allele have been reported to show similar phenotypes as those of Tet2-null mice, suggesting that haploinsufficiency of Tet2 plays a role in the development of myeloid malignancies. However, little is known about long-term cell autonomous effects of Tet2 loss in hematopoietic cells: most of the reports were based on relatively short-term observations or did not exclude the influence of Tet2 loss in the niche cells. To study long-term cell autonomous effect of Tet2 loss in hematopoietic cells, we analyzed the hematopoiesis of wild-type recipient mice reconstituted with fetal liver cells from Tet2 hypomorphic mice for a longer period up to 1 year.Tet2 gene trap mice (Tet2trap/trap), in which the gene trap vector was inserted into the exon 2 of Tet2 just before the first coding exon, express Tet2 mRNA at the level approximately 20% of that of the wild-type (WT) mice (Shide et al. Leukemia 2012). We transplanted fetal liver cells from E14.5 WT or Tet2trap/trap mice into lethally irradiated recipient mice. At 4 months after transplantation, the recipient mice reconstituted with Tet2trap/trap cells showed a significantly increased proportion of monocytes in peripheral blood (PB) compared with those with WT cells (WT=5.59±2.57%, Tet2trap/trap=12.67±7.45%, p=0.01). While there were no significant differences between the two groups in the bone marrow (BM) compartments including the numbers of Lineage−Sca-1+c-Kit+ (LSK) hematopoietic stem/progenitor cells, extramedullary hematopoiesis in the spleen was markedly enhanced in the recipients with Tet2trap/trap cells. The proportion of LSK, granulocyte/macrophage progenitors (GMPs) and megakaryocyte/erythroid progenitors (MEPs) in the spleen of recipient mice reconstituted with WT and Tet2trap/trap cells were 0.002±0.001% vs 0.006±0.001% (p<0.01), 0.007±0.004% vs 0.029±0.01% (p=0.026) and 0.084±0.024% vs 0.25±0.044% (p<0.01), respectively. These findings were compatible with those reported previously and indicated that recipient mice reconstituted with Tet2trap/trapcells induce chronic myelomonocytic leukemia (CMML)-like disease.Of note, after a long observation period, particularly after 9 months post-transplantation, mice reconstituted with Tet2trap/trap cells developed advanced hematological disease and 53.3% (8 of 15) died or were killed because of their moribund condition by 11 months after transplantation. Detailed analysis on moribund as well as surviving mice reconstituted with Tet2trap/trap cells (n=3 each) revealed that the 2/3 of the mice developed CMML-like disease with monocytosis in PB and evident extramedullary hematopoiesis harboring increased number of LSK and GMPs in spleen, whereas the remaining 1/3 of mice developed MDS/MPN-like disease with severe anemia. The latter mice did not show monocytosis in PB and BM, but displayed dyserythropoiesis accompanied by massive extramedullary erythropoiesis, as we saw increased number of LSK and MEPs, but not GMPs, in spleen. The proportion of Annexin V+ cells in Ter119highCD71higherythroblasts in the BM of WT mice, CMML-like disease-carrying mice and MDS/MPN-like disease-carrying mice were 9.46±0.53%, 10.71±0.36%, and 19.04±0%, respectively, suggesting that the enhanced apoptosis led to the severe anemia seen in the MDS/MPN-like disease-carrying mice. Thus, decreased expression of Tet2 is sufficient to promote not only CMML-like disease, but also an MDS/MPN-like disease as well.Our findings confirmed a long-term cell autonomous effect from insufficient function of Tet2 in the development of hematological diseases. Interested in the molecular mechanism of disease progression, we have identified 1,642 differentially methylated regions (DMRs) in Tet2trap/trap GMPs compared with WT GMPs by ChIP-sequencing. We are now working to understand how these DMRs are involved in the development of the two distinct diseases associated with hypomorphic Tet2. Disclosures:No relevant conflicts of interest to declare.

Tet2 disruption leads to enhanced self-renewal and altered differentiation of fetal liver hematopoietic stem cells

Somatic mutation of ten-eleven translocation 2 (TET2) gene is frequently found in human myeloid malignancies. Recent reports showed that loss of Tet2 led to pleiotropic hematopoietic abnormalities including increased competitive repopulating capacity of bone marrow (BM) HSCs and myeloid transformation. However, precise impact of Tet2 loss on the function of fetal liver (FL) HSCs has not been examined. Here we show that disruption of Tet2 results in the expansion of Lin−Sca-1+c-Kit+ (LSK) cells in FL. Furthermore, Tet2 loss led to enhanced self-renewal and long-term repopulating capacity of FL-HSCs in in vivo serial transplantation assay. Disruption of Tet2 in FL also led to altered differentiation of mature blood cells, expansion of common myeloid progenitors and increased resistance for hematopoietic progenitor cells (HPCs) to differentiation stimuli in vitro. These results demonstrate that Tet2 plays a critical role in homeostasis of HSCs and HPCs not only in the BM, but also in FL.

Disruption of Tet2 Leads to Enhanced Self-Renewal and Competitive Repopulating Capacity of Fetal Liver Hematopoietic Stem Cells

Abstract 2331TET2 (ten-eleven-translocation 2) gene has been reported to be frequently mutated in various human myeloid malignancies, including myeloproliferative neoplasms, myelodysplastic syndromes, acute myeloid leukemia, and chronic myelomonocytic leukemia. These observations suggest critical roles of TET2 dysfunction in molecular pathogenesis of myeloid malignancies. Recent studies using conditional knockout mouse model indicated that mouse Tet2 loss leads to clonal dominance of adult hematopoietic stem cells (HSCs) in competitive repopulation assay. However, self-renewal capacity of adult HSCs has never been addressed precisely by serial transplantation assay. In addition, the effect of Tet2 loss on hematopoietic stem/ progenitor cells was examined only in the BM, but not in the fetal livers (FLs). Since FL HSCs and adult HSCs differ in several aspects of their phenotypes and functions, we speculated that Tet2 might be involved differently in the regulation of FL and adult hematopoiesis.To address this issue, we analyzed E14.5 FL cells from Tet2 gene-trap (Tet2gt) mice. In these mice, gene trap-cassette was inserted into the second intron, just before the first coding exon. RT-PCR analysis showed that over 99% of Tet2 mRNAs from endogenous promoter were trapped by the gene-trap cassette in Tet2gt/gt mice, showing that Tet2gt allele can be considered as a null allele. Initial analysis showed that Tet2gt/gt embryos developed normally, but most Tet2gt/gt mice were perinatally lethal. Total numbers of FL cells and the numbers of committed progenitors in FLs as revealed by colony assays were not significantly different between each genotype. Interestingly, Tet2gt/gt embryos displayed significant increase in lineage (Lin)(-)Sca-1(+)c-Kit(+)(LSK) fraction compared to wild type (WT) (Tet2+/+) littermate (2.42±0.66% vs. 1.17±0.18%, p=0.02). In addition, common myeloid progenitor (CMP) fraction (IL7Rα(-), Lin(-), Sca-1(-), C-Kit(+), CD34(+), FcgRII/ III(low)) was significantly increased in Tet2gt/gt FLs compared to WT (9.04±1.09% vs. 6.26±0.53%, p=0.008). In serial transplantation assays, donor cells derived from Tet2+/gt and Tet2gt/gt FLs showed significantly higher peripheral blood chimerism in secondary and tertiary recipient mice as compared to that of WT cells, showing that disruption of Tet2 leads to the enhanced self-renewal capacity of FL HSCs. Moreover, donor-derived HSC fraction (CD34−LSK cells) was significantly expanded in the recipients of Tet2gt/gt FL cells, suggesting that increased self-renewal capacity is cell intrinsic to Tet2gt/gt HSCs. We have also examined differentiation of Tet2-mutant FL cells in the recipients’ peripheral blood, and found that Tet2gt/gt cells displayed impaired differentiation to Gr-1(+)CD11b(+) mature granulocytes (WT vs. Tet2gt/gt = 5.02±1.35% vs. 11.5±3.09% in the primary recipients) and slight, but significant increase of B cells. Liquid culture of FL cells with cocktails of cytokines in vitro demonstrated that Tet2gt/gt FL cells retained higher percentage and number of LSK, Lin- and c-Kit+ cells after the culture for 7-days compared to WT cells, showing enhanced resistance of Tet2gt/gt cells to differentiative stimuli in in vitro culture. It is of note that Tet2+/gt mice showed a significant increase in hematopoietic stem/progenitor fraction (LSK) in the BM compared to wild type littermate (0.48±0.11% vs. 0.32±0.04%, p=0.04). However, they presented no signs of extramedullary hematopoiesis such as splenomegaly and expansion of LSK cells in spleens during an observation up to 35-weeks.Taken together, we demonstrate that Tet2 critically regulates self-renewal and long-term repopulating capacity of FL HSCs and has pleiotropic functions in myeloid and lymphoid differentiation. These data strongly indicate that Tet2 is an essential regulator of BM and FL hematopoiesis. In addition, enhanced HSC self-renewal, expansion of HPC and myeloid progenitors and perturbed myeloid differentiation induced by TET2 ablation likely to set molecular basis for myeloid transformation, which explains high incidence of loss-of-function mutations of TET2 in myeloid malignancies. Disclosures:No relevant conflicts of interest to declare.