ALBERT

Description: Abstract 462 In myelodysplastic syndrome (MDS), mutations in genes affecting epigenetic regulation constitute a link between genomic and epigenetic instability. Previously, we and others described mutations in TET2, coding for a 2-oxyglutarate-dependent methylcytosine dioxygenase, which converts 5-methycytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC). Subsequently, dysfunction of wild type TET2 was mechanistically linked to neomorphic IDH mutations which deplete 2-oxyglutarate and produce a competitive inhibitor, 2-hydroxyglutarate. Previously, we established analytic tools to indirectly quantify 5-hmC content in leukemic genomes: in patients with myeloid malignancies 5-hmC levels are decreased as compared to healthy controls (p=1.8e-09). A decrease in 5-hmC levels correlated with dysfunction of TET2 as a consequence of inactivating hypomorphic mutations. Nevertheless, while in a majority of patients with decreased 5hmC levels TET2 mutations can be found, in a substantial minority of cases no explanation for the 5hmC deficiency has been found; down-modulation of TET2 mRNA and protein expression was absent and mutations in TET1 and TET3 have not been identified. Thus, other currently unidentified proteins may be directly or indirectly (via regulation of TET activity) involved in the deregulation of 5hmC levels in TET2 and IDH1/2-mutation-negative cases with low 5-hmC. To further investigate this issue we first characterized on a molecular levels patients with low 5-hmC using various approaches. SNP-A karyotyping failed to identify recurrent chromosomal defects in these patients that could point towards defects in pathogenic genes involved in the regulation of 5-hmC levels. We also screened 107 MDS patients to correlate of genomic 5-hmC content and the presence of recurrent mutations including IDH1/2, DNMT3A, ASXL1 and RUNX1 genes (as well as TET2). Within these genes, except for an association with TET2 mutations, a positive correlation with low 5-hmC levels was found only for IDH1/2 mutant cases (p=.05, n=5), whereas no correlation has been established for DNMT3A (p=.119, n=12), ASXL1 (p=.434, n=21) and RUNX1 (p=.602, n=22) mutant cases. While TET2 and IDH mutations were rarely seen together (n=1), none of the other studied gene mutations were mutually exclusive with TET2, suggesting contributions of defects in novel yet not identified genes. Several other genes similar to TET or IDH proteins, or hypothetically linked to DNA demethylation pathways could, at least theoretically, affect 5-hmC content, including for instance D2HGDH and the ELP gene family. However, no mutations were identified in these patients, except for identification of yet unknown SNPs in D2HGDH and ELP4 in some patients with unexplained low 5-hmC levels. In addition to the targeted approach we have also applied next generation sequencing technologies and sequenced whole exomes of malignant and non-affected cells (paired-end (2×100) Illumina HiSeq 2000) to identify novel acquired determinants of 5-mC hydroxymethylation in two representative patients. By using a selective algorithm, 18 overlapping potential somatic alterations in these patients were found in genes which could functionally affect 5-hmC content. In addition, several other mutated genes have been identified in each patient; these are being further investigated in other patients with low 5-hmC levels. Sanger sequencing was applied to confirm the presence of previously detected mutations in NF1 and KRAS, as well as all novel mutations, for instance in BRCC3 and SF3B1, in these patients. In sum, our results provide novel insights into the molecular mechanisms underlying MDS pathophysiology and describe the possibility that the TET family enzymes can act together with other putative proteins linked to DNA demethylation pathways. The use of high throughput sequencing technologies increase the probability of identification of novel changes which can be linked to functional consequences in these patients, ultimately furthering the understanding its role in genomic stability in MDS. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1 TET2 mutations are frequently found across broad spectrum of myeloid malignancies but how these mutations contribute to diseases is still unknown. Preliminary results from our laboratory have suggested that TET2 converts 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and consequently, the levels of 5-hmC may be lower in genomes of mutant bone marrow cells. To facilitate study of TET2 function we developed a blot assay to detect 5-hmC in genomic DNA with a specific antiserum to 5-hmC. In a second improved assay with increased sensitivity and precision, we treated genomic DNA with bisulfite in order to convert 5-hmC to cytosine 5-methylenesulfonate (CMS) and measured 5-hmC levels indirectly using a specific anti-CMS serum. Based on the results of this technique we demonstrate here for the first time that indeed TET2 mutations in predicted catalytic residues and other positions compromised TET2 function. We studied 102 patients with various myeloid malignancies (4/28 MDS, 14%, 26/48 MDS/MPN, 54% and 1/4 MPN, 2% and primary 2/11 AML 18% and 3/11 sAML, 27% TET2 mutants, respectively) and compared to wt cases or controls (N=17). Mutations were found throughout the entire coding region and were mostly inactivating (33/45 TET2 mutations). The levels of 5-hmC in genomic DNA from TET2 mutants were significantly decreased in comparison to wt cases and controls (p=4.5e-08 and p=1.8e-09, respectively). Particularly low levels of 5-hmC were found in patients with homozygous (UPD)/hemizygous (deletion) TET2 mutations and those with biallelic mutations. Surprisingly, 18% of all TET2 WT patients also showed low levels of genomic 5-hmC (despite normal TET2 mRNA expression), suggesting that these patients may carry not yet identified variants/lesions in TET2 or other partner proteins involved in TET2-mediated catalysis. To further investigate the impact of TET2 mutations associated with myeloid malignancies we also introduced 9 different missense mutations corresponding to those found in patients into murine Tet2 cells; severe loss of enzymatic activity was observed in 7/9 cases as measured by greatly diminished 5-hmC levels. To study the role of Tet2 in normal hematopoiesis we depleted Tet2 in C57BL/6 mice by retrovirus-mediated transduction of shRNA against Tet2. Tet2 depletion is associated with skewing of hematopoietic differentiation towards the monocyte/macrophage lineage. To further investigate the function of TET2 we transduced the myeloid THP-1 cell line with lentiviral vector containing TET2 cDNA (TET2+) or an empty vector. This manipulation allowed us to select clones showing 19-fold increase in TET2 mRNA expression without significantly alterations of proliferation kinetics. Using this model we studied the impact of TET2 overexpression on resultant methylation pattern of CpG sites. We have applied Illumina Infinium HumanMethylation27 arrays (27,5K CpG sites/14.4K genes). Overexpression of TET2 resulted in a distinct promoter methylation patterns with 169 altered CpG sites with difference of averaged β〉0.5 (considered significant as compared to control). Among these differentially methylated loci, 27 promoters were significantly hypomethylated while 42 were hypermethylated as compared to control cells. Change in methylation pattern observed through overexpression of TET2 in vitro prompted us to analyze methylation patterns in patients with and without TET2 mutations or those with decreased 5-hmC levels. Using methylation arrays a total of 62 cases were analyzed. When patients were grouped based on the levels of 5hmC, an associated methylation signature can be clearly discerned with 2512 differentially methylated loci and distinct skewing towards hypomethylation (2510 sites; e.g., TMEM102, ABCC11) vs. hypermethylation (2 sites, AIM2 and SP140), consistent with the observation made in the TET2+ cells line. In sum, our results provide strong evidence for TET2 as the first mutated gene in myeloid malignancies that is involved in conversion of 5-mC to 5-hmC in DNA, indicating the novel role of TET2 in a substantial component of epigenetic deregulation in myeloid malignancies. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2354 Epigenetic alterations in cancer cells include aberrant DNA methylation and histone modifications. Specifically, cancer cells display global hypomethylation associated with genomic instability as well as promoter hypermethylation associated with inactivation of tumor suppressor, cell cycle or repair-related genes. In the hematopoietic system, whole-genome sequencing and other genetic analyses have led to the discovery of recurrent somatic alterations that contribute to the pathogenesis of a variety of myeloid malignancies by perturbing the epigenetic landscape of cancer cells. Ten-Eleven-Translocation (TET) family enzymes, TET1, TET2, and TET3 modify DNA methylation status by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in a 2-oxoglutarate and Fe2+-dependent manner. TET2 is located in chromosome 4q24, a region undergoing frequent microdeletions and uniparental disomy in patients with a wide spectrum of myeloid malignancies. Somatic mutations in TET2 are some of the most prevalent acquired mutations in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), MDS/MPN overlap syndromes including chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML) and secondary AML (sAML). We previously showed that missense mutations of TET2 in myeloid malignancies are loss-of-function mutations that compromise its dioxygenase activity. TET2 mutations correlate with decreased levels of 5hmC in patients. Tet2-depleted mouse hematopoietic precursor cells are preferentially committed to differentiation towards monocyte/ macrophage lineages in culture. The levels of DNA methylation in patients with high 5hmC versus healthy controls are similar, however, samples from patients with low 5hmC show hypomethylation relative to controls at the majority of differentially methylated CpG sites. Although it is postulated that impaired TET2 activity may potentiate myeloid transformation by influencing hematopoietic stem/progenitor cells (HSPCs), it has yet to be directly tested whether Tet2 mutations or deletions are implicated in abnormal hematopoiesis in vivo. To clarify the function of Tet2 in hematopoietic development, we generated mice with targeted disruption of the Tet2 catalytic domain and found that Tet2 is critical for self-renewal and differentiation of hematopoietic stem cells (HSCs). Ablation of Tet2 specifically repressed Tet2 expression with no effect on the other Tet family members, Tet1 and Tet3. Dot blot analysis showed that Tet2-deficient cells contain significantly diminished levels of genomic 5hmC in several organs examined. Tet2 deficiency augmented the frequency and absolute number of HSPC compartment in a cell-autonomous manner. In competitive transplantation assays, Tet2-deficient HSCs were capable of multi-lineage reconstitution and possessed a competitive advantage over wild type HSCs, resulting in enhanced hematopoiesis into both lymphoid and myeloid lineages. In vitro differentiation assays showed that Tet2 restrains HSCs from undergoing differentiation, as assessed by expression of lineage markers upon differentiation. Despite this antagonizing effect, however, the number of monocyte/ macrophage cells was greater in Tet2−/− cultures compared with controls, and immature Tet2−/− progenitor cells differentiated prematurely into the monocyte/macrophage lineage. These results indicate that Tet2 deficiency alters stem/progenitor cell properties to delay HSC differentiation and induce developmental skewing towards the monocyte/macrophage lineage. Taken together, these studies indicate that Tet2 has a critical role in regulating the expansion and self-renewal of HSCs. Our data suggest that cell fate decisions of HSPC are affected by TET2 mutations that decrease enzymatic activity, and that this phenomenon has a crucial role in the pathogenesis of diverse myeloid malignancies. We are testing whether Tet2 deficiency synergises with other recurrent mutations to promote myeloid malignancies. Because loss-of-function mutations in TET1 or TET3 have not been reported in most TET2-mutated cancer and TET2 loss-of-function seems to facilitate myeloid transformation because of impaired 5hmC production, it might be beneficial from the perspective of cancer therapies to develop strategies to activate the enzymatic activity of other TET proteins. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract SCI-32 TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. Somatic TET2 mutations are frequently observed in myeloid neoplasms in humans. Bone marrow samples from patients with mutant TET2, as well as some patients with wild type TET2, display low levels of 5hmC in genomic DNA compared to healthy controls. Measurement of 5hmC levels in myeloid malignancies may prove valuable as a diagnostic and prognostic tool to tailor therapies and assess responses to anticancer drugs. We have developed novel and specific approaches to profile the genomic localization of 5hmC and will describe their application to profiling 5hmC in mouse hematopoietic progenitor cells that express or lack Tet2. Disclosures: No relevant conflicts of interest to declare.

Description: Identification of recurrent leukemia-associated mutations in genes encoding regulators of DNA methylation such as DNMT3A and TET2 have underscored the critical importance of DNA methylation in maintenance of normal physiology. To gain insight into how DNA methylation exerts the central role, we sought to determine the genome-wide pattern of DNA methylation in the normal precursors of leukemia cells: the hematopoietic stem cell (HSC), and investigate the factors that affect alterations in DNA methylation and gene expression. We performed whole genome bisulfite sequencing (WGBS) on purified murine HSCs achieving a total of 1,121M reads, resulting in a combined average of 40X coverage. Using Hidden Markov Model we identified 32,325 under-methylated regions (UMRs) with average proportion of methylation ≤ 10% and by inspecting the UMR size distribution, we discovered exceptionally large “methylation Canyons” which span highly conserved domains frequently containing transcription factors and are quite distinct from CpG islands and shores. Methylation Canyons are a distinct genomic feature that is stable, albeit with subtle differences, across cell-types and species. Canyon-associated genes showed a striking pattern of enrichment for genes involved in transcriptional regulation (318 genes, P=6.2 x 10-123), as well as genes containing a homeobox domain (111 genes, P=3.9 x 10-85). We compared Canyons with TF binding sites as identified from more than 150 ChIP-seq data sets across a variety of blood lineages (〉10)19 and found that TF binding peaks for 10 HSC pluripotency TFs are significantly enriched in entirety of Canyons compared with their surrounding regions. Low DNA methylation is usually associated with active gene expression. However, half of Canyon genes associated with H3K27me3 showed low or no expression regardless of their H3K4me3 association while H3K4me3-only Canyon genes were highly expressed. Because DNMT3A is mutated in a high frequency of human leukemias24, we examined the impact of loss of Dnmt3a on Canyon size. Upon knockout of Dnmt3a, the edges of the Canyons are hotspots of differential methylation while regions inside of Canyon are relatively resistant. The methylation loss in Dnmt3a KO HSCs led Canyon edge erosion, Canyon size expansion and addition of 861 new Canyons for a total of 1787 Canyons. Canyons marked with H3K4me3 only were most likely to expand after Dnmt3a KO and the canyons marked only with H3K27me3 or with both marks were more likely to contract. This suggests Dnmt3a specifically is acting to restrain Canyon size where active histone marks (and active transcription) are already present. WGBS cannot distinguish between 5mC and 5hmC, so we determined the genome-wide distribution of 5hmC in WT and Dnmt3a KO HSCs using the cytosine-5-methylenesulphonate (CMS)-Seq method in which sodium bisulfate treatment convert 5hmC to CMS; CMS-containing DNA fragments are then immunoprecipitated using a CMS specific antiserum. Strikingly, 5hmC peaks were enriched specifically at the borders of Canyons. In particular, expanding Canyons, typically associated with highest H3K4me3 marking, were highly enriched at the edges for the 5hmC signal suggesting a model in which Tet proteins and Dnmt3a act concomitantly on Canyon borders opposing each other in alternately effacing and restoring methylation at the edges, particularly at sites of active chromatin marks. Using Oncomine data, we tested whether Canyon-associated genes were likely to be associated with hematologic malignancy development and found Canyon genes were highly enriched in seven signatures of genes over-expressed in Leukemia patients compared to normal bone marrow; in contrast, four sets of control genes were not similarly enriched. Further using TCGA data, we found that expressed canyon genes are significantly enriched for differentially expressed genes between patients with and without DNMT3A mutation (p value