Tet2 Loss Research Articles

Enantiomer-Specific Transformation by 2HG Is Linked to Opposing Effects on α-Ketoglutarate-Dependent Dioxygenases

Abstract LBA-4 Somatic mutations in IDH1 and IDH2 occur frequently in clonal myeloid disorders and result in the neomorphic ability of IDH to convert α-ketoglutarate (2-OG) to the R-enantiomer of 2-hydroxyglutarate (R-2HG) (Dang, et al Nature 462: 739, 2009). 2OG is an essential cofactor for many metabolic enzymes, including the TET family of 5-methylcytosine hydroxylases and the EglN family of prolyl-4-hydroxylases, and 2HG has been shown to inhibit several 2OG-dependent dioxygenases in vitro, including TET2 (Xu, et al Cancer Cell 19: 17, 2011; Figueroa, et al Cancer Cell 18: 1, 2010). We recently showed that the (S) enantiomer of 2HG (S-2HG), but not the (R) enantiomer of 2HG (R-2HG), inhibits the EglN prolyl-4-hydroxylases (Koivunen, et al. Submitted for publication). Moreover, we found that R-2HG can act as a cofactor to promote the hydroxylase activity of EglN1, EglN2 and EglN3. We hypothesized that the qualitatively different effects of R- and S-2HG on the EglN prolyl-4-hydroxylases might influence their transforming activities. In order to elucidate the role of mutant IDH, and R- and S-2HG, in myeloid leukemia, we developed a myeloid transformation assay using TF-1 cells. TF-1 is a human erythroleukemia cell line that requires GM-CSF for growth and undergoes erythrocytic differentiation when stimulated with erythropoietin (EPO). We expressed wild-type IDH1 (WTIDH1), a tumor-derived mutant IDH1 (IDH1R132H), or a catalytically inactive IDH1R132H variant (IDH1R132H/3DN) in TF-1 cells. As expected, cells expressing IDH1R132H, but not cells expressing WTIDH1 or IDH1R132H/3DN, had dramatically elevated levels of 2HG. Furthermore, we found that expression of IDH1R132H, but not WTIDH1 or IDH1R132H/3DN, conferred growth factor-independence to TF-1 cells (Figure 1a), and blocked their EPO-induced differentiation (Figure 1b). In order to determine whether transformation of TF-1 cells by IDH1R132H is mediated by 2HG, we treated TF-1 cells with cell-permeable esterified R-2HG or S-2HG. R-2HG recapitulated the growth and differentiation phenotypes of IDH1R132H expression in a dose-dependent manner. In contrast, S-2HG did not induce these phenotypes at any concentration tested. Next, we examined the effect of loss of TET2 on TF-1 cells. We infected TF-1 cells with shRNAs targeting TET1 or TET2 and found that knockdown of TET2, but not TET1, induced growth factor-independence and blocked EPO-induced differentiation similarly to expression of IDH1R132H or treatment with R-2HG. Interestingly, we found that transformation by IDH1R132H and TET2 knockdown were reversed by inhibition of EglN1 (Figure 2), suggesting that R-2HG, but not S-2HG, transforms leukemic cells by inhibiting targets such as TET2 while preserving, and possibly enhancing, EglN activity. These findings further suggest that therapeutic targeting of EglN prolyl-4-hydroxylase activity might be effective in the treatment of IDH1-mutant and TET2-mutant myeloid leukemias. Disclosures: Kaelin: Fibrogen: Consultancy, Equity Ownership.

Enantiomer-Specific Transformation by 2HG Is Linked to Opposing Effects on α-Ketoglutarate-Dependent Dioxygenases

Abstract LBA-4Somatic mutations in IDH1 and IDH2 occur frequently in clonal myeloid disorders and result in the neomorphic ability of IDH to convert α-ketoglutarate (2-OG) to the R-enantiomer of 2-hydroxyglutarate (R-2HG) (Dang, et al Nature 462: 739, 2009). 2OG is an essential cofactor for many metabolic enzymes, including the TET family of 5-methylcytosine hydroxylases and the EglN family of prolyl-4-hydroxylases, and 2HG has been shown to inhibit several 2OG-dependent dioxygenases in vitro, including TET2 (Xu, et al Cancer Cell 19: 17, 2011; Figueroa, et al Cancer Cell 18: 1, 2010). We recently showed that the (S) enantiomer of 2HG (S-2HG), but not the (R) enantiomer of 2HG (R-2HG), inhibits the EglN prolyl-4-hydroxylases (Koivunen, et al. Submitted for publication). Moreover, we found that R-2HG can act as a cofactor to promote the hydroxylase activity of EglN1, EglN2 and EglN3. We hypothesized that the qualitatively different effects of R- and S-2HG on the EglN prolyl-4-hydroxylases might influence their transforming activities.In order to elucidate the role of mutant IDH, and R- and S-2HG, in myeloid leukemia, we developed a myeloid transformation assay using TF-1 cells. TF-1 is a human erythroleukemia cell line that requires GM-CSF for growth and undergoes erythrocytic differentiation when stimulated with erythropoietin (EPO). We expressed wild-type IDH1 (WTIDH1), a tumor-derived mutant IDH1 (IDH1R132H), or a catalytically inactive IDH1R132H variant (IDH1R132H/3DN) in TF-1 cells. As expected, cells expressing IDH1R132H, but not cells expressing WTIDH1 or IDH1R132H/3DN, had dramatically elevated levels of 2HG. Furthermore, we found that expression of IDH1R132H, but not WTIDH1 or IDH1R132H/3DN, conferred growth factor-independence to TF-1 cells (Figure 1a), and blocked their EPO-induced differentiation (Figure 1b). In order to determine whether transformation of TF-1 cells by IDH1R132H is mediated by 2HG, we treated TF-1 cells with cell-permeable esterified R-2HG or S-2HG. R-2HG recapitulated the growth and differentiation phenotypes of IDH1R132H expression in a dose-dependent manner. In contrast, S-2HG did not induce these phenotypes at any concentration tested. Next, we examined the effect of loss of TET2 on TF-1 cells. We infected TF-1 cells with shRNAs targeting TET1 or TET2 and found that knockdown of TET2, but not TET1, induced growth factor-independence and blocked EPO-induced differentiation similarly to expression of IDH1R132H or treatment with R-2HG. Interestingly, we found that transformation by IDH1R132H and TET2 knockdown were reversed by inhibition of EglN1 (Figure 2), suggesting that R-2HG, but not S-2HG, transforms leukemic cells by inhibiting targets such as TET2 while preserving, and possibly enhancing, EglN activity. These findings further suggest that therapeutic targeting of EglN prolyl-4-hydroxylase activity might be effective in the treatment of IDH1-mutant and TET2-mutant myeloid leukemias. [Display omitted] Disclosures:Kaelin:Fibrogen: Consultancy, Equity Ownership.

Epigenetic Effects of IDH1/IDH2 Mutations

Abstract SCI-33Epigenetic deregulation of gene expression through aberrant DNA methylation or histone modification plays an important role in the malignant transformation of hematopoietic cells. In particular, acute myeloid leukemias (AMLs) can be classified according to epigenetic signatures affecting DNA methylation or histone modifications affecting specific gene sets. Heterozygous somatic mutations in the loci encoding isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in ∼20% of AMLs and are accompanied by global DNA hypermethylation and hypermethylation and silencing of a number of specific gene promoters. IDH1/2 mutations are almost completely mutually exclusive with somatic loss-of-function mutations in TET2, which hydroxylates methylcytosine (mCpG). DNA hydroxymethylation can function as an intermediate step in mCpG demethylation. TET2 mutant de novo AMLs also display global and promoter specific hypermethylation partially overlapping with IDH1/2 mutant cases. Mutations in the IDH1/2 loci result in a neomorphic enzyme that generates the aberrant oncometabolite 2-hydroxyglutarate (2HG) using α-ketoglutarate (αKG) as a substrate. 2HG can disrupt the activity of enzymes that use αKG as a cofactor, including TET2 and the jumonji family of histone demethylases. Expression of mutant IDH isoforms inhibits TET2 hydroxymethylation and jumonji histone demethylase functions. IDH and TET2 mutant AMLs accordingly exhibit reduced levels of hydroxymethylcytosine and a trend towards increased histone methylation. Mutant IDH or TET2 loss of function causes differentiation blockade and expansion of hematopoietic stem cells and TET2 knockout results in a myeloproliferative phenotype in mice. Hydroxymethylcytosine is in abundance in hematopoietic stem cells and displays specific distribution patterns, yet the function of this covalent modification is not fully understood. Recent data link TET2 with the function of cytosine deaminases as a pathway towards DNA demethylation, which has implications as well for B cell lymphomas and CML lymphoid blast crisis, which are linked with the actions of activation induced cytosine deaminase. Altogether, the available data implicate mutations in IDH1/2 and TET2 in promoting malignant transformation in several tissues, by disrupting epigenomics programming and altering gene expression patterning. Disclosures:Thompson:Agios Pharmaceuticals: Consultancy.

Mutations and deletions of the SUZ12 polycomb gene in myeloproliferative neoplasms

Mutations and deletions of the SUZ12 polycomb gene in myeloproliferative neoplasms

Tet2 Loss Leads to Increased Hematopoietic Stem Cell Self-Renewal and Myeloid Transformation

Somatic loss-of-function mutations in the ten-eleven translocation 2 (TET2) gene occur in a significant proportion of patients with myeloid malignancies. Although there are extensive genetic data implicating TET2 mutations in myeloid transformation, the consequences of Tet2 loss in hematopoietic development have not been delineated. We report here an animal model of conditional Tet2 loss in the hematopoietic compartment that leads to increased stem cell self-renewal in vivo as assessed by competitive transplant assays. Tet2 loss leads to a progressive enlargement of the hematopoietic stem cell compartment and eventual myeloproliferation in vivo, including splenomegaly, monocytosis, and extramedullary hematopoiesis. In addition, Tet2(+/-) mice also displayed increased stem cell self-renewal and extramedullary hematopoiesis, suggesting that Tet2 haploinsufficiency contributes to hematopoietic transformation in vivo.