Tet2 Loss Research Articles

AML Xenografts Undergo Extensive Clonal Competition and Unmask Rare Clones in Patient Samples

The biology of acute myeloid leukemia (AML) is largely dictated by the complex combinations of mutations that initiate and propagate the disease. It is therefore critical that models accurately reflect the mutation patterns of the specific leukemias under investigation. In recent years, patient-derived xenografts have emerged as one of the most faithful models to study AML and evaluate potential treatments in vivo. However, little is still known about how leukemias are affected by transplantation and consecutive passaging in vivo. To address this issue, we characterized the mutational patterns and clonal dynamics in 19 AML patient samples and 65 corresponding xenografts from primary and secondary recipients by whole-exome sequencing.We found that xenografts from each patient generally displayed the same mutational profile but with striking differences from the corresponding patient sample as a result of extensive clonal competition. Only 1 of 19 patient samples (5%) retained its mutation pattern in vivo and an additional 3 of 19 samples (16%) displayed gains or losses of non-recurrent presumed passenger mutations. However, the remaining 15 of 19 samples (79%) were shown to contain multiple clones with different recurrent AML mutations that engrafted according to one of the four identified patterns of clonal dynamics: loss, expansion, burst and stable. In 5 of 19 samples (26%), we observed a loss or reduction of subclones containing either NRAS, KRAS, CDKN2A, RUNX1T1 or del(19)(p13), giving way to the respective parental clones, despite allele frequencies in the diagnostic samples suggesting these mutations to be present in the entire cell populations. Seven of 19 samples (37%) showed an expansion of a minor subclone in the patient sample to the point where it made up the entire xenograft. This was paralleled by the complete loss of parental clones as well as sibling clones carrying other recurrent AML mutations such as BCOR, NRAS and WT1. Furthermore, two of the expanding subclones (with TET2 loss of heterozygosity and monosomy 7) were present but not detected by sequencing (100x) of the diagnostic samples, showing that xenografts can unmask unexpected clonal complexity and rare subclones in patient samples. Notably, certain expanding subclones were defined by mutations in NRAS and CDKN2A, which were both lost in other patient samples, suggesting that the interplay between driver mutations is complex and that engraftment cannot be predicted solely based on single mutations. In an additional 2 of 19 samples (11%), a subclonal burst was observed. In these cases, subclones with mutations in SMC3 and PTPN11, respectively, expanded in primary recipients but were lost upon secondary transplantation, indicating that subclones may differ not only in their ability to initiate but also to sustain leukemia in vivo. This notion is further supported by the observation that certain subclones that decreased in vivo only did so in secondary recipients. Notably, only 1 of 19 patient samples (5%) displayed a pattern of stable clonal dynamics, where a subclone was retained at its original frequency throughout primary and secondary passaging.In conclusion, patient-derived xenografts are highly representative of the corresponding patient samples, in that the cells maintain their mutation patterns and gain very few, if any, novel driver mutations throughout consecutive passaging in vivo. However, most AML patient samples comprise multiple clones that differ in their ability to initiate and propagate leukemia in vivo. This leads to xenografts with clonal compositions and mutation patterns that are significantly different from the patient samples, demonstrating the need for genetic profiling of individual xenografts for precise studies of AML biology and treatment. DisclosuresFioretos:Cantargia: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

RETRACTED ARTICLE: TET2 inhibits tumorigenesis of breast cancer cells by regulating caspase-4

Epigenetic regulators have been shown to influence breast cancer progression. However, the detailed mechanism by which TET2 plays the suppressive role in tumorigenesis remains not completely understood. We employed RT-qPCR and westernblot to examine genes expression. Next, the bisulphite sequencing PCR was used to determine the methylation level at CASP4 promoter in the cells. Phenotypically, we utilized growth curve analysis, colony formation in soft agar and xenograft tumor assay to assess tumorigenesis of MCF-7 cell. We found that TET2 knockout enhanced colony formation ability and in vivo tumor formation ability of MCF-7 cell, whereas TET2 depletion not affected the growth rate of MCF-7 cell in the culture. Mechanistically, TET2 loss led to a significant decrease in caspase-4 expression possibly via increasing DNA methylation of CASP4 promoter in MCF-7 cell. To validate, TET2 overexpression led to higher level of caspase-4 in MDA-MB-231 and 293T cells, which was dependent on TET2 enzymatic activity. Finally, we observed that caspase-4 could revert, at least partially, TET2 deletion-induced tumorigenesis of MCF-7. In summary, we reveal a novel mechanism that TET2 suppresses tumorigenesis of breast cancer cells through caspase-4. Our findings will facilitate development of new diagnostic markers or therapeutical therapies for breast cancer.

TET2 Loss Dysregulates the Behavior of Mesenchymal Stem Cells and Increases Their Ability to Promote TET2−/−-Driven Myeloid Malignancy Progression

Background: TET2 is a methylcytosine dioxygenase that regulates cytosine hydroxymethylation. Although there are extensive data implicating a pivotal role of TET2 in hematopoietic stem/progenitor cell (HSPCs), the importance of TET2 in bone marrow mesenchymal stromal cells (BMSCs) remains unknown. Aim: Tet2 loss may dysregulate the integrity of bone marrow niche, by which affects the malignant progression. Methods: Generation and maintenance of Tet2 conditional knockout MiceAnalysis of 5-hmC and 5-mC levels using dot blotFlow cytometry analysis, cell sorting, and hematopoietic progenitor cell (HPC) assayMSC Culture and long-term coculture with HSPCsReal-time PCR and RAN-Seq AnalysisMeDIP-seq to profile the genome-wide distribution of 5hmC. Results: Here, we show that while both Tet2 f/f ;Mx1Cre (conditional Tet2-inactivation in hematopoietic cells) and Tet2 f/f ;MxfCre (germ line Tet2-inactivation) developed myeloid malignancies in mice, Tet2 f/f ;MxfCre mice had a significantly shortened survival compared with Tet2 f/f ;Mx1Cre mice. Interestingly, Tet2 −/− recipient mice exhibited a higher incident of myeloid malignancies and a significantly reduced survival rate compare with WT recipient mice. These data indicate that Tet2 −/− bone marrow niche might promote the progression of myeloid malignancies in Tet2 −/− mice. Strikingly, deletion of Tet2 in mesenchymal stem cells (MSCs) using Prx1-cre is associated with a significantly accelerated malignancy progression and shortened survival, suggesting that MSCs are the cell components in Tet2 −/− mice play a role in the initiation/progression of Tet2 −/−-driven myeloid malignancies. Furthermore, Tet2 −/− MSCs displayed a significantly increased self-renewal, proliferating and differentiation capability as assayed by the frequency of CFU-F and commitment toward osteoblasts. In addition, Tet2 −/− but not WT MSCs exhibited a significantly increased supportive capacity to Tet2 −/− HSC/HPC proliferation. RNA-sequencing analysis revealed that Tet2 −/− MSCs exhibited a distinct gene expression profiles with 468 dysregulated genes as compared with WT MSCs. Furthermore, the number of 5-hmC peaks were significantly decreased in Tet2 −/− MSCs compared with WT MSCs based on whole genomic 5-hmC profiling. The majority of TET2-dependent 5hmC modifications in MSCs are located within genes. We then examined TET2 gene expression in MSCs derived from human myeloproliferative neoplasms (MPN) patients and healthy individuals and found that TET2 and 5-hmC was moderately down-regulated in MPN MSCs as compared with healthy controls. Conclusion: These results highlight the critical role of TET2 in the maintenance of BMSC functions and osteoblast differentiation, and provide evidence that dysregulation of epigenetic modifier in BMSCs contributes to the progression of myeloid malignancies.

Disordered haematopoiesis and cardiovascular disease: a focus on myelopoiesis.

Cardiovascular (CV) diseases (CVD) are primarily caused by atherosclerotic vascular disease. Atherogenesis is mainly driven by recruitment of leucocytes to the arterial wall, where macrophages contribute to both lipid retention as well as the inflammatory milieu within the vessel wall. Consequently, diseases which present with an enhanced abundance of circulating leucocytes, particularly monocytes, have also been documented to accelerate CVD. A host of metabolic and inflammatory diseases, such as obesity, diabetes, hypercholesteraemia, and rheumatoid arthritis (RA), have been shown to alter myelopoiesis to exacerbate atherosclerosis. Genetic evidence has emerged in humans with the discovery of clonal haematopoiesis of indeterminate potential (CHIP), resulting in a disordered haematopoietic system linked to accelerated atherogenesis. CHIP, caused by somatic mutations in haematopoietic stem and progenitor cells (HSPCs), consequently provide a proliferative advantage over native HSPCs and, in the case of Tet2 loss of function mutation, gives rise to inflammatory plaque macrophages (i.e. enhanced interleukin (IL)-1β production). Together with the recent findings of the CANTOS (Canakinumab Anti-inflammatory Thrombosis Outcomes Study) trial that revealed blocking IL-1β using Canakinumab reduced CV events, these studies collectively have highlighted a pivotal role of IL-1β signalling in a population of people with atherosclerotic CVD. This review will explore how haematopoiesis is altered by risk-factors and inflammatory disorders that promote CVD. Further, we will discuss some of the recent genetic evidence of disordered haematopoiesis in relation to CVD though the association with CHIP and suggest that future studies should explore what initiates HSPC mutations, as well as how current anti-inflammatory agents affect CHIP-driven atherosclerosis.

GBX2 Methylation Is a Novel Prognostic Biomarker and Improves Prediction of Biochemical Recurrence Among Patients with Prostate Cancer Negative for Intraductal Carcinoma and Cribriform Architecture

BackgroundTumor intraductal carcinoma/cribriform architecture (IDC/C) is associated with an unfavorable prognosis and biochemical recurrence (BCR) in prostate cancer (PCa). Up to 70% of PCa patients are IDC/C-negative, but it is estimated that 20% of these cases still experience BCR. Thus, biomarkers for better detection of aggressive disease in IDC/C-negative patients are required. ObjectiveTo investigate tumor-specific methylation of the transcription factor GBX2 as a novel prognosticator and predictor of BCR in PCa patients stratified by histopathologic features including IDC/C. Design, setting, and participantsUsing genome-wide methylome profiling, we identified higher GBX2 methylation in grade group (GG) 4 tumors compared to GG1 (discovery cohort). The prognostic nature of GBX2 methylation was validated in silico using The Cancer Genome Atlas data (n=478) and a quantitative methylation assay for radical prostatectomy samples (n=254). Regulation of GBX2 methylation was investigated in prostate cells using methyl-CpG-binding domain sequencing and methylation analysis in functional knockouts of TET2, a key epigenetic player in prostate carcinogenesis. Outcome measurements and statistical analysisThe association of GBX2 methylation with Gleason score (GS), pathologic stage (pT), IDC/C, and BCR was analyzed using Kruskal-Wallis and Mann-Whitney tests. Univariate and multivariate Cox regression analyses were used to predict BCR. ResultsGBX2 methylation was associated with GS (p<0.05), pT (p<0.01), and BCR (p<0.05). GBX2 methylation (p=0.004), GS (p<0.001), pT (p=0.012), and prostate-specific antigen (p=0.005) were independent predictors of BCR. Among IDC/C-negative patients, GBX2 methylation improved prediction of BCR (p=0.002). Loss of TET2 in prostate cells resulted in greater GBX2 methylation. ConclusionsWe identified GBX2 methylation as a novel prognostic factor in PCa and an independent predictor of BCR. We demonstrated the additive value of GBX2 methylation in predicting BCR among IDC/C-negative patients and elucidated a novel TET2-mediated upstream epigenetic regulatory mechanism of GBX2. Patient summaryWe identified GBX2 methylation as a promising prognostic biomarker that could improve the identification of prostate cancer patients at higher risk of biochemical recurrence.

Abstract B075: The role of TET1 and hydroxymethylation in high-risk prostate cancer

Abstract The Ten-eleven translocation (TET) proteins are dioxygenases that catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) as part of the DNA demethylation pathway. Gene silencing through cytosine methylation contributes to cancer formation, but how hydroxymethylation affects gene expression is largely unknown. In this work, TET1 and genomic hydroxymethylation were investigated in a high-risk prostate cancer cohort for which exome sequencing data are available (1). Immunohistochemical analysis of tumor versus nontumor prostate biopsies from high-risk prostate cancer showed a strong reduction of genome-wide 5hmC and milder but still significant reduction of 5mC levels in prostate cancer tissue compared to control tissue. Copy number analysis of high-risk prostate cancer showed a loss of the TET1 genomic region in 6 out of 39 samples of our cohort. As expected, the mRNA levels of TET1 were decreased in these tumor samples. Recently, an alternative promoter of TET1 was discovered. This promoter generates a novel isoform lacking the CXXC-domain, which is called TET1ALT (2). We therefore performed functional analyses of the two TET1 promoters in prostate cancer cells. Interestingly, the promoter controlling expression of the endogenous full-length TET1 is less active in prostate cancer cell line PC-3, while the alternative promoter generating TET1ALT is more active in these cells. This is shown by luciferase constructs driven by the two promoter regions. In conclusion, our findings indicate that there is a drop in (hydroxy)methylation levels in high-risk prostate cancer that correlates with decreased TET1 expression levels and in some cases by a copy number loss of TET1. We are currently working on a more detailed analysis of TET1 promoter usage and control. This will unravel the regulation of hydroxymethylation in prostate cancer, which is crucial for the understanding of prostate cancer development and progression.

3199 - TET2 Regulates Osteoclast Differentiation by Interacting with RUNX1 and Maintaining Genomic 5-Hydroxymethylcytosine

TET2 protein catalyzes subsequent steps of 5-Methylcytosine oxidation. Loss-of-function mutations in TET2 are frequent in both myeloid and lymphoid malignancies. TET2 mutations also occur in elderly individuals with clonal hematopoiesis. The role of TET2 in hematopoietic stem cells was extensively investigated, but its impact on mature hematopoietic cells is not well-characterized. Here we show that TET2 plays an essential role in osteoclastogenesis. We found that Tet2-/- and Tet2f/f;LysMCre mice showed mild osetopetrosis, accompanied by decreased osteoclast numbers in vivo. Deletion of Tet2 impairs osteoclast precursor cell differentiation and maturation into osteoclasts. Tet2 loss in osteoclast precursor cells altered the expression of a set of genes implicated in osteoclast differentiation, such as Cebpa and Nfkbiz (through inhibition of NFB activity), both may contribute to the impaired osteoclast differentiation and the osteopetrotic bone phenotype in Tet2-/- mice. Tet2 deletion also led to a genome-wide alteration of 5hmC in osteoclast precursor cells with enrichment of differential hydroxymethylated regions in specific sets of genes implicated in osteoclast differentiation. However, there was no overall linear correlation between gene expression changes and 5hmC alteration in Tet2-/- vs WT osteoclast precursor cells. Furthermore, TET2 interacts with RUNX1 as revealed by FLAG-TET2-IP followed by LC-MS/MS and confirmed by reciprocal co-IP. It was shown that Runx1 loss accelerates osteoclastogenesis. RUNX1 binding motifs are enriched in the FLAG-TET2 ChIP-seq peak regions. Indeed, luciferase reporter assays showed that TET2 negatively modulates RUNX1 transcriptional activity. These data suggests that Tet2 loss activates RUNX1 transcriptional activity, contributing to the impaired osteoclast differentiation. In, conclusion, our studies demonstrate a novel molecular mechanism controlling osteoclast differentiationby TET2, through interactions with RUNX1 and the maintenance of genomic 5-hmC. Targeting TET2 and its pathway could be a novel therapeutic strategy for the prevention and treatment of abnormal bone mass caused by osteoclast activity deregulation.

3147 - Loss-of-Function Mutation in TET2 in Zebrafish Leads to Early MDS Like Phenotype

Abstract Myelodysplastic syndrome (MDS) is a pre-leukemic state characterized by the failure of the bone marrow to produce mature and functional blood cells. Nearly one-third of MDS patients progress to acute myeloid leukemia (AML). AML is the most common form of acute leukemia in adults and accounts for a high level of mortality in pediatric leukemia. Loss-of-function mutations in the Ten-11 Translocation 2 (TET2) gene are implicated in MDS and AML, but evidence that TET2 mutations are also found in healthy individuals as a result of clonal hematopoiesis calls into question the contribution of TET2 to leukemogenesis. Many studies have tried to mutate the DNA binding domain in TET2 both in mouse and zebrafish, but the blood phenotypes observed have been inconsistent. We created a tet2 zebrafish mutant using CRISPR-Cas9 technology, where we deleted 2.1 kb from exon 2, confirmed by cDNA analysis. A recent publication suggests the importance of Ser99 in the stability of the TET2 protein (Wu et al., 2018) and this conserved serine residue is contained in the region of deletion in our zebrafish tet2 mutant. These tet2 mutant fish were incrossed to create a maternal-zygotic mutant with no tet2 expression. Importantly, quantitative PCR demonstrated that there was no compensatory effect from tet1 and tet3 in the context of tet2 loss. Whole mount in situ hybridization performed on 24-48h tet2 mutant zebrafish embryos provided evidence of a significant reduction of early and mature myeloid and erythroid cells through staining for spi1/pu.1 (myeloid), gata1 (erythroid) myeloperoxidase (mpx; neutrophils), carboxypeptidase A5 (cpa5; mast cells) and l-plastin (lcp1; macrophages). The stem cell compartment was not impacted, as shown by wild-type runx1/c-myb expression. Further, o-dianisidine staining revealed low hemoglobin content in tet2 mutants compared with control embryos. Overall, the early embryos elucidate a refractory anemia like phenotype. Flow cytometry analysis of kidney marrow from 3-month-old adult tet2 mutant fish similarly demonstrated a decrease in the myeloid and erythroid compartments, in keeping with characteristics typical of low stage MDS. We are currently employing RNAseq and MeDIP analysis on blood cell populations from tet2 mutants to determine the genetic and epigenetic signatures associated with this mutation. This zebrafish model of tet2 loss uniquely provides with a dynamic phenotype that starts with refractory anemia with progression through low stage MDS. Thus this model will be beneficial in elucidating the mechanisms underlying how tet2 contributes to MDS and AML and provide a preclinical platform to screen for therapeutic interventions. Disclosures No relevant conflicts of interest to declare.

Abstract 1130: The role of TET2 in mammary stem cell fate decision

Abstract Epigenetic mechanisms, including DNA methylation, has an essential role in governing the stem cell fate decision. The Ten-Eleven-Translocation (TET) protein family serves as a primary epigenetic regulator responsible for catalyzing the conversion of the modified genomic base 5-methylcytosine into 5-hydroxymethylcytosine to mediate DNA demethylation. It has been shown that loss of TET2 promotes aberrant stem cell self-renewal in hematopoietic malignancy. Interestingly, our recent study has revealed that expression of TET2 directs the breast cancer stem cell-like population to the differentiation cell fate in vitro. Since aberrant stem cell fate is often associated with tumorigenesis, delineating the role that a critical epigenetic regulator (e.g., TET2) plays to govern the mammary stem cell (MaSC) fate will likely provide new strategies used for breast cancer treatment. Thus, to determine the physiological and pathological roles of TET2 in the regulation of MaSC fate and mammary gland development using a mouse model with mammary specific deletion of the Tet2 gene (MMTV-Cre; Tet2fl/fl). Our data show that deletion of Tet2 in mouse mammary gland leads to disrupted mammary epithelial morphology, defective lobular-alveolar development, along with aberrant mammary stem cell fate decision, contributing to an expansion of the MaSC population and development of mammary lesions. Together, the data demonstrate that TET2 plays an important role in controlling MaSC fate decision to maintain epithelial homeostasis, and loss of TET2 may contribute to the development of mammary malignancies. Citation Format: Meng-Ju Wu, Chun-Ju Chang. The role of TET2 in mammary stem cell fate decision [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1130.

Oncogenic N-Ras and Tet2 haploinsufficiency collaborate to dysregulate hematopoietic stem and progenitor cells

AbstractConcurrent genetic lesions exist in a majority of patients with hematologic malignancies. Among these, somatic mutations that activate RAS oncogenes and inactivate the epigenetic modifier ten-eleven translocation 2 (TET2) frequently co-occur in human chronic myelomonocytic leukemias (CMMLs) and acute myeloid leukemias, suggesting a cooperativity in malignant transformation. To test this, we applied a conditional murine model that endogenously expressed oncogenic NrasG12D and monoallelic loss of Tet2 and explored the collaborative role specifically within hematopoietic stem and progenitor cells (HSPCs) at disease initiation. We demonstrate that the 2 mutations collaborated to accelerate a transplantable CMML-like disease in vivo, with an overall shortened survival and increased disease penetrance compared with single mutants. At preleukemic stage, N-RasG12D and Tet2 haploinsufficiency together induced balanced hematopoietic stem cell (HSC) proliferation and enhanced competitiveness. NrasG12D/+/Tet2+/− HSCs displayed increased self-renewal in primary and secondary transplantations, with significantly higher reconstitution than single mutants. Strikingly, the 2 mutations together conferred long-term reconstitution and self-renewal potential to multipotent progenitors, a pool of cells that usually have limited self-renewal compared with HSCs. Moreover, HSPCs from NrasG12D/+/Tet2+/− mice displayed increased cytokine sensitivity in response to thrombopoietin. Therefore, our studies establish a novel tractable CMML model and provide insights into how dysregulated signaling pathways and epigenetic modifiers collaborate to modulate HSPC function and promote leukemogenesis.

Tet2 Regulates Osteoclast Differentiation by Interacting with Runx1 and Maintaining Genomic 5-Hydroxymethylcytosine (5hmC)

As a dioxygenase, Ten-Eleven Translocation 2 (TET2) catalyzes subsequent steps of 5-methylcytosine (5mC) oxidation. TET2 plays a critical role in the self-renewal, proliferation, and differentiation of hematopoietic stem cells, but its impact on mature hematopoietic cells is not well-characterized. Here we show that Tet2 plays an essential role in osteoclastogenesis. Deletion of Tet2 impairs the differentiation of osteoclast precursor cells (macrophages) and their maturation into bone-resorbing osteoclasts in vitro. Furthermore, Tet2−/− mice exhibit mild osteopetrosis, accompanied by decreased number of osteoclasts in vivo. Tet2 loss in macrophages results in the altered expression of a set of genes implicated in osteoclast differentiation, such as Cebpa, Mafb, and Nfkbiz. Tet2 deletion also leads to a genome-wide alteration in the level of 5-hydroxymethylcytosine (5hmC) and altered expression of a specific subset of macrophage genes associated with osteoclast differentiation. Furthermore, Tet2 interacts with Runx1 and negatively modulates its transcriptional activity. Our studies demonstrate a novel molecular mechanism controlling osteoclast differentiation and function by Tet2, that is, through interactions with Runx1 and the maintenance of genomic 5hmC. Targeting Tet2 and its pathway could be a potential therapeutic strategy for the prevention and treatment of abnormal bone mass caused by the deregulation of osteoclast activities.

Abstract 018: TET2 Loss of Function-driven Clonal Hematopoiesis Promotes Age-related Vascular and Systemic Inflammation and Metabolic Dysfunction in Mice

Background: Acquired mutations in the hematopoietic system and subsequent clonal hematopoiesis are emerging as potential new drivers of atherosclerotic cardiovascular disease. TET2 was the first gene reported to exhibit acquired mutations in blood cells of cancer-free elderly individuals and we recently reported that the clonal expansion of TET2-mutant hematopoietic cells accelerates atherogenesis in mouse models by exacerbating inflammatory responses in the atherosclerotic plaque. The objective of this study was to examine whether TET2 loss of function-driven clonal hematopoiesis also has systemic effects on age-related inflammation and metabolic function beyond its direct actions on the plaque. Methods: An adoptive transfer technique was used to introduce a small percentage of Tet2-/- hematopoietic cells in non-irradiated C57Bl/6J mice, which were monitored for 16 months following transfer. Flow cytometry techniques were used to follow the expansion of Tet2-/- cells in the blood. Results: Tet2-/- hematopoietic cells expanded progressively over time after adoptive transfer, with a mild myeloid bias. This was paralleled by increased expression of the pro-inflammatory cytokines IL-1β and IL-6 in the white adipose tissue and the aortic wall, as well as by increased serum levels of IL-6. Tet2-/- expansion also led to exacerbated systemic insulin resistance, as revealed by insulin tolerance tests. Conclusion: TET2-deficient hematopoietic cell expansion promotes vascular and systemic inflammation, as well as metabolic dysfunction in aging mice. These results support a causal role for TET2 mutation-driven clonal hematopoiesis in age-related cardiometabolic disease, and suggest that somatic TET2 mutations may contribute to atherosclerosis by promoting inflammation both systemically and locally in the atherosclerotic plaque.

Loss of epigenetic regulator TET2 and oncogenic KIT regulate myeloid cell transformation via PI3K pathway.

Mutations in KIT and TET2 are associated with myeloid malignancies. We show that loss of TET2-induced PI3K activation and -increased proliferation is rescued by targeting the p110α/δ subunits of PI3K. RNA-Seq revealed a hyperactive c-Myc signature in Tet2-/- cells, which is normalized by inhibiting PI3K signaling. Loss of TET2 impairs the maturation of myeloid lineage-derived mast cells by dysregulating the expression of Mitf and Cebpa, which is restored by low-dose ascorbic acid and 5-azacytidine. Utilizing a mouse model in which the loss of TET2 precedes the expression of oncogenic Kit, similar to the human disease, results in the development of a non-mast cell lineage neoplasm (AHNMD), which is responsive to PI3K inhibition. Thus, therapeutic approaches involving hypomethylating agents, ascorbic acid, and isoform-specific PI3K inhibitors are likely to be useful for treating patients with TET2 and KIT mutations.

RHOA G17V Induces T Follicular Helper Cell Specification and Promotes Lymphomagenesis

Angioimmunoblastic Tcell lymphoma (AITL) is an aggressive tumor derived from malignant transformation of T follicular helper (Tfh) cells. AITL is characterized by loss-of-function mutations in Ten-Eleven Translocation 2 (TET2) epigenetic tumor suppressor and a highly recurrent mutation (p.Gly17Val) in the RHOA small GTPase. Yet, the specific role of RHOA G17V in AITL remains unknown. Expression of Rhoa G17V in CD4+ Tcells induces Tfh cell specification; increased proliferation associated with inducible co-stimulator (ICOS) upregulation and increased phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase signaling. Moreover, RHOA G17V expression together with Tet2 loss resulted in development of AITL in mice. Importantly, Tet2-/-RHOA G17V tumor proliferation invivo can be inhibited by ICOS/PI3K-specific blockade, supporting a driving role for ICOS signaling in Tfh cell transformation.

The Loss of TET2 Promotes CD8+ T Cell Memory Differentiation.

T cell differentiation requires appropriate regulation of DNA methylation. In this article, we demonstrate that the methylcytosine dioxygenase ten-eleven translocation (TET)2 regulates CD8+ T cell differentiation. In a murine model of acute viral infection, TET2 loss promotes early acquisition of a memory CD8+ T cell fate in a cell-intrinsic manner without disrupting Ag-driven cell expansion or effector function. Upon secondary recall, TET2-deficient memory CD8+ T cells demonstrate superior pathogen control. Genome-wide methylation analysis identified a number of differentially methylated regions in TET2-deficient versus wild-type CD8+ T cells. These differentially methylated regions did not occur at the loci of differentially expressed memory markers; rather, several hypermethylated regions were identified in known transcriptional regulators of CD8+ T cell memory fate. Together, these data demonstrate that TET2 is an important regulator of CD8+ T cell fate decisions.

TET2 Loss Dysregulates the Behavior of Bone Marrow Mesenchymal Stromal Cells and Accelerates Tet2-/--Driven Myeloid Malignancy Progression.

SummaryTET2 is a methylcytosine dioxygenase that regulates cytosine hydroxymethylation. Although there are extensive data implicating a pivotal role of TET2 in hematopoietic stem/progenitor cells (HSPCs), the importance of TET2 in bone marrow mesenchymal stromal cells (BMSCs) remains unknown. In this study, we show that loss of TET2 in BMSCs increases cell proliferation and self-renewal and enhances osteoblast differentiation potential of BMSCs, which may in turn alter their behavior in supporting HSPC proliferation and differentiation. In addition, Tet2 loss alters BMSCs in promoting Tet2-deficiency-mediated myeloid malignancy progression. Tet2 loss in BMSCs also dysregulates hydroxylation of 5-methylcytosine (5mC) and the expression of genes that are key for BMSC proliferation and osteoblast differentiation, leading to alteration of biological characteristics in vivo. These results highlight the critical role of TET2 in the maintenance of BMSC functions and osteoblast differentiation and provide evidence that dysregulation of epigenetic modifiers in BMSCs contributes to the progression of myeloid malignancies.

Cooperative Epigenetic Remodeling by TET2 Loss and NRAS Mutation Drives Myeloid Transformation and MEK Inhibitor Sensitivity

Mutations in epigenetic modifiers and signaling factors often co-occur in myeloid malignancies, including TET2 and NRAS mutations. Concurrent Tet2 loss and NrasG12D expression in hematopoietic cells induced myeloid transformation, with a fully penetrant, lethal chronic myelomonocytic leukemia (CMML), which was serially transplantable. Tet2 loss and Nras mutation cooperatively led to decrease in negative regulators of mitogen-activated protein kinase (MAPK) activation, including Spry2, thereby causing synergistic activation of MAPK signaling by epigenetic silencing. Tet2/Nras double-mutant leukemia showed preferential sensitivity to MAPK kinase (MEK) inhibition in both mouse model and patient samples. These data provide insights into how epigenetic and signaling mutations cooperate in myeloid transformation and provide a rationale for mechanism-based therapy in CMML patients with these high-risk genetic lesions.

Abstract IA26: Role of mutations in epigenetic regulators in pathogenesis of myeloid malignancies

Abstract Clinical, cytogenetic, and gene-based studies have been used to inform biology and improve prognostication for patients with acute myeloid leukemia (AML), myelodysplasia (MDS), and myeloroliferative neoplasms (MPN). Most recently, a series of candidate gene and whole-genome studies have identified recurrent somatic mutations in AML patients including TET2, ASXL1, DNMT3A, and cohesin complex mutations. Moreover, these mutations can be used to improve risk stratification in AML independent of established clinical parameters. Integrating mutational data with dose-intensity revealed that high-dose daunorubicin improved survival in patients with DNMT3A/NPM1 mutations or MLL translocations relative to treatment with standard-dose daunorubicin, but not in patients wild-type for these alterations. These data provide important clinical implications of genetic alterations in AML by delineating mutation combination genotypes that predict outcome in AML and improve AML risk stratification. Of biologic importance, the TET family of proteins have been shown to place a hydroxyl mark on methylated DNA and lead to DNA demethylation. We and others have found that TET2/IDH mutations lead to loss of DNA hydroxymethylation and a hypermethylation phenotype in leukemia patients. In addition, in vitro and in vivo studies show that TET2 loss or neomorphic IDH1/2 mutations lead to impaired hematopoietic differentiation, increased stem cell self-renewal, and myeloid transformation in vivo. We have also investigated the role of mutant DNMT3A in AML pathogenesis, and shown that these mutations cooperate with other disease alleles to induce leukemic transformation and to confer chemoresistance due to impaired DNA damage sensing. We will present novel data showing how these mutations coopt the epigenetic state of hematopoietic stem/progenitor cells in order to contribute to transformation and that these mutations have biologic and prognostic relevance. Citation Format: Ross Levine. Role of mutations in epigenetic regulators in pathogenesis of myeloid malignancies [abstract]. In: Proceedings of the Second AACR Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; May 6-9, 2017; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(24_Suppl):Abstract nr IA26.

TET1 deficiency attenuates the DNA damage response and promotes resistance to DNA damaging agents

ABSTRACTRecent studies have shown that loss of TET1 may play a significant role in the formation of tumors. Because genomic instability is a hallmark of cancer, we examined the potential involvement of 10-11 translocation 1 (TET1) in the DNA damage response (DDR). Here we demonstrate that, in response to clinically relevant doses of ionizing radiation (IR), human glial cells made TET1-deficient with lentiviral vectors displayed greater numbers of colony forming units and lower levels of apoptotic markers compared with glial cells transduced with control vectors; yet, they harbored greater DNA strand breaks. The G2/M check point and expression of cyclin B1 were greatly diminished in TET1-deficient cells, and TET1-deficient cells displayed lower levels of γH2A.x following exposure to IR. Levels of DNA-PKcs, which are DNA-PK complex members, were lower in TET1-deficient cells compared with control cell lines. However, levels of ATM were similar in both cell lines. Cyclin B1, DNA-PKcs, and γH2A.x levels were each rescued by reintroduction of the TET1 catalytic domain. Finally, cytosine methylation within intron 1 of PRKDC, the gene encoding DNA-PKcs, was significantly higher upon depletion of TET1. Taken together, this study illustrates the involvement of TET1 in the different arms of the DDR and suggests its loss results in the continued survival of cells with genomic instability.

Role of mutations in epigenetic regulators in the pathogenesis of AML

Clinical, cytogenetic, and gene-based studies have been used to inform biology and improve prognostication for patients with acute myeloid leukemia (AML). Candidate gene and whole genome studies have identified recurrent somatic mutations in AML patients including TET2, ASXL1, DNMT3A, and cohesin complex mutations. We and others have found that TET2/IDH mutations leads to loss of DNA hydroxymethylation and a hypermethylation phenotype in leukemia patients. In addition, in vitro and in vivo studies show that TET2 loss or neomorphic IDH1/2 mutations leads to impaired hematopoietic differentiation, increased stem cell self-renewal, and myeloid transformation in vivo. We have also investigated the role of cohesin mutations in AML pathogenesis, and shown these mutations cooperate with other disease alleles to induce leukemic transformation and to alter gene regulatory networks which impact self-renewal and differentiation. We will present novel data showing how these mutations coopt the epigenetic state of hematopoietic stem/progenitor cells in order to contribute to transformation and that these mutations have biologic and prognostic relevance.