Expression Of Mutant IDH1 Research Articles

Abstract 5853: (R)-2-hydroxyglutarate inhibits KDM5 histone lysine demethylases to drive tumorigenesis in IDH-mutant cancers

Abstract Gain-of-function mutations in isocitrate dehydrogenase enzymes IDH1 and IDH2 occur in ∼10% of acute myeloid leukemias (AML) and >80% of gliomas. The mutant enzymes convert 2-oxoglutarate (2OG) to the oncometabolite R-2-hydroxyglutarate (R-2HG). R-2HG promotes cellular transformation by modulating the activities of 2OG-dependent dioxygenases (2OGDDs). The only functionally validated direct target of R-2HG is TET2, a 2OGDD myeloid tumor suppressor that catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Interestingly, in clonal myeloid disorders the patterns of IDH and TET2 mutations are vastly different. TET2 mutations occur at similar frequencies in both low- and high-grade disease. IDH mutations, on the other hand, are associated with higher-grade and blast-phase MPN and MDS and with de novo AML and are rare in CHIP and low-grade MDS. This suggests that mutant IDH promotes a more aggressive disease phenotype and that R-2HG has additional targets other than TET2 that contribute to its leukemogenic activity. To identify other 2OGDD myeloid tumor suppressors that could be contributing to R-2HG-mediated transformation, we performed a positive-selection CRISPR-Cas9 screen under cytokine-poor conditions in TF-1 cells. We identified three H3K4 histone lysine demethylases, KDM5A, KDM5C and KDM5D, as genes whose sgRNAs were enriched upon cytokine withdrawal. Triple knockout of KDM5A, KDM5C and KDM5D (TKO) in TF-1 cells induces robust cytokine independence as does treatment of TF-1 cells with KDM5c70, a KDM5 inhibitor. We further found that R-2HG is a more potent inhibitor of KDM5A, KDM5C and KDM5D than of TET2. We then assessed the effect of mutant IDH1 expression, TKO, R-2HG treatment and KDM5c70 treatment on H3K4 trimethylation by ChIP-seq and found that each of these perturbations results in a significant enrichment in H3K4me3 peaks relative to controls, with the same sites being enriched. We then asked if mutant IDH positivity is associated with increased levels of H3K4 methylation in glioma. We performed ChIP-seq on a panel of IDH wild-type and IDH mutant glioma MCTS lines and found H3K4me peaks to be highly enriched in the IDH mutant lines when compared to IDH wild-type lines. Trimethyl-H3K4 levels were likewise increased in isogenic normal human astrocyte (NHA) cells ectopically expressing mutant IDH1. Expression of mutant IDH or treatment KDM5 inhibitor was sufficient to cause increased colony formation in NHA cells relative to controls. Collectively, these data suggest that R-2HG inhibits KDM5 histone lysine demethylases to promote mutant IDH-mediated transformation in AML and glioma. These studies identify a novel direct target of R-2HG in IDH mutant tumors and provide a functional link between IDH mutations and dysregulated histone lysine methylation in cancer. Citation Format: Kathryn E. Gunn, Matti Myllykoski, John Cao, Bofu Huang, Betty Rouaisnel, Bill Diplas, Michael M. Levitt, Ryan Looper, John G. Doench, Keith L. Ligon, Harley I. Kornblum, Hai Yan, Samuel McBrayer, Lucy A. Godley, Peppi Koivunen, Julie-Aurore Losman. (R)-2-hydroxyglutarate inhibits KDM5 histone lysine demethylases to drive tumorigenesis in IDH-mutant cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5853.

Integrative multi-omic analysis reveals neurodevelopmental gene dysregulation in CIC-knockout and IDH1-mutant cells.

Capicua (CIC)'s transcriptional repressor function is implicated in neurodevelopment and in oligodendroglioma (ODG) aetiology. However, CIC's role in these contexts remains obscure, primarily from our currently limited knowledge regarding its biological functions. Moreover, CIC mutations in ODG invariably co‐occur with a neomorphic IDH1/2 mutation, yet the functional relationship between these two genetic events is unknown. Here, we analysed models derived from an E6/E7/hTERT‐immortalized (i.e. p53‐ and RB‐deficient) normal human astrocyte cell line. To examine the consequences of CIC loss, we compared transcriptomic and epigenomic profiles between CIC wild‐type and knockout cell lines, with and without mutant IDH1 expression. Our analyses revealed dysregulation of neurodevelopmental genes in association with CIC loss. CIC ChIP‐seq was also performed to expand upon the currently limited ensemble of known CIC target genes. Among the newly identified direct CIC target genes were EPHA2 and ID1, whose functions are linked to neurodevelopment and the tumourigenicity of in vivo glioma tumour models. NFIA, a known mediator of gliogenesis, was discovered to be uniquely overexpressed in CIC‐knockout cells expressing mutant IDH1‐R132H protein. These results identify neurodevelopment and specific genes within this context as candidate targets through which CIC alterations may contribute to the progression of IDH‐mutant gliomas. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd on behalf of The Pathological Society of Great Britain and Ireland.

The KDM5 Family of Histone Lysine Demethylases Are Targets of R-2-Hydroxyglutarate in IDH Mutant Tumors

Gain-of-function mutations in isocitrate dehydrogenase enzymes IDH1 and IDH2 occur in ∼10% of acute myeloid leukemias (AML) and >80% of gliomas. The mutant enzymes convert 2-oxoglutarate (2OG) to the oncometabolite R-2-hydroxyglutarate (R-2HG). R-2HG promotes cellular transformation by modulating the activities of 2OG-dependent dioxygenases (2OGDDs). The only functionally validated direct target of R-2HG is TET2, a 2OGDD myeloid tumor suppressor that catalyzes the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC).Interestingly, in clonal myeloid disorders the patterns of IDH and TET2 mutations are vastly different. TET2 mutations occur at similar frequencies in clonal hematopoiesis of unknown significance (CHIP), lower- and higher-grade myeloproliferative (MPN) and myelodysplastic (MDS) disorders, and primary and secondary AML. IDH mutations, on the other hand, are associated with higher-grade and blast-phase MPN and MDS and with de novo AML and are rare in CHIP and low-grade MDS. This suggests that mutant IDH promotes a more aggressive disease phenotype and that R-2HG has additional targets other than TET2 that contribute to its leukemogenic activity.To ask if the in vitro transforming activity of R-2HG directly correlates with TET2 inhibition, we treated TF-1 cells, a cytokine-dependent human AML cell line, with a dose range of cell-permeable esterified R-2HG. We found that R-2HG induces cytokine independence at concentrations that have no effect on 5hmC levels. To identify other 2OGDD myeloid tumor suppressors that could be contributing to R-2HG-mediated transformation, we performed a positive-selection CRISPR-Cas9 screen under cytokine-poor conditions in TF-1 cells. We identified three H3K4 histone lysine demethylases, KDM5A, KDM5C and KDM5D, as genes whose sgRNAs were enriched upon cytokine withdrawal. Triple knockout of KDM5A, KDM5C and KDM5D (TKO) in TF-1 cells induces robust cytokine independence. Likewise, treatment of TF-1 cells with KDM5c70, a specific inhibitor of KDM5 enzymes, strongly induces TF-1 cytokine independence. Of note, KDM5 inhibition has no effect on TET2 expression or 5hmC levels. We further found that R-2HG is a more potent inhibitor of KDM5A, KDM5C and KDM5D than of TET2. We then assessed the effect of mutant IDH1 expression, TKO, R-2HG treatment and KDM5c70 treatment on H3K4 trimethylation by ChIP-seq and found that each of these perturbations results in a significant enrichment in H3K4me3 peaks relative to controls.TET enzymes are not recurrently mutated in glioma and although there is a strong correlation between mutant IDH status and the CpG island methylator phenotype (CIMP), direct inhibition of TET2 by R-2HG has not been reproducibly demonstrated in glioma. To ask if TET2 activity is suppressed in IDH mutant glioma, we quantified 5hmC levels in a panel of primary IDH wild-type and IDH mutant glioma and AML samples by mass spectrometry. We found that, unlike in AML, in glioma there is no correlation between IDH1 mutation status and loss of 5hmC. We likewise saw no correlation between 5hmC levels and either IDH mutation status or intracellular R-2HG levels in patient derived xenograft (PDX) models of glioma. Given the lack of evidence that TET enzymes are tumor suppressor targets of R-2HG in IDH mutant glioma, we asked if mutant IDH positivity is associated with increased levels of H3K4 methylation in glioma. We performed ChIP-seq on a panel of IDH wild-type and IDH mutant glioma PDX lines and found H3K4me peaks to be highly enriched in the IDH mutant lines when compared to IDH wild-type lines. Trimethyl-H3K4 levels were likewise increased in isogenic normal human astrocyte (NHA) cells ectopically expressing mutant IDH1.Collectively, these data suggest that R-2HG inhibits KDM5 histone lysine demethylases to promote mutant IDH-mediated transformation in AML and glioma. These studies identify a novel direct target of R-2HG in IDH mutant tumors and provide a functional link between IDH mutations and dysregulated histone lysine methylation in cancer. DisclosuresNo relevant conflicts of interest to declare.

Isocitrate dehydrogenase 1 mutation enhances 24(S)-hydroxycholesterol production and alters cholesterol homeostasis in glioma.

Isocitrate dehydrogenase (IDH) mutation is the most important initiating event in gliomagenesis, and the increasing evidence shows that IDH mutation is associated with the metabolic reprogramming in the tumor. Dysregulated cholesterol metabolism is a hallmark of tumor cells, but the cholesterol homeostasis in IDH-mutated glioma is still unknown. In this study, we found that astrocyte-specific mutant IDH1(R132H) knockin reduced the cholesterol contents and damaged the structure of myelin in mouse brains. In U87 and U251 cells, the expression of mutant IDH1 consistently reduced the cholesterol levels. Furthermore, we found that IDH1 mutation enhanced the production of 24(S)-hydroxycholesterol (24-OHC), which is not only the metabolite of cholesterol elimination, but also functions as an endogenous ligand for the liver X receptors (LXRs). In IDH1-mutant glioma cells, the elevated 24-OHC activated LXRs, which consequently accelerated the low-density lipoprotein receptor (LDLR) degradation by upregulating the inducible degrader of the LDLR (IDOL). The reduced LDLR expressions in IDH1-mutant glioma cells abated the uptakes of low-density lipoprotein (LDL) to decrease the cholesterol influx. In addition, the activated LXRs also promoted the cholesterol efflux by elevating the ATP-binding cassette transporter A1 (ABCA1), ABCG1, and apolipoprotein E (ApoE) in both IDH1-mutant astrocytes and glioma cells. As a feedback, the reduced cholesterol levels stimulated the cholesterol biosynthesis, which made IDH1-mutated glioma cells more sensitive to atorvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase. The altered cholesterol homeostasis regulated by mutant IDH provides a pivotal therapeutical strategy for the IDH-mutated gliomas.

The oncometabolite 2-hydroxyglutarate produced by mutant IDH1 sensitizes cells to ferroptosis

Ferroptosis is a non-apoptotic form of cell death characterized by the iron-dependent lipid peroxidation and is implicated in several human pathologies, such as tissue ischemia, neurodegeneration, and cancer. Ferroptosis appears to be high cell-context dependent and the regulation of ferroptosis by physiological or pathological conditions are unclear. Here, we report that tumor-derived IDH1 mutation sensitizes cells to ferroptosis. Deletion of the mutant IDH1 allele in IDH1 heterozygous tumor cells or pharmacological inhibition of mutant IDH1 to produce the oncometabolite D-2-hydroxyglutarate (D-2-HG) confers resistance to erastin-induced ferroptosis. Conversely, ectopic expression of mutant IDH1 or treatment of cells with cell-permeable D-2-HG promotes the accumulation of lipid reactive oxygen species (ROS) and subsequently ferroptosis. Mechanistically, mutant IDH1 reduces the protein level of the glutathione peroxidase 4 (GPX4), a key enzyme in removing lipid ROS and ferroptosis, and promotes depletion of glutathione. Our results uncover a new role of mutant IDH1 and 2-HG in ferroptosis.

Correlation between SOX2 and Mutant IDH1 Protein Expression in Astrocytomas and their Clinicopathologic Significance

AbstractBackground: Recent advances in genomic analyses provide a comprehensive view of the tumour-to-tumour complexity of glioma. Subgroups have been defined based on distinct genetic and epigenetic alterations and gene expression profiles. IDH1gene mutation is among the first genetic alterations observed during the development of the glioma. SOX2 is a transcriptional co-factors that are associated with various developmental milestones and is over-expressed in tumours, It plays a role in maintaining pluripotency in several cancers.Aim of Study: Evaluate the correlation between IHC expression of SOX2 and mutant IDH1 protein in astrocytomas and their clinicopathological significance.Material and Methods: The study was conducted with 50 patients diagnosed as Astrocytomas at the pathology laboratory, Suez Canal University Hospital between 2003 to 2015. The clinicopathological data were obtained from medical records Each case was studied for IHC expression of SOX2 and mutant IDH1(R132H antibody) and correlate expression results with variable clinicopathological features.Results: A statistically significant correlation between the expression of SOX2 and mutant IDH1 protein in astrocytomas and their expressions and grades of astrocytoma as, Glioblas-toma multiform (GBM) showed prominent expression of (SOX2 and mutant IDH1 protein) than other astrocytoma grades. A statistically significant relation between mutant IDH1 protein expression and tumour location with more expression among a tumour at the frontal lobe but no a statistically significant relation between SOX2 expression and tumour location. A higher SOX2 expression was among female patients compared to male patients, with a statistically significant correlation. No a statistically significant correlation between the expression of mutant IDH1 protein and gender. No a statistically significant correlation between the expression of both SOX2 & mutant IDH1 protein and age of the studied patients. A positive relationship between the expression of SOX2 and mutant IDH1 protein in the astrocytomas.Conclusion: Expression of SOX2 and mutant IDH1 protein in astrocytomas mainly GBM suggest a role in tumour invasion and dedifferentiation of tumour cells in astrocytomas.

Isocitrate dehydrogenase 1 mutation sensitizes intrahepatic cholangiocarcinoma to the BET inhibitor JQ1.

Cholangiocarcinoma is a life‐threatening disease with a poor prognosis. Although genome analysis unraveled some genetic mutation profiles in cholangiocarcinoma, it remains unknown whether such genetic abnormalities relate to the effects of anticancer drugs. Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are exclusively found in almost 20% of intrahepatic cholangiocarcinoma (ICC). Recently, the anticancer effects of BET inhibitors including JQ1 have been shown in various tumors. In the present study, we report that the antigrowth effect of JQ1 differs among ICC cells and IDH1 mutation sensitizes ICC cells to JQ1. RBE cells harboring IDH1 mutation was more sensitive to JQ1 than HuCCT1 or HuH28 cells with wild‐type IDH1. JQ1 induced apoptosis only in RBE cells through the upregulation of proapoptotic genes BAX and BIM. We found that the antigrowth effect was not attributed to downregulation of the MYC gene as a well‐known target of JQ1 in various cancer cells. Notably, the forced expression of mutant IDH1 successfully sensitized HuCCT1 cells to JQ1. In addition, AGI‐5198, a selective inhibitor of mutant IDH1 partially reversed the decrease in viability after JQ1 treatment and also suppressed the JQ1‐induced apoptosis in RBE cells. These data suggest that IDH1 mutation contributed to the growth inhibitory effect of JQ1 in RBE cells. Furthermore, given that the effect of mutant IDH1 was not recapitulated in glioblastoma cells, the enhancement of JQ1 sensitivity by IDH1 mutation seems to be specific for ICC cells. Our findings propose a new stratified therapeutic strategy based on IDH1 mutation in ICC.

Mutant IDH1 Cooperates with ATRX Loss to Drive the Alternative Lengthening of Telomere Phenotype in Glioma.

A subset of tumors use a recombination-based alternative lengthening of telomere (ALT) pathway to resolve telomeric dysfunction in the absence of TERT. Loss-of-function mutations in the chromatin remodeling factor ATRX are associated with ALT but are insufficient to drive the process. Because many ALT tumors express the mutant isocitrate dehydrogenase IDH1 R132H, including all lower grade astrocytomas and secondary glioblastoma, we examined a hypothesized role for IDH1 R132H in driving the ALT phenotype during gliomagenesis. In p53/pRb-deficient human astrocytes, combined deletion of ATRX and expression of mutant IDH1 were sufficient to create tumorigenic cells with ALT characteristics. The telomere capping complex component RAP1 and the nonhomologous DNA end joining repair factor XRCC1 were each downregulated consistently in these tumorigenic cells, where their coordinate reexpression was sufficient to suppress the ALT phenotype. RAP1 or XRCC1 downregulation cooperated with ATRX loss in driving the ALT phenotype. RAP1 silencing caused telomere dysfunction in ATRX-deficient cells, whereas XRCC1 silencing suppressed lethal fusion of dysfunctional telomeres by allowing IDH1-mutant ATRX-deficient cells to use homologous recombination and ALT to resolve telomeric dysfunction and escape cell death. Overall, our studies show how expression of mutant IDH1 initiates telomeric dysfunction and alters DNA repair pathway preferences at telomeres, cooperating with ATRX loss to defeat a key barrier to gliomagenesis.Significance: Studies show how expression of mutant IDH1 initiates telomeric dysfunction and alters DNA repair pathway preferences at telomeres, cooperating with ATRX loss to defeat a key barrier to gliomagenesis and suggesting new therapeutic options to treat low-grade gliomas. Cancer Res; 78(11); 2966-77. ©2018 AACR.

Highlights of This Issue

Follicular dendritic cell (FDC) sarcomas are rare soft tissue sarcomas with significant metastatic potential. In addition, diagnosis is a challenge due to morphologic and immunophenotypic heterogeneity. To better understand this rare tumor type and uncover potential diagnostic and therapeutic targets, genomic analysis was performed on FDC sarcomas compared to other rare mesenchymal tumors. Based on differential gene expression and pathway enrichment, a number of critical transcriptional programs were identified: signal transduction, cell cycle, metabolism, extracellular matrix (ECM), chromatin organization, and immune system. Further investigation revealed that the PD-1 receptor and its ligands were upregulated in FDC sarcomas and suggests the potential for targeted immunotherapy.Mutations in isocitrate dehydrogenase 1 (IDH1) occur in the majority of low-grade gliomas and lead to the production of D-2HG. To determine the effects mutant IDH1 has on neural stem cells (NSCs) and on the microenvironment, Pirozzi and colleagues generated a genetically faithful conditional knock-in mouse model. The data demonstrate that mutant Idh1 confers reduced proliferation of NSCs both in vitro and in vivo, and results in a disorganized subventricular zone (SVZ). Additionally, this mouse glioma model shows that expression of mutant Idh1 leads to distinct molecular and histological features, and alters the course of tumor progression. Collectively, these findings provide new insights into IDH1-R132H-driven tumorigenesis, and establish a platform for future preclinical investigations focused on mutant IDH1-targeted therapy.Increased nucleolar size and number are common in many cancers. These increases partly reflect increased activity of RNA polymerase I, which catalyzes the synthesis of the precursor rRNA (45S rRNA). Guner and colleagues describe and validate a chromogenic in situ hybridization (CISH) assay for the 5′-external transcribed spacer (ETS) of the 45S rRNA in formalin-fixed, paraffin-embedded (FFPE) tissues as a measure of Pol I activity–a therapeutic target in cancer. This 5′ETS/45S signal, restricted to the nucleolus, was attenuated after pharmacological inhibition or RNAi depletion of Pol I. Most invasive prostatic adenocarcinomas and high-grade prostatic intraepithelial neoplasia had increased 5′ETS/45S signals. 5′ETS/45S CISH is a novel, potentially predictive and pharmacodynamic biomarker for Pol I inhibitors in tissue specimens.Bone marrow mononuclear cells from multiple myeloma patients overexpress tumor necrosis factor receptor-associated factor 6 (TRAF6). This protein has been implicated in polyubiquitin-mediated IL1R/TLR signaling through activation of IκB kinase which regulates the NF-κB and JNK signaling pathways. Inhibition of TRAF6 reduces tumor growth and enhances the anti-myeloma effects of proteasome inhibitors. TRAF6 dominant negative peptides also downregulate the RANKL/RANK signaling pathway leading to inhibition of osteoclast cell formation and a reduction in bone resorption. Taken together, these data indicate that targeting TRAF6 is a potential strategy for the treatment of multiple myeloma and its related disease-associated bone loss.

Isocitrate dehydrogenase mutations suppress STAT1 and CD8+ T cell accumulation in gliomas.

Mutations in the isocitrate dehydrogenase genes IDH1 and IDH2 are among the first genetic alterations observed during the development of lower-grade glioma (LGG). LGG-associated IDH mutations confer gain-of-function activity by converting α-ketoglutarate to the oncometabolite R-2-hydroxyglutarate (2HG). Clinical samples and gene expression data from The Cancer Genome Atlas (TCGA) demonstrate reduced expression of cytotoxic T lymphocyte-associated genes and IFN-γ-inducible chemokines, including CXCL10, in IDH-mutated (IDH-MUT) tumors compared with IDH-WT tumors. Given these findings, we have investigated the impact of IDH mutations on the immunological milieu in LGG. In immortalized normal human astrocytes (NHAs) and syngeneic mouse glioma models, the introduction of mutant IDH1 or treatment with 2HG reduced levels of CXCL10, which was associated with decreased production of STAT1, a regulator of CXCL10. Expression of mutant IDH1 also suppressed the accumulation of T cells in tumor sites. Reductions in CXCL10 and T cell accumulation were reversed by IDH-C35, a specific inhibitor of mutant IDH1. Furthermore, IDH-C35 enhanced the efficacy of vaccine immunotherapy in mice bearing IDH-MUT gliomas. Our findings demonstrate a mechanism of immune evasion in IDH-MUT gliomas and suggest that specific inhibitors of mutant IDH may improve the efficacy of immunotherapy in patients with IDH-MUT gliomas.

Abstract 2675: Metabolic addiction in IDH1 mutant cancers

Abstract Introduction: Recent efforts have revealed IDH1 mutation not only results in marked accumulation of 2-hydroxyglutarate (2-HG), it generates genomic chromatin alterations, altered HIF activity and reprogrammed metabolic flux. We characterized the metabolic perturbations caused by heterozygous mutant IDH1 by comparing endogenous IDH1 mutant glioma cells treated with direct inhibitors of mutant IDH1 (IDH1i) to control cells without treatment, with the overall aim of identifying novel metabolic therapeutic targets. Experimental Procedures: We tested the effect of IDH1i on the in vitro and in vivo phenotype of a panel of patient-derived endogenous IDH1 mutant solid cancer cells (including gliomas, melanoma, and sarcomas). We then assessed the effect of IDH1i on the metabolome of endogenous IDH1 mutant glioma cells using liquid chromatography-mass spectrometry. We further investigated specifically perturbed metabolic pathways using lentiviral knockdown and overexpression systems. Results: We found that IDH1i exposure and the resulting 2-HG depletion had a mixed effect in 12 endogenous mutant IDH1 lines studied both in vitro or in vivo, including an orthotopic glioma xenograft model. For 10 of 12 lines, IDH1i treatment had no demonstrable effect on proliferation or survival, while 2 lines displayed growth inhibition after IDH1i treatment. However, no significant changes were detected in the genomic DNA methylation profiles or the global levels of histone tail marks including H3K4me3, H3K9me2, H3K9me3 and H3K27me3 in IDH1 mutant glioma lines, even after inhibition of mutant IDH1 for 12 months in vitro. Broader metabolite profiling revealed that inhibition of mutant IDH1 significantly altered levels of the canonical metabolite NAD+. We tested the effect of NAD+ depletion using NAD+ biosynthesis inhibitors, and found that endogenously mutant IDH1 cells were highly dependent on NAD+ for survival, whereas proliferation and survival of IDH1/2 wild-type cancer cells were unaffected by NAD+ depletion. Using an inducible mutant IDH1 expression system we discovered that mutant IDH1 reduced intracellular NAD+ levels, rendering mutant IDH1 cells susceptible to further NAD+ depletion. Lack of sufficient NAD+ induced metabolic crisis with reduced ATP levels in IDH1 mutant cells, triggering the intracellular energy sensor AMPK and autophagy. In vivo, NAD+ depletion significantly extended the survival of mice bearing IDH1 mutant xenograft tumors, including intracerebral gliomas. Conclusions: Although most IDH1 mutant cell lines derived from solid cancers were resistant to direct IDH1 inhibition, a subset of IDH1 mutant cancers were found to be sensitive. Mutant IDH1 reprograms metabolism and renders cancer cells highly dependent on NAD+ for survival. This metabolic addiction presents a potential opportunity for metabolically targeted therapeutic development. Citation Format: Andrew S. Chi, Kensuke Tateishi, Wakimoto Hiroaki, Tracy T. Batchelor, Anthony J. Iafrate, Daniel P. Cahill. Metabolic addiction in IDH1 mutant cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2675.

Abstract B32: FOXOs support the metabolic requirements of normal and tumor cells by regulating IDH1 expression

Abstract FOXO transcription factors are considered bona fide tumor suppressors, however recent studies showed FOXOs are also required for tumor survival. Here we identify FOXOs as transcriptional regulators of IDH1. FOXOs regulate IDH1 expression and thereby maintain the cytosolic levels of α-ketoglutarate and NADPH. In cancer cells carrying mutant IDH1, FOXOs likewise regulate mutant IDH1 expression and maintain the levels of the oncometabolite 2-hydroxyglutarate, which stimulates cancer cell proliferation likely by repressing differentiation. Combined, our data provide a new paradigm for the paradoxical role of FOXOs in both tumor suppression and promotion. Furthermore these data suggest a role for FOXOs in maintaining the de-differentiated state by regulating α-ketoglutarate and/or 2-hydroxyglutarate levels. Indeed, in models of differentiation (e.g. 3T3-L1 differentiation towards adipocytes) loss of FOXO, as well as loss of IDH1 expression repress differentiation and we are currently further exploring the molecular underpinnings. Note: This abstract was not presented at the conference. Citation Format: Paraskevi Charitou, Maria Rodriguez-Colman, Johan Gerrits, Nanda Verhoeven-Duif, Boudewijn Burgering. FOXOs support the metabolic requirements of normal and tumor cells by regulating IDH1 expression. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr B32.

FOXOs support the metabolic requirements of normal and tumor cells by promoting IDH1 expression.

FOXO transcription factors are considered bona fide tumor suppressors; however, recent studies showed FOXOs are also required for tumor survival. Here, we identify FOXOs as transcriptional activators of IDH1. FOXOs promote IDH1 expression and thereby maintain the cytosolic levels of α-ketoglutarate and NADPH. In cancer cells carrying mutant IDH1, FOXOs likewise stimulate mutant IDH1 expression and maintain the levels of the oncometabolite 2-hydroxyglutarate, which stimulates cancer cell proliferation and inhibits TET enzymes and histone demethylases. Combined, our data provide a new paradigm for the paradoxical role of FOXOs in both tumor suppression and promotion.

Isocitrate Dehydrogenase Mutations Induce BCL-2 Dependence in Acute Myeloid Leukemia through Inhibition of Cytochrome C Oxidase Function

IDH1 and IDH2 are two of the most frequently mutated genes in acute myeloid leukemia at an overall frequency of about 15-20%. The genes encode enzymes in the citric acid cycle that normally catalyze the oxidative decarboxylation of isocitrate, producing α-ketoglutarate (α-KG). The mutant enzymes gain a neomorphic activity that catalyzes the conversion of α-KG to (R)-2-hydroxyglutarate (2-HG). The intracellular concentration of (R)-2-HG is over 100-fold higher in IDH-mutated cells than in wildtype cells. (R)-2-HG has been shown to be a competitive inhibitor of multiple α-KG dependent dioxygenases including TET2, and the Jumonji-C domain containing histone demethylases. These enzymes are thought to be the main targets through which (R)-2-HG exerts its effects on leukemogenesis.We previously reported that inhibition of the anti-apoptotic BCL-2 protein is synthetic lethal against mutant IDH which we discovered through a large-scale pooled lentiviral RNA interference screen. We confirmed that expression of mutant IDH1 or IDH2 strikingly sensitized AML cells to shRNA-mediated BCL-2 knockdown and pharmacologic BCL-2 inhibition with ABT-199, a highly specific BH3 mimetic. Importantly, we found that primary human AML blasts harboring IDH mutations were significantly more sensitive to ABT-199 than blasts with wildtype IDH ex vivo and in xenograft transplant models. Furthermore, we showed that ABT-199 was able to target the leukemic stem cell compartment in IDH-mutated samples.Here, we present our work to uncover the synthetic lethal mechanism. An important clue to the mechanism surfaced with the finding that treatment with a cell-permeable precursor of (R)-2-HG sensitized AML cells to BCL-2 inhibition, indicating that the intracellular accumulation of (R)-2-HG found in IDH-mutated cells is sufficient to mediate the phenotype. In addition, we found that (R)-2-HG was able to sensitize isolated mitochondria to ABT-199 with collapse of the mitochondrial transmembrane potential as a surrogate marker for commitment to apoptosis. This finding indicates that 1) the target mediating the synthetic lethal phenotype is localized to the mitochondria, and 2) changes in the epigenome and expression of nuclear-encoded genes are not required for the synthetic lethal phenotype.To identify the potential mitochondrial molecular target, we focused our analysis on the effect of (R)-2-HG on the enzymatic activity of individual complexes in the electron transport chain (ETC), given that ETC dysfunction can potentially alter the threshold for apoptosis. We found that (R)-2-HG at concentrations found in IDH mutated cells inhibited the in vitro enzymatic activity of complex IV (cytochrome C oxidase (COX)) in a dose-dependent manner, but had no effect on the remaining ETC complexes. This finding has in vivo significance as COX activity in intact IDH-mutated primary AML cells was found to be significantly decreased compared with non-mutated AML cells.Next, we investigated the possibility that COX inhibition is sufficient to induce BCL-2 dependence. We found that suppression of COX activity with chemical inhibitors or genetically through shRNA-mediated knockdown of a COX subunit (COX-IV) was sufficient to sensitize AML cells to ABT-199. Furthermore, treatment with tigecycline, a FDA-approved antibiotic that has previously been shown to disrupt ETC function through inhibition of mitochondrial translation resulting in decreased expression of mitochondrial-encoded proteins including the catalytic subunits of the COX complex, reproduced the sensitization effect in non-IDH mutated AML cells. Based on the above findings, we propose a mechanistic model in which (R)-2-HG accumulation in IDH mutant cells directly inhibits COX, thereby lowering the mitochondrial threshold for triggering apoptosis upon BCL-2 inhibition.In summary, we discovered that in addition to the previously described inhibition of α-KG dependent dioxygenases through (R)-2-HG production, IDH mutations also affect mitochondrial bioenergetics. This finding opens up the intriguing possibility that IDH mutations contribute to leukemogenesis not only through epigenetic changes but also through metabolic dysregulation. Lastly, our findings form the rational basis for combining agents that disrupt ETC function such as tigecycline with ABT-199 to target resistant cancer cells and maximize the clinical utility of this promising drug. DisclosuresMedeiros:Agios: Consulting - Ad board Other.

DN-03 * MUTANT IDH1-DRIVEN CELLULAR TRANSFORMATION INCREASES RAD51-MEDIATED HOMOLOGOUS RECOMBINATION AND TEMOZOLOMIDE RESISTANCE

BACKGROUND: IDH1 mutation is noted in roughly 75% of all lower-grade glioma and is considered to be an early and driving event in the genesis of these tumors. In clinical studies IDH1 mutation correlates with increased overall survival, progression-free survival, and upfront response to TMZ implying that mutant IDH1 directly contributes to drug sensitivity. IDH1 mutant gliomas, however, have a different underlying biology than comparable IDH1 WT glioma, complicating any analysis of the contribution of IDH1 mutation to drug sensitivity. To more accurately determine if and how IDH1 mutation alters drug sensitivity, we employed a system widely for the study of cellular transformation and IDH biology. In this system normal human astrocytes immortalized by introduction of virally-encoded E6, E7 and hTERT can be transformed by additional expression of oncogenic H-Ras or by mutant IDH1, the latter of which induces epigenetic changes similar to those noted in lower-grade glioma. We therefore used these cells to determine if mutant IDH1 expression and/or the transformative events it sets in place were sufficient to alter TMZ sensitivity. METHODS: E6/E7/hTERT-immortalized, untransformed human astrocytes, and the same cells transformed by prior introduction of oncogenic V12H-Ras, were infected with lentiviral constructs encoding WT or mutant (R132H) IDH1, then monitored for transformation status and, following TMZ exposure (100 uM, 3hrs), for DNA damage and repair, cell cycle response, and TMZ sensitivity. RESULTS: The TMZ-sensitive immortalized, untransformed parental cells exhibited DNA damage (H2AX foci) and a prolonged G2 cell cycle arrest that began 2 days after TMZ exposure and persisted for 5 days. The same cells transformed by mutant IDH1 exhibited the same levels of initial DNA damage and cell cycle arrest as controls, although both events resolved significantly faster in the IDH1 mutant cells in association with increased clonagenic survival. The increases in DNA damage processing, cell cycle progression, and clonagenicity were unique to cells transformed by mutant IDH1, and were not noted following introduction of mutant IDH1 into Ras-transformed or established GBM cells. The changes were, however, associated with increased homologous recombination (HR) and could be reversed by the genetic suppression of Rad51, the rate limiting protein in HR. CONCLUSIONS: Mutant IDH1 drives a unique set of transformative events that indirectly enhance HR and increase repair of TMZ-induced DNA damage and TMZ resistance. Inhibitors of HR may therefore be a viable means to further enhance TMZ response in IDH1 mutant glioma. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

IDH1 mutation and MGMT expression in astrocytoma and the relationship with prognosis after radiotherapy

To study the correlation between IDH1 mutation, MGMT expression, clinicopathologic features and post-radiotherapy prognosis in patients with astrocytoma. Detection of IDH1 mutation and MGMT expression was carried out in 48 cases of astrocytoma (WHO grade II to III) by EnVision method with immunohistochemical staining. Follow-up data, including treatment response and overall survival time, were analyzed. The rates of IDH1 mutation and MGMT expression in astrocytomas were 62.7% (30/48) and 47.9% (23/48), respectively. There was a negative correlation between IDH1 mutation and MGMT expression (r = -0.641, P < 0.01). The age of patients with IDH1 mutation was younger at disease onset. The IDH1 mutation rate in patients with WHO grade II astrocytoma was higher than that in patients with WHO grade III tumor (P < 0.05). The age at onset was an independent factor affecting the expression of mutant IDH1. After radiotherapy, patients with IDH1 mutation+/MGMT- tumor carried a longer overall survival time than patients with IDH1 mutation-/MGMT+ tumor (P < 0.05). There is a correlation between IDH1 mutation and MGMT expression in WHO grade II to III astrocytoma. Age at onset is an independent factor affecting the expression of mutant IDH1. Tumors with IDH1+/MGMT- pattern show better response to radiotherapy than tumors with IDH1-/MGMT+ pattern. Detection of IDH1 mutation and MGMT protein expression can provide some guidance in choice of treatment modalities in patients with astrocytoma.

Abstract 63: Driving brain tumorigenesis: Generation of a mutant IDH1 mouse model of progressive glioma

Abstract Despite decades worth of research, glioblastoma remains one of the most lethal cancers. The identification of IDH1 as a major cancer gene in glioblastoma provides a great opportunity for improving our understanding, diagnostics and treatment of this disease. Recent studies from our laboratory characterized the genetic landscape of gliomas and have shown the cooperation between IDH1 mutations and other oncogenic alterations including TP53 mutations. Normally, IDH1 functions in the oxidative decarboxylation of isocitrate to α-ketoglutarate, however, the mutant form confers neomorphic enzymatic activity by producing D-2-hydroxyglutarate, an oncometabolite responsible for aberrant methylation in IDH1-mutated tumors. To determine the role of mutant IDH1 in vivo, we generated a conditional knockin mouse model that produces D-2-hydroxyglutarate. This genetically faithful system is biologically and clinically relevant and will promote the understanding of mutant IDH1-mediated tumorigenesis while offering a route for therapeutic targeting. We observed that broad expression of mutant IDH1 throughout the brain leads to hydrocephalus in 80% of animals. To obviate these deleterious effects, we restricted expression of mutant IDH1 to the neural stem cell population. Animals were sacrificed after one year, at which time we observed mild hydrocephalus in several animals. Recapitulating the genetics of human tumors, we sought to determine the synergistic effects between mutant IDH1 and deletion of P53. We observed development of low- and high-grade gliomas in approximately 14% of animals. Tumors developed in both P53 deleted animals as well as in mutant IDH1/P53 deleted animals, with mutant IDH1 expressed in tumors of the latter cohort. Differences in latency, penetrance, differentiation, and epigenetic modifications are being assessed. Additionally, we are determining the cell fate of IDH1-mutant cells over time to assess the biological effects of the mutation on the behavior and differentiation of neural stem cells. Lastly, to decrease the latency and increase the penetrance of tumor formation, an orthotopic intracranial injection model is being generated to allow for visualization of tumor formation and development, as well as investigation of therapeutic modalities. Understanding the biological effects of the most common mutations found in gliomas will bring us strides closer to determining mechanisms and therapeutic targets for IDH1-mutated cancers. Citation Format: Christopher J. Pirozzi, Catherine Y. Wang, Austin B. Carpenter, Huishan Zhu, Paula K. Greer, Roger E. McLendon, Darell D. Bigner, Yiping He, Hai Yan. Driving brain tumorigenesis: Generation of a mutant IDH1 mouse model of progressive glioma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 63. doi:10.1158/1538-7445.AM2014-63

Mutant IDH1-driven cellular transformation increases RAD51-mediated homologous recombination and temozolomide resistance.

Isocitrate dehydrogenase 1 (IDH1) mutations occur in most lower grade glioma and not only drive gliomagenesis but are also associated with longer patient survival and improved response to temozolomide. To investigate the possible causative relationship between these events, we introduced wild-type (WT) or mutant IDH1 into immortalized, untransformed human astrocytes, then monitored transformation status and temozolomide response. Temozolomide-sensitive parental cells exhibited DNA damage (γ-H2AX foci) and a prolonged G2 cell-cycle arrest beginning three days after temozolomide (100 μmol/L, 3 hours) exposure and persisting for more than four days. The same cells transformed by expression of mutant IDH1 exhibited a comparable degree of DNA damage and cell-cycle arrest, but both events resolved significantly faster in association with increased, rather than decreased, clonogenic survival. The increases in DNA damage processing, cell-cycle progression, and clonogenicity were unique to cells transformed by mutant IDH1, and were not noted in cells transformed by WT IDH1 or an oncogenic form (V12H) of Ras. Similarly, these effects were not noted following introduction of mutant IDH1 into Ras-transformed cells or established glioma cells. They were, however, associated with increased homologous recombination (HR) and could be reversed by the genetic or pharmacologic suppression of the HR DNA repair protein RAD51. These results show that mutant IDH1 drives a unique set of transformative events that indirectly enhance HR and facilitate repair of temozolomide-induced DNA damage and temozolomide resistance. The results also suggest that inhibitors of HR may be a viable means to enhance temozolomide response in IDH1-mutant glioma.

MUTANT IDH1-DRIVEN CELLULAR TRANSFORMATION INCREASES RAD51-MEDIATED HOMOLOGOUS RECOMBINATION AND TEMOZOLOMIDE (TMZ) RESISTANCE.

BACKGROUND: IDH1 mutation is noted in roughly 75% of all lower-grade glioma and is considered to be an early and driving event in the genesis of these tumors. In clinical studies IDH1 mutation correlates with increased overall survival, progression-free survival, and upfront response to TMZ implying that mutant IDH1 directly contributes to drug sensitivity. IDH1 mutant gliomas, however, have a different underlying biology than comparable IDH1 WT glioma, complicating any analysis of the contribution of IDH1 mutation to drug sensitivity. To more accurately determine if and how IDH1 mutation alters drug sensitivity, we employed a system widely for the study of cellular transformation and IDH biology. In this system normal human astrocytes immortalized by introduction of virally-encoded E6, E7 and hTERT can be transformed by additional expression of oncogenic H-Ras or by mutant IDH1, the latter of which induces epigenetic changes similar to those noted in lower-grade glioma. We therefore used these cells to determine if mutant IDH1 expression and/or the transformative events it sets in place were sufficient to alter TMZ sensitivity. METHODS: E6/E7/hTERT-immortalized, untransformed human astrocytes, and the same cells transformed by prior introduction of oncogenic V12H-Ras, were infected with lentiviral constructs encoding WT or mutant (R132H) IDH1, then monitored for transformation status and, following TMZ exposure (100 uM, 3hrs), for DNA damage and repair, cell cycle response, and TMZ sensitivity. RESULTS: The TMZ-sensitive immortalized, untransformed parental cells exhibited DNA damage (H2AX foci) and a prolonged G2 cell cycle arrest that began 2 days after TMZ exposure and persisted for 5 days. The same cells transformed by mutant IDH1 exhibited the same levels of initial DNA damage and cell cycle arrest as controls, although both events resolved significantly faster in the IDH1 mutant cells in association with increased clonagenic survival. The increases in DNA damage processing, cell cycle progression, and clonagenicity were unique to cells transformed by mutant IDH1, and were not noted following introduction of mutant IDH1 into Ras-transformed or established GBM cells. The changes were, however, associated with increased homologous recombination (HR) and could be reversed by the genetic suppression of Rad51, the rate limiting protein in HR. CONCLUSIONS: Mutant IDH1 drives a unique set of transformative events that indirectly enhance HR and increase repair of TMZ-induced DNA damage and TMZ resistance. Inhibitors of HR may therefore be a viable means to further enhance TMZ response in IDH1 mutant glioma. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

Lactate dehydrogenase A silencing in IDH mutant gliomas

Mutations of the isocitrate dehydrogenase 1 and 2 gene (IDH1/2) were initially thought to enhance cancer cell survival and proliferation by promoting the Warburg effect. However, recent experimental data have shown that production of 2-hydroxyglutarate by IDH mutant cells promotes hypoxia-inducible factor (HIF)1α degradation and, by doing so, may have unexpected metabolic effects. We used human glioma tissues and derived brain tumor stem cells (BTSCs) to study the expression of HIF1α target genes in IDH mutant ((mt)) and IDH wild-type ((wt)) tumors. Focusing thereafter on the major glycolytic enzyme, lactate dehydrogenase A (LDHA), we used standard molecular methods and pyrosequencing-based DNA methylation analysis to identify mechanisms by which LDHA expression was regulated in human gliomas. We found that HIF1α-responsive genes, including many essential for glycolysis (SLC2A1, PDK1, LDHA, SLC16A3), were underexpressed in IDH(mt) gliomas and/or derived BTSCs. We then demonstrated that LDHA was silenced in IDH(mt) derived BTSCs, including those that did not retain the mutant IDH1 allele (mIDH(wt)), matched BTSC xenografts, and parental glioma tissues. Silencing of LDHA was associated with increased methylation of the LDHA promoter, as was ectopic expression of mutant IDH1 in immortalized human astrocytes. Furthermore, in a search of The Cancer Genome Atlas, we found low expression and high methylation of LDHA in IDH(mt) glioblastomas. To our knowledge, this is the first demonstration of downregulation of LDHA in cancer. Although unexpected findings, silencing of LDHA and downregulation of several other glycolysis essential genes raise the intriguing possibility that IDH(mt) gliomas have limited glycolytic capacity, which may contribute to their slow growth and better prognosis.