Ten-eleven Translocation 2 Mutations Research Articles

Analyzing Differences in Hematological and Immunological Characteristics Related to Common Gene Mutations in Myelodysplastic Syndromes.

Genetic mutations play a crucial role in the development and progression of myelodysplastic syndromes (MDS), impacting the immune microenvironment and influencing the choice of treatment regimen, as well as the efficacy and prognosis of patients. The objective of this study was to examine variations in hematological and immunological characteristics associated with common gene mutations in MDS patients and establish a foundation for the precise treatment of MDS. The hematological, immunological, and other clinical features of 71 recently diagnosed MDS patients from January 1, 2019, to July 31, 2023, were retrospectively analyzed. These patients were categorized based on their gene mutations, and the variances in hematological and immunological characteristics among distinct groups were compared. Hematological variances were observed among different gene mutation groups. Specifically, platelet counts in the splicing factor 3B subunit 1 (SF3B1) mutation group were notably higher compared to the wild-type group (p = 0.009). Conversely, in the additional sex combs like 1 (ASXL1) mutation groups, monocyte ratios were significantly elevated in comparison to the wild-type group (p = 0.046), and in the ten-eleven translocation 2 (TET2) mutation group, lymphocyte ratios were significantly lower (p = 0.022). Additionally, the leukocyte (p = 0.005), neutrophil ratio (p = 0.002), and lymphocyte ratio (p = 0.001) were significantly higher in the Runt-related transcription factor 1 (RUNX1) mutation group. Regarding immunological distinctions, the Natural Killer (NK) cell ratio demonstrated a significant increase in the SF3B1 mutation group (p = 0.005). Moreover, the TET2 mutation group exhibited a significantly higher Interleukin-8 (IL-8) level (p = 0.017). In contrast, the U2 small nuclear RNA auxiliary factor 1 (U2AF1) group displayed significantly lower levels of IL-1β (p = 0.033), IL-10 (p = 0.033), and Tumour Necrosis Factor-α (TNF-α) (p = 0.009). Distinct variations exist in the immune microenvironment of MDS associated with different genetic mutations. Further studies are imperative to delve into the underlying mechanisms that drive these differences.

STING activation in TET2-mutated hematopoietic stem/progenitor cells contributes to the increased self-renewal and neoplastic transformation

Somatic loss-of-function mutations of the dioxygenase Ten-eleven translocation-2 (TET2) occur frequently in individuals with clonal hematopoiesis (CH) and acute myeloid leukemia (AML). These common hematopoietic disorders can be recapitulated in mouse models. However, the underlying mechanisms by which the deficiency in TET2 promotes these disorders remain unclear. Here we show that the cyclic guanosine monophosphate-adenosine monophosphate synthase (cGAS)-stimulator of interferon genes (STING) pathway is activated to mediate the effect of TET2 deficiency in dysregulated hematopoiesis in mouse models. DNA damage arising in Tet2-deficient hematopoietic stem/progenitor cells (HSPCs) leads to activation of the cGAS-STING pathway which in turn promotes the enhanced self-renewal and development of CH. Notably, both pharmacological inhibition and genetic deletion of STING suppresses Tet2 mutation-induced aberrant hematopoiesis. In patient-derived xenograft (PDX) models, STING inhibition specifically attenuates the proliferation of leukemia cells from TET2-mutated individuals. These observations suggest that the development of CH associated with TET2 mutations is powered through chronic inflammation dependent on the activated cGAS-STING pathway and that STING may represent a potential target for intervention of relevant hematopoietic diseases.

The Long Non-Coding RNA MALAT1 Is Necessary for Hematopoietic Stem and Progenitor Cell Expansion and Preleukemic Myeloproliferation in TET2 Deficient Myeloid Neoplasms

Ten Eleven Translocation 2 (TET2) is a dioxygenase which regulates gene expression by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine. Loss of function mutations in TET2 occur in up to 58% and 30% of myeloid malignancies and clonal hematopoiesis respectively. Loss of TET2 function also occurs with isocitrate dehydrogenase 2 (IDH2) mutations in Acute Myeloid Leukemia (AML) via 2-hydroxyglutarate (2-HG). Genetically engineered murine models (GEMM) of Tet2 deficiency produce a Chronic Myelomonocytic Leukemia (CMML)-like phenotype with splenomegaly, granulomonocytic expansion and increased bone marrow hematopoietic stem and progenitors (LSK). Further, bone marrow mononuclear cell (BMNC) transplant models of Tet2-/- clonal hematopoiesis suggest that this myeloid expansion leads to enhanced inflammation and increased cardiovascular disease risk. However, the downstream effects of TET2 loss of function leading to this phenotype are unknown. Here we show that TET2-deficient granulomonocytic expansion and inflammation are mediated via MALAT1, a long non-coding RNA overexpressed in several malignancies. MALAT1 expression was increased in BMNCs from IDH2 mutated AML patients (n=152 WT n=17 mutant IDH2, p=0.04). Increased MALAT1 expression was also seen in human and murine models of TET2 deficiency including isogenic IDH2 mutated TF-1 cells (5 biological replicates p=0.005), 2-HG treated leukemia cell lines (2 biological replicates p<0.0001) and BMNCs from Tet2f/f mice (n= 7/grp p=0.005). To evaluate the impact of Malat1 on normal and Tet2 deficient hematopoiesis we utilized the Malat1tg overexpression and Tet2-/-Malat1-/-GEMM models. Similar to Tet2-/- mice, bone marrow (n=4/grp p=0.02) and spleen (n=8/grp p=0.004) LSK numbers were increased in Malat1tg mice compared to control. However, unlike Tet2-/-LSKs, MALAT1tgLSKs did not show increased self-renewal in serial in vivo competitive transplants. Further, depleting Malat1 in Tet2-/- mice did not rescue the increased self-renewal capacity seen in Tet2-/- LSKs. Collectively, these data suggest that Malat1 overexpression is sufficient for LSK expansion but does not impact LSK function. Splenomegaly, myeloproliferation and extramedullary hematopoiesis is a phenotype termed Preleukemic Myeloproliferation (PMP) and is predictive of myeloid neoplasm development in Tet2-/- models (Meisel M et al, Nature 2018). Consistent with a role of Malat1 in PMP (Fig A), Malat1 overexpression recapitulated PMP at 8 (n=10/grp p<0.0001) and 20 (n=10/grp p=0.0005) weeks. Malat1 overexpression was also associated with an increase in classical monocytes in the peripheral blood- a hallmark of CMML (8 weeks n= 7/grp p<0.0001, 20 weeks n=10/grp p=0.0007). This was recapitulated in Tet2-/-mice (n=3/grp p=0.001) and CMML patients with TET2 mutations (n=27 mutant TET2 v 109 WT, odds ratio 5.6 p= 0.001). Finally, depleting Malat1 in a Tet2-deficient GEMM rescued PMP (n=4 Tet2-/-Malat1-/-v 5 Tet2-/-p=0.01) and classical monocytosis (n=2/grp p=0.002) suggesting that Malat1 overexpression is both sufficient and necessary for PMP in Tet2 deficient leukemia. This impact was also observed in recipients in BMNC competitive transplant experiments (Fig B). Initial insights into the molecular mechanism underlying the role of Malat1 in PMP were derived from analyzing competitor and host cells during in vivo competitive transplants. Interestingly, transplant recipients with Malat1tg LSK demonstrated myeloid and granulomonocytic expansion in both donor and non-donor cells suggesting that the effects of Malat1 overexpression are, in part, cell extrinsic. Consistent with this hypothesis and the impact of NF-kB on inflammatory cytokine secretion and Tet2-/- PMP, we show that MALAT1 deletion in human leukemia attenuates NF-kB activity by enhancing the phosphatase activity of PP2A. Further, Interleukin 6 (IL-6), a major downstream target of NF-kB and potent activator of myelopoiesis in response to inflammatory stimuli, was increased in the plasma of MALAT1tg mice (n=8/grp p=0.01) suggesting a possible cell-extrinsic mechanism for Malat1 dependent PMP. Ongoing studies are focused on demonstrating that IL-6 is necessary for Malat1 dependent PMP. Our current data provides hitherto unknown insights establishing MALAT1 as a major effector of TET2 loss of function which may lead to novel therapeutic options in TET2 deficient myeloid neoplasms. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

JAK2 and TET2 Mutation in Polycythemia Vera

The Ten-Eleven Translocation-2 (TET2) gene, located on chromosome 4q24, has been implicated in hematological malignancies. The TET2 gene shows mutations in variable myeloid malignancies with the involvement of 15% of myeloproliferative neoplasms (MPNs). The inactivation of the TET2 gene in both mice and humans has shown a high degree of deregulation of the hematopoiesis process leading to hematological malignancies. Polycythemia vera (PV), an MPN characterized by increased red blood cell mass, has been associated with the TET2 gene. Furthermore, TET2 genes have been found to facilitate Janus kinase-2 and signal transducer activator of transcription 5, as well as modulate the epigenetic composition of genomic DNA. However, little is known about the role of TET2 mutations in patients with PV. Several studies have been conducted to further assess the significant role of TET2 gene function in various disease processes and prognoses to enhance the management and care of these patients.

TET2: A cornerstone in normal and malignant hematopoiesis.

Regulation of genome‐wide DNA methylation is fundamental for a variety of biological processes such as mammalian development, stem cell function, cellular proliferation/differentiation, and oncogenesis. Among the regulators of DNA methylation, ten‐eleven translocation 2 (TET2) is one of the most frequently mutated genes in clonal hematopoiesis of indeterminate potential and in various hematological malignancies, underscoring a pivotal role for TET2 in blood homeostasis and hematopoietic transformation. TET2 oxidizes methylated cytosines to further modify cytosines, which behave as intermediates in active/passive DNA demethylation processes. TET2 itself associates with histone modifiers, thereby regulating histone modifications and expression of target genes. A number of studies have reported pleiotropic effects of TET2 on hematopoietic stem cell self‐renewal, hematopoietic differentiation, genome instability and inflammatory response. Recent single‐cell genomics studies have identified gene promoters as well as transcription factor binding sites as TET2‐targeted genetic loci in which disruption of DNA methylation can fundamentally modify hematopoietic differentiation and promote leukemogenesis. TET2 mutations show convergent cooperativity with other disease alleles in signaling molecules, epigenetic modifiers, and spliceosome factors in hematopoietic transformation. Future studies focusing on the molecular basis of stem cell and immune regulation by TET2 loss will further deepen our understanding of the entire landscape of pathophysiology and molecular vulnerabilities of TET2‐mutated hematological malignancies.

Mutations in genes affecting DNA methylation enhances responses to decitabine in patients with myelodysplastic syndrome.

Background/AimsIn this study, we tested whether mutations in the methylation pathway genes ten-eleven-translocation 2 (TET2) and DNA methyltransferase gene 3A (DNMT3A) improve the responses of patients with myelodysplastic syndrome (MDS) to decitabine.MethodsWe retrospectively sequenced the TET2 and DNMT3A genes from 70 patients diagnosed with de novo MDS between June 2008 and December 2011 and treated with a 5-day regimen of decitabine (290 cycles). We then analyzed treatment outcomes.ResultsPatients with hematological improvement survived longer than those without hematological improvement (22.9 months vs. 10.9 months, p = 0.006). Among the 70 patients, 12 (17.1%) carried TET2 or DNMT3A mutations. The baseline characteristics of patients with wild type or mutated genes were similar. Patients with mutations in TET2 or DNMT3A had a higher overall response rate than those with the wild type genes (82.3% vs. 46.6%, p = 0.023). Multivariate analysis demonstrated that the TET2 or DMNT3A mutation status was associated with improved treatment responses and better overall survival among patients receiving decitabine.ConclusionsThese results demonstrate that TET2 mutations enhance the treatment response of MDS patients to hypomethylating agents like decitabine.

Loss of Tet2 affects platelet function but not coagulation in mice.

Ten-eleven translocation 2 (TET2) functions as a methylcytosine dioxygenase that catalyzes the iterative oxidation of 5-methylcytosine to 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine. TET2 has been shown to be crucial for the maintenance and differentiation of hematopoietic stem cells, and its deletion and/or mutations results in the expansion of HSPCs, and leads to hematological malignancies. TET2 mutations were found in a variety of hematological disorders such as CMML (60%), MDS (30%), MPN (13%) and AML (20%). Interestingly, it was shown that CMML patients with TET2 mutation exhibited fewer platelets than CMML patients without TET2 mutation. However, the role and function of TET2 in platelet hemostasis and thrombogenesis is not well defined. Here in this study, using a genetically engineered Tet2 deletion mouse model, we found that the absence of Tet2 caused a decrease in the proportion of MEP cells and hyperploid megakaryocytes. Additionally, Tet2-deficient mice displayed impaired platelet activation and aggregation under stimulation of ADP and low concentrations of thrombin, although the modestly compromised platelet function and MEP differentiation in Tet2-deficient mice could be compensated without affecting blood coagulation function. Our study indicate that Tet2 deficiency leads to mild impairment of platelet function and thrombopoiesis in mice.

FGFR3\u25b37\u20139 promotes tumor progression via the phosphorylation and destabilization of ten-eleven translocation-2 in human hepatocellular carcinoma

Overexpression of fibroblast growth factor receptor 3 (FGFR3) correlates with more severe clinical features of hepatocellular carcinoma (HCC). Our previous study has shown that FGFR3∆7–9, a novel splicing mutation of FGFR3, contributes significantly to HCC malignant character, but the epigenetic mechanism is still elusive. In this study, through mass spectrometry and co-immunoprecipitation studies, we discover a close association between FGFR3∆7–9 and the DNA demethylase Ten-Eleven Translocation-2 (TET2). Unlike other certain types of cancer, mutation of TET2 is rare in HCC. However, activation of FGFR3∆7–9 by FGF1 dramatically shortens TET2 half-life. FGFR3∆7–9, but not wild-type FGFR3, directly interacts with TET2 and phosphorylates TET2 at Y1902 site, leading to the ubiquitination and proteasome-mediated degradation of TET2. Overexpression of a phospho-deficient mutant TET2 (Y1902F) significantly reduces the oncogenic potential of FGFR3∆7–9 in vitro and in vivo. Furthermore, FGFR3∆7–9 significantly enhances HCC cell proliferation through the TET2-PTEN-AKT pathway. Specifically, TET2 offsets the elevation of p-AKT level induced by FGFR3∆7–9 through directly binding to PTEN promoter and increasing 5-hmC. Therefore, through phosphorylation and inhibition of TET2, FGFR3∆7–9 reduces PTEN expression and substantiates AKT activation to stimulate HCC proliferation. Together, this study identifies TET2 as a key regulator of the oncogenic role of FGFR3∆7–9 in HCC carcinogenesis and sheds light on new therapeutic strategies for HCC treatment.

Changes in DNA 5-Hydroxymethylcytosine Levels and the Underlying Mechanism in Non-functioning Pituitary Adenomas.

Epigenetic factors have been proven to contribute to pituitary adenoma formation. 5-hydroxymethylcytosine (5hmC), which is catalyzed by ten-eleven translocation 2 (TET2), is related to DNA demethylation. In order to explore the pathogenesis of non-functioning pituitary adenomas (NFPAs), we detected genomic 5hmC levels in 57 NFPAs and 5 normal pituitary glands, and TET2 expression, distribution and TET2 alteration. Genomic 5hmC levels in NFPAs were significantly lower than those in normal pituitary glands (0.38‰ (0.24‰, 0.61‰) vs. 2.47‰ (1.56‰, 2.83‰), P < 0.0001). There was positive correlation of 5hmC levels with TET2 total and nuclear expression in NFPAs (r = 0.461, P = 0.018; r = 0.458, P = 0.019). Genomic 5hmC levels in NFPAs with TET2 p.P29R were significantly lower than those in wild type NFPAs (0.33 ± 0.18‰ vs. 0.51 ± 0.25‰, P = 0.021). We found genomic 5hmC loss in human NFPAs for the first time. Genomic 5hmC levels may be affected by TET2 expression, subcellular localization and TET2 mutation.

Characteristics of myeloid sarcoma in mice and patients with TET2 deficiency.

Myeloid sarcoma (MS) carries a poor prognosis, and information on epigenetic modifications in MS is currently limited. In the present study, 214 ten-eleven translocation-2 (TET2)−/− mice were successfully constructed. In addition, 436 patients with myelodysplastic syndrome (MDS) and 354 with acute myeloid leukemia (AML) patients were recruited. The incidence of MS in mice and patients with TET2 deficiency was examined, and the efficiency of hypomethylating agents (HMAs) was also be evaluated. A total of 93% of the TET2−/− mice developed myeloid malignancies, 5.5% of which were accompanied by MS (n=11). The survival of these TET2−/− mice ranged between 3 and 25 months. No significant difference was observed between the survival of MS and non-MS mice with TET2 loss (P>0.05). In addition, MS cells were transplantable, and their recipients exhibited myeloproliferative characteristics, such as increased white blood cell counts, monocytosis, low erythrocyte counts and hepatosplenomegaly. Their median survival duration was 94.8 days. In the clinical setting, 9.7% of MDS and 11.6% of AML patients with TET2 deficiency developed MS, which was higher compared with previous reports (1.5–9.1%). The median age of the MS patients was 44 years old. 5-Aza-2′-deoxycytidine (5-Aza-dC) reduced the incidence of MS in TET2−/− mice, and decitabine was a suitable treatment strategy for MS patients. These data indicate that TET2 deficiency plays a key role in MS and its prognostic significance requires further investigation. HMAs may be a useful treatment for MS patients with TET2 mutations.

Molecular pathogenesis of progression to myeloid leukemia from TET-insufficient status

AbstractLoss-of-function mutations in ten-eleven translocation-2 (TET2) are recurrent events in acute myeloid leukemia (AML) as well as in preleukemic hematopoietic stem cells (HSCs) of age-related clonal hematopoiesis. TET3 mutations are infrequent in AML, but the level of TET3 expression in HSCs has been found to decline with age. We examined the impact of gradual decrease of TET function in AML development by generating mice with Tet deficiency at various degrees. Tet2f/f and Tet3f/f mice were crossed with mice expressing Mx1-Cre to generate Tet2f/wtTet3f/fMx-Cre+ (T2ΔT3), Tet2f/fTet3f/wtMx-Cre+ (ΔT2T3), and Tet2f/fTet3f/fMx-Cre+ (ΔT2ΔT3) mice. All ΔT2ΔT3 mice died of aggressive AML at a median survival of 10.7 weeks. By comparison, T2ΔT3 and ΔT2T3 mice developed AML at longer latencies, with a median survival of ∼27 weeks. Remarkably, all 9 T2ΔT3 and 8 ΔT2T3 mice with AML showed inactivation of the remaining nontargeted Tet2 or Tet3 allele, respectively, owing to exonic loss in either gene or stop-gain mutations in Tet3. Recurrent mutations other than Tet3 were not noted in any mice by whole-exome sequencing. Spontaneous inactivation of residual Tet2 or Tet3 alleles is a recurrent genetic event during the development of AML with Tet insufficiency.

Coexistence of ten-eleven translocation 2 and calreticulin mutations in myeloproliferative neoplasms: Possible prognostic value

Background: Mutations in the genes regulating epigenetic factors such as Ten-Eleven Translocation 2 (TET2) along with other mutations are highly effective in the patient's prognosis. The aim of this study was to evaluate the prevalence of TET2 and calreticulin (CALR) mutations among myeloproliferative neoplasms (MPNs) patients to determine if there is a prognostic value for these mutations coexistence. Materials and Methods: Blood sample was collected from patients. For the evaluation of mutations, polymerase chain reaction (PCR) was performed on the patient's DNA and PCR products were subsequently sequenced. Results: No significant correlation between coexistence of TET2 and CALR mutations with complete blood count parameters in patients was seen. However, platelet count was lower in patients with TET2 and CALR mutations compared with patients with CALR mutations only. Conclusion: The coexistence of CALR and TET2 mutations in MPN patients can be used as a prognostic factor.

Impact of Mutations in Epigenetic Modifiers in Acute Myeloid Leukemia: A Systematic Review and Meta-Analysis

Objectives: Mutations in epigenetic modifiers including IDH (Isocitrate dehydrogenase), DNMT3A (DNA Methyltransferase 3 Alpha), TET2 (Ten-Eleven Translocation 2), ASXL1 (Additional Sex Combs-Like 1), and EZH2 (Enhancer of Zeste Homolog 2) have been recently recognized in patients with Acute Myeloid Leukemia (AML). The DNMT3A and IDH were found to be associated with poor prognosis in most studies; however, there is no meta-analysis evaluating other mutations in the epigenetic modifiers. We performed a systematic review and meta-analysis to evaluate the prognostic significance of mutations in the epigenetic modifiers on the Overall Survival (OS) of patients with AML. Methods: We searched for studies evaluating epigenetic mutations in AML (up to August 2018) in PubMed. Two reviewers independently screened all citations and later abstracted data using a common data collection form. Risk of bias was assessed using Newcastle-Ottawa Scale (NOS) and Cohen kappa coefficient (k) was calculated to assess the agreement. Hazard ratio (HR) and 95% confidence interval (CI) of outcomes were extracted and random effects model was used to pool the results. Statistical heterogeneity was calculated using Cochran I2 and tested using chi-squared tests. Results: A total of 9543 citations were retrieved from the search strategy; of which 9415 were excluded after title screening and 37 articles were excluded after abstract screening. A review of full text excluded additional 49 articles for incomplete data and missing analysis on OS. Forty-two articles were identified for the systematic review and meta-analysis (IDH n=15, DNMT3A n=12, TET2 n=8, ASXL1 =7), all of which were fair to good quality studies based on the NOS. The analysis of IDH mutation included 15 articles covering 5100 patients with a median age of 55 years. The frequency of IDH mutations ranged from 7 to 33%. The pooled HR indicated that IDH mutation conferred significantly worse OS (HR = 1.54; 95% CI: 1.15-2.06) though with a substantial heterogeneity (I2 = 85%). Mutation analysis of DNMT3A was based on 12 articles including 5418 patients with a median age of 52 years. The proportion of patients with this mutation ranged from 14 to 34%. Results indicated a worse OS with a pooled HR of 1.35; 95% CI 1.16-1.56, and a substantial heterogeneity (I2=71%). For the ASXL1 mutation, our analysis included 4115 adult and pediatric patients from 7 studies. The pooled HR indicated that ASXL1 mutation conferred a significantly worse OS (HR = 1.88; 95% CI: 1.49‒2.36); however, with a moderate heterogeneity (I2 = 43%). The TET2 mutation analysis was based on eight studies with a total of 3286 patients. The pooled HR indicated that TET2 mutation conferred a significantly worse OS (HR = 1.39; 95% CI: 1.18‒1.63); with a low heterogeneity (I2 = 28%). Conclusions: Epigenetic mutations in IDH, DNMT3A, ASXL1 and TET2 adversely impact OS in patients with AML. These mutations in the epigenetic modifiers should be considered in the initial evaluation of patients with AML and in the decision on post induction management. Disclosures Al-Khabori: Shire (Takeda): Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; SOBI: Honoraria; AstraZeneca: Honoraria; NovoNardisk: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Servier: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Mutation of ten-eleven translocation-2 is associated with increased risk of autoimmune disease in patients with myelodysplastic syndrome.

Background/AimsMyelodysplastic syndrome (MDS) is caused by genetic and epigenetic alteration of hematopoietic precursors and immune dysregulation. Approximately 20% of patients with MDS develop an autoimmune disease (AID). Here, we investigated whether particular genetic mutations are associated with AID in patients with MDS.MethodsEighty-eight genetic mutations associated with myeloid malignancy were sequenced in 73 MDS patients. The association between these mutations and AID was then analyzed.ResultsThe median age of the 73 MDS patients was 70 years (interquartile range, 56 to 75), and 49 (67.1%) were male. AID was observed in 16 of 73 patients (21.9%). Mutations were detected in 57 (78.1%) patients. The percentage (68.8% vs. 80.7%, p = 0.32) and the mean number of mutations (1.8 ± 1.6 vs. 2.2 ± 1.8, p = 0.34) in MDS patients with or without AID were similar. However, the ten-eleven translocation- 2 (TET2) mutation rate was significantly higher in patients with AID than in those without (31.3% vs. 5.3%, respectively; p = 0.001). All TET2 mutations were variants of strong clinical significance.ConclusionsMutation of TET2 in patients with MDS may be associated with increased risk of developing AID.

TET2 Function in Hematopoietic Malignancies, Immune Regulation, and DNA Repair.

Over the last decade, investigation of Ten-Eleven Translocation 2 (TET2) gene function and TET2 mutation have become of increasing interest in the field of hematology. This heightened interest was sparked by the seminal discoveries that (1) TET2 mutation is associated with development of hematological malignancies and that (2) the TET family of proteins is critical in promoting DNA demethylation and immune homeostasis. Since then, additional studies have begun to unravel the question “Does TET2 have additional biological functions in the regulation of hematopoiesis?” Here, we present a mini-review focused on the current understanding of TET2 in hematopoiesis, hematological malignancies, and immune regulation. Importantly, we highlight the critical function that TET2 facilitates in maintaining the stability of the genome. Based on our review of the literature, we provide a new hypothesis that loss of TET2 may lead to dysregulation of the DNA repair response, augment genome instability, and subsequently sensitize myeloid leukemia cells to PARP inhibitor treatment.

Abstract B170: Therapeutic implications of altered epigenetics and DNA damage responses in IDH2-mutated hematologic diseases

Abstract Acute myeloid leukemia (AML) and angioimmunoblastic T-cell lymphoma (AITL), are hematologic diseases requiring novel approaches to patient selection and therapy. In AITL, neoplastic cells express many markers of T follicular helper (TFH) cells, while in AML, myeloid stem and progenitor cells are affected. Tumor cells of AML and AITL patients frequently bear mutations affecting genes involved in epigenetic regulation, including isocitrate dehydrogenase (IDH) and ten-eleven translocation-2 (TET2). IDH mutations drive production of the rare metabolite D-2-hydroxyglutarate (2HG), which competitively inhibits α-ketoglutarate (α-KG)-dependent dioxygenases such as the TET proteins (affecting DNA methylation) and Jumonji histone demethylases (altering histone methylation). IDH mutations occur in 30% of AML and AITL cases but the spectrum of these mutations differs. In AML, IDH1R132 (41%), IDH2R140 (44%) and IDH2R172 (15%) dominate, and IDH and TET2 mutations are mutually exclusive. In AITL, IDH2R140 (2%) and IDH2R172 (98%) dominate (no IDH1 mutations), with co-mutation of TET2 in 82.1% of cases. In AML, loss of these dioxygenase activities causes epigenetic alterations to DNA and histones, leads to abnormal gene transcription that affects hematopoietic cell differentiation, and drives myeloid disease. However, in AITL disease, the impact of the IDH2 mutation is completely unknown. My goal is to dissect the effects of IDH2 and TET2 mutations in hematologic diseases to understand how IDH2 and TET2 mutations collaborate to drive malignancy in AITL, and why they cooperate differently in myeloid and lymphoid diseases. I focus on DDR signaling and epigenetics analysis to found novel therapeutic vulnerabilities arise from the altered epigenetic regulation and DDR linked to IDH2 and TET2 mutations. I used a CD4-Cre mouse model to introduce the IDH2R172K and TET2 mutations in the T-cell compartment. This would allow me to study the collaboration between IDH2 and TET2 mutations in the context of T-cells. Mice bearing both IDH2 and TET2 mutations show decreased survival, with a median survival of approximately 8 months accompanied by splenomegaly and lymphadenopathy. This is significantly different than CD4+ control mice or single mutant mice. Double mutant mice (DM) present a disruption of spleen and lymph node architecture. To understand this phenotype, I performed comprehensive flow cytometry staining and, surprisingly, I found that the increased spleen size is not due to T-cell infiltration, but is due to increased erythropoiesis and expansion of immature erythropoietic cells (CD71+, cKit+). We can explain this result by the fact that CD4-Cre is also expressed in some progenitor cells leading to stress induced erythropoiesis. However, if we focus on the T-cell population, we see that DM mice present a T-cell phenotype including a decrease of CD4+ naïve cells and an increase of CD4+ effector memory cells as early as 5 months. So, there is an imbalance in T-cell homeostasis, but only when both the IDH2 and TET2 mutations are present, suggesting a cooperative role for both mutations in T-cell development. Since both IDH2 and TET2 affect epigenetic regulation, I want to conduct experiments to understand how these changes modulate T-cells homeostasis. Moreover, emerging data support the hypothesis that connections exist between epigenetic regulators and DDR signaling in hematologic diseases. My final aim will be to attempt to evaluate the efficacy of treatment with IDH2 inhibitors, hypomethylating agents, or DDR-targeting drugs, alone or in combination in AITL disease and identify factors involved in responses to these therapies or in the development of resistance. Finally, I will compare results obtained in AML versus AITL mouse models and clinical samples to identify mechanisms that are shared, and those that are unique to each disease. Citation Format: Julie Leca. Therapeutic implications of altered epigenetics and DNA damage responses in IDH2-mutated hematologic diseases [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B170.

Diverse Landscape of TET2 Variants in MDS and AML

INTRODUCTION The TET2 (Ten‐Eleven Translocation 2) gene, located at chromosome 4q24.1, belongs to TET family of proteins that possesses the capacity of catalyzing the conversion of 5‐methylcytosine into 5‐hydroxymethylcytosine. It is considered to be a putative tumor suppressor gene during cancer initiation and development. TET2 mutations are extensively studied and reported in a variety of human hematological malignancies including acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS), suggesting a crucial role of TET2 in the pathogenesis of blood cancers. The landscape of rare TET2 variants which may be of potential clinical relevance is not fully described. The landscape of TET2 variants from a large cohort of MDS and AML patients was explored. METHODS We analyzed data from bone marrow samples of 2634 patients with AML or MDS who presented at MD Anderson between the years 2012-2016. Seven hundred seventy seven patients were found to have TET2 variants that were not classifiable as known mutations. Two hundred sixty-three patients were diagnosed with MDS, 144 with secondary AML transformed from MDS, and 370 with AML without prior history of MDS. The coding regions of TET2 gene was analyzed by next-generation sequencing in the molecular diagnostics laboratory. Non-mutation variants were classified into 2 groups: 1) those reported as germline polymorphisms (GP) in population studies, literature or matched tumor-normal analysis on patients at MDACC; and 2) variants for which a germline versus somatic origin could not be determined unequivocally (variants of uncertain origin, VUO). Very common GP variants (defined as those with a population frequency of over 20% in laboratory sample cohort) were not evaluated. Variants9 predicted frequency in the population according to gnomAD Exomes 2.0.2, and classification according to American College of Medical Genetics and Genomics (ACMG) guidelines for interpretation of sequence variants, were assessed using the VarSome platform by Saphetor. RESULTS Two-hundred and twenty-eight unique variants were identified. The amino acid substitutions from missense variants were widely distributed throughout TET2. Fifty-four unique variants were classified as GP and several of these recurrent variants were found to be present in our population at a significantly higher frequency (p CONCLUSIONS The landscape of rare TET2 variants in a large group of MDS and AML patients was frequent, diverse, and included both GP and VUO. GP variants were more likely benign by ACMG classification and found in both MDS and AML patients. A large number of singleton VUO were found among MDS patients, and many were predicted to be pathogenic or had uncertain significance. VUO9s also contained many fs and nonsense variants. These result support the idea that rare pathogenic variants in TET2 are currently underrecognized as contributory, although they are likely to help drive leukemogenesis. Greater understanding of the importance of individual TET2 variants and additional classification metrics are needed. Disclosures DiNardo:Karyopharm: Honoraria; Abbvie: Honoraria; Agios: Consultancy; Medimmune: Honoraria; Celgene: Honoraria; Bayer: Honoraria. Daver:Karyopharm: Research Funding; Incyte: Consultancy; Novartis: Consultancy; ImmunoGen: Consultancy; Sunesis: Consultancy; Pfizer: Consultancy; Daiichi-Sankyo: Research Funding; Kiromic: Research Funding; Alexion: Consultancy; Otsuka: Consultancy; Sunesis: Research Funding; Incyte: Research Funding; ARIAD: Research Funding; BMS: Research Funding; Karyopharm: Consultancy; Novartis: Research Funding; Pfizer: Research Funding. Kadia:Celgene: Research Funding; Takeda: Consultancy; BMS: Research Funding; Abbvie: Consultancy; Novartis: Consultancy; Novartis: Consultancy; Amgen: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Abbvie: Consultancy; Pfizer: Consultancy, Research Funding; Takeda: Consultancy; Amgen: Consultancy, Research Funding; BMS: Research Funding; Pfizer: Consultancy, Research Funding; Jazz: Consultancy, Research Funding; Celgene: Research Funding. Konopleva:Stemline Therapeutics: Research Funding; abbvie: Research Funding; cellectis: Research Funding; Immunogen: Research Funding. Andreeff:Amgen: Consultancy, Research Funding; Astra Zeneca: Research Funding; Jazz Pharma: Consultancy; Oncolyze: Equity Ownership; Aptose: Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Oncoceutics: Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; SentiBio: Equity Ownership; United Therapeutics: Patents & Royalties: GD2 inhibition in breast cancer ; Reata: Equity Ownership; Celgene: Consultancy; Eutropics: Equity Ownership, Membership on an entity9s Board of Directors or advisory committees; Daiichi-Sankyo: Consultancy, Patents & Royalties: MDM2 inhibitor activity patent, Research Funding.

Successful Treatment of Cytogenetically Normal Acute Myeloid Leukemia With Ten-Eleven Translocation 2–Isocitrate Dehydrogenase 2 and Additional Sex Comb-like 1–Nucleophosmin Co-mutations by HLA Haploidentical Stem Cell Transplantation: A Case Report and Literature Review

The presence of recurrent gene mutations is increasingly important in acute myeloid leukemia (AML) and sheds new insights into the understanding of leukemogenesis, prognostic evaluation, and clinical therapeutic efficacy. Until now, ten-eleven translocation 2 (TET2) and isocitrate dehydrogenase 2 (IDH2) mutations were reported to be mutually exclusive in AML patients. Similarly, nucleophosmin (NPM1) and additional sex comb-like 1 (ASXL1) mutations were rarely coexisted in AML. A 47-year-old man diagnosed with high-risk AML presented simultaneous mutations of TET2–IDH2 and NPM1–ASXL1 revealed by next-generation sequencing. After successful treatment with chemotherapy followed by HLA haploidentical transplantation, he achieved a clinical complete remission without evidence of overt graft-versus-host disease. This case highlights that HLA haploidentical transplantation might be a safe and feasible therapy for AML patients who are characterized by TET2–IDH2 and NPM1–ASXL1 co-mutations.

TET2 expression is a potential prognostic and predictive biomarker in cytogenetically normal acute myeloid leukemia.

TET2 (Ten-Eleven Translocation 2) gene is a member of TET family that can modify DNA through catalyzing the conversion of 5-methylcytosine (5-mC) into 5-hydroxymethylcytosine (5-hmC). Although TET2 mutation has been disclosed in a variety of hematopoietic malignancies, the prognostic implication of TET2 expression in acute myeloid leukemia (AML) remains largely elusive. In this study, real-time quantitative PCR was carried out to detect the level of TET2 transcript in 134 de novo AML patients and 35 healthy donors. TET2 mRNA level was significantly down-regulated in AML patients compared with controls (p = 0.010). Among the French-American-British (FAB) subtypes, the incidence of TET2 under-expression in M0/M1 subtypes was significantly higher than in the other subtypes M2/M3/M4/M5/M6 (p = 0.017), and also markedly higher than in the other granulocyte subtypes M2/M3 (p = 0.005). TET2 low-expressed patients showed a significantly higher frequency of NPM1 mutations than TET2 high-expressed patients. Although there was no significant difference in complete remission rate between two groups (low and high TET2 expression), patients with low TET2 expression had markedly shorter overall survival (OS) in both non-M3 and cytogenetically normal AML (CN-AML) (p = 0.016 and 0.044, respectively). Furthermore, multivariate analysis confirmed the prognostic value of TET2 expression on OS among CN-AML patients (p = 0.049). Importantly, TET2 expression in complete remission (CR) time was significantly higher than newly diagnosis time (p = 0.001), and was returned to lower level when in relapse time (p < 0.001). These findings indicated that down-regulation of TET2 expression was a common event and acted as a prognostic and predictive biomarker in CN-AML patients.

Determination of CEBPA mutations in pediatric acute leukemia

The CCAAT/enhancer-binding protein-alpha (CEBPA) is lineage-specific transcription factor in the hematopoietic system. Several studies have reported the presence and frequency of CEBPA mutations.In this study, we aimed the clinical features and the prognostic significance associated with CEBPA mutations in 30 pediatric patients with acute leukemia.In addition, association between found variants and mutations of Ten-Eleven-Translocation 2 (TET2), Kirsten rat sarcoma viral oncogene homolog (KRAS), and Casitas B-cell lymphoma (CBL), Fms-Related Tyrosine Kinase (FLT3), Januse Kinase-2 (JAK2) and Nucleophosmin 1 (NPM1) were analyzed which are important prognostic risk factors for pediatric acute leukemia. The entire CEBPA coding region was screened using the Next Generation Sequencing (NGS) method. Furthermore, TET2, KRAS and CBL genes were screened using the NGS method, FLT3 gene was analyzed by Real Time Polymerase chain Reaction (Real Time-PCR).JAK2 and NPM1 genes were screened by Sanger DNA sequencing. CEBPA mutations were detected in 16 (53.3%) of 30 patients. In total, ten distinct of nucleotide changes were identified in 30 patients including 6 novel and 4 known mutations by sequencing the entire CEBPA gene. We found 6 frame shift mutations, 1 missense mutation, 3 synonymous variant. The most common mutation was the c.487del G resulting p.Glu163Ser in 5 cases. Three patients carried CEBPA double mutations. The detected variants in this article seem to be the first screening results of genes studied by NGS in pediatric acute leukemia patients. Our results also showed some degree of association between FLT3-ITD, TET2, KRAS, CBL and CEBPA mutations.