IDH1-mutant Cell Lines Research Articles

Abstract B069: Targeting vulnerabilities in double strand break repair in cancer with polymerase theta inhibitors

Abstract Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in approximately 80% of secondary glioblastomas and low-grade gliomas and to a lesser extent in AML, cholangiocarcinoma, colorectal cancer, and chondrosarcomas. Mutations in IDH1/2 lead to the overproduction of 2-hydroxyglutarate (2HG), which has been named an “oncometabolite” and is implicated in cancer progression. Work by our group has shown that 2HG induced hypermethylation suppresses homologous recombination repair. As a result, cells with mutations in IDH1/2 accumulate double strand breaks (DSBs) that must be addressed by other modes of DSB repair, such as canonical NHEJ or polymerase theta mediated end joining (TMEJ). Furthermore, TMEJ is understudied in IDH1/2-mutant cancers but has been studied extensively in other HR-deficient cancer contexts, such as BRCA-mutant cancers. Conflicting reports remain on whether TMEJ promotes genomic stability or instability in these contexts, due to its error prone repair. To better understand how IDH1/2-mutant cells utilize TMEJ and polymerase theta’s impact on genomic stability, we studied the effects of polymerase theta inhibitors on isogenic cell line models of the IDH1R132H mutation. We find that mRNA levels of polymerase theta are increased in IDH1-mutant cell lines. Using a polymerase theta inhibitor, ART558, we also observed reduced viability in IDH1-mutant cells compared to parental cells. This loss of viability in IDH1-mutant cells is likely a result of the observed increase of gamma H2AX subnuclear foci in IDH1-mutant cells treated with ART558, which is a marker of unresolved DNA damage. Preliminary data suggests that this unrepaired damage is contributing to an overall increase in micronuclei formation in IDH1-mutant cells treated with ART558 as well. Taken together, these results suggest that IDH1-mutant cells upregulate polymerase theta expression to promote genomic stability through repair of persistent DSBs. This study on TMEJ in IDH1/2 mutant cancers has identified a therapeutic vulnerability in these HR-deficient cancers that can be targeted with polymerase theta inhibitors. Citation Format: Katelyn Noronha, Samantha Liu, Karlie Lucas, Ranjit S. Bindra. Targeting vulnerabilities in double strand break repair in cancer with polymerase theta inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B069.

P02.21.A A BIOBANK OF IDH1-MUTANT CELL LINES FOR STUDYING TUMOR HETEROGENEITY, PROGRESSION AND TARGET IDENTIFICATION

Abstract BACKGROUND Diffuse low grade gliomas are grade II brain tumors mainly affecting young patients. Most of these tumors have a mutation in the IDH1 gene (isocitrate dehydrogenase 1) gene causing differentiation defects and cell accumulation in the brain. These tumors are classified as astrocytomas or oligodendrogliomas and often progress into high grade gliomas (i.e IDH1-mutant grade IV astrocytomas and grade III oligodendrogliomas). We and others have shown that IDH1-mutant diffuse low grade gliomas contain 3 types of tumoral cells, resembling astrocytes, oligodendrocytes and neural progenitors, the formation of which is regulated by Notch signaling (Augustus et al, 2021, Cells, Suvà et al, 2020, Cancer Cell). There is a significant lack of cellular tools to study these tumors in vitro, hindering the development of new therapies. We aimed to address this by creating a fully annotated biobank of IDH1-mutant cell lines derived from different grades of tumors and carrying various rare mutations. MATERIAL AND METHODS We derived and collected 8 IDH1-mutant cell lines (4 astrocytomas grade II/III/IV and 4 grade III oligodendrogliomas). These cell lines were characterized using bulk RNA sequencing under different culture conditions, single-cell RNA sequencing to determine their cellular heterogeneity, and immunofluorescence and western blotting. RESULTS Our findings show that these IDH1-mutant cell lines exhibit different growth rates and morphologies and are phenotypically related to brain radial glial cells, oligodendrocyte progenitor cells, or more mature oligodendrocytes. Some of these cell lines are multipotent and contain astrocyte-like and oligodendrocyte-like cells. Our current work aims to purify different cell subpopulations to study their properties and plasticity. Additionally, we are analyzing pathways and differentially expressed genes involved in IDH1-mutant cell quiescence and malignant progression. CONCLUSION In summary, we present a novel and deeply-annotated biobank of 8 IDH1-mutant cell lines derived from different grades of gliomas, providing an invaluable resource to study the cellular and molecular heterogeneity of IDH1-mutant gliomas, their progression to high-grade gliomas, and the identification of new therapeutic targets. We anticipate that our biobank may facilitate the development of personalized medicine for patients with IDH1-mutant gliomas and accelerate the discovery of new therapeutic strategies.

DDRE-03. IDH1-MUTANT GBM CELLS ARE HIGHLY SENSITIVE TO COMBINATION OF KDM6A/B AND HDAC INHIBITORS

Mutations in IDH1 and IDH2 genes are common in low grade gliomas and secondary GBM and are known to cause a distinct epigenetic landscape in these tumors. To interrogate the epigenetic vulnerabilities of IDH-mutant gliomas, we performed a chemical screen with inhibitors of chromatin modifiers and identified 5-azacytidine, Chaetocin, GSK-J4 and Belinostat as potent agents against primary IDH1-mutant cell lines. Testing the combinatorial efficacy of these agents, we demonstrated GSK-J4 and Belinostat combination as a very effective treatment for the IDH1-mutant glioma cells. Engineering established cell lines to ectopically express IDH1R132H, we showed that IDH1R132H cells adopted a different transcriptome with changes in stress-related pathways that were reversible with the mutant IDH1 inhibitor, GSK864. The combination of GSK-J4 and Belinostat was highly effective on IDH1R132H cells, but not on wt glioma cells or nonmalignant fibroblasts and astrocytes. The cell death induced by GSK-J4 and Belinostat combination involved the induction of cell cycle arrest and apoptosis. RNA sequencing analyses revealed activation of inflammatory and unfolded protein response pathways in IDH1-mutant cells upon treatment with GSK-J4 and Belinostat conferring increased stress to glioma cells. Specifically, GSK-J4 induced ATF4-mediated integrated stress response and Belinostat induced cell cycle arrest in primary IDH1-mutant glioma cells; which were accompanied by DDIT3/CHOP-dependent upregulation of apoptosis. Moreover, to dissect out the responsible target histone demethylase, we undertook genetic approach and demonstrated that CRISPR/Cas9 mediated ablation of both KDM6A and KDM6B genes phenocopied the effects of GSK-J4 in IDH1-mutant cells. Finally, GSK-J4 and Belinostat combination significantly decreased tumor growth and increased survival in an orthotopic model in mice. Together, these results suggest a potential combination epigenetic therapy against IDH1-mutant gliomas.

EXTH-10. COMBINATION OF EPIGENETIC ENZYME INHIBITORS, GSK-J4 AND BELINOSTAT, REVEALS HIGH EFFICACY IN IDH1 MUTANT GLIOMAS

Abstract Mutations in IDH1 and IDH2 genes are common in low grade gliomas and secondary GBM and are known to cause a distinct epigenetic landscape in these tumors. To interrogate the epigenetic vulnerabilities of IDH-mutant gliomas, we performed a chemical screen with inhibitors of chromatin modifiers and identified 5-azacytidine, Chaetocin, GSK-J4 and Belinostat as potent agents against primary IDH1-mutant cell lines. Testing the combinatorial efficacy of these agents, we demonstrated GSK-J4 and Belinostat combination as a very effective treatment for the IDH1-mutant glioma cells. Engineering established cell lines to ectopically express IDH1R132H, we showed that IDH1R132H cells adopted a different transcriptome with changes in stress-related pathways that were reversible with the mutant IDH1 inhibitor, GSK864. The combination of GSK-J4 and Belinostat was highly effective on IDH1R132H cells, but not on wt glioma cells or nonmalignant fibroblasts and astrocytes. The cell death induced by GSK-J4 and Belinostat combination involved the induction of cell cycle arrest and apoptosis. RNA sequencing analyses revealed activation of inflammatory and unfolded protein response pathways in IDH1-mutant cells upon treatment with GSK-J4 and Belinostat conferring increased stress to glioma cells. Specifically, GSK-J4 induced ATF4-mediated integrated stress response and Belinostat induced cell cycle arrest in primary IDH1-mutant glioma cells; which were accompanied by DDIT3/CHOP-dependent upregulation of apoptosis. Moreover, to dissect out the responsible target histone demethylase, we undertook genetic approach and demonstrated that CRISPR/Cas9 mediated ablation of both KDM6A and KDM6B genes phenocopied the effects of GSK-J4 in IDH1-mutant cells. Finally, GSK-J4 and Belinostat combination significantly decreased tumor growth and increased survival in an orthotopic model in mice. Together, these results suggest a potential combination epigenetic therapy against IDH1-mutant gliomas.

Metabolic plasticity of IDH1-mutant glioma cell lines is responsible for low sensitivity to glutaminase inhibition

BackgroundTargeting glutamine metabolism in cancer has become an increasingly vibrant area of research. Mutant IDH1 (IDH1mut) gliomas are considered good candidates for targeting this pathway because of the contribution of glutamine to their newly acquired function: synthesis of 2-hydroxyglutarate (2HG).MethodsWe have employed a combination of 13C tracers including glutamine and glucose for investigating the metabolism of patient-derived IDH1mut glioma cell lines through NMR and LC/MS. Additionally, genetic loss-of-function (in vitro and in vivo) approaches were performed to unravel the adaptability of these cell lines to the inhibition of glutaminase activity.ResultsWe report the adaptability of IDH1mut cells’ metabolism to the inhibition of glutamine/glutamate pathway. The glutaminase inhibitor CB839 generated a decrease in the production of the downstream metabolites of glutamate, including those involved in the TCA cycle and 2HG. However, this effect on metabolism was not extended to viability; rather, our patient-derived IDH1mut cell lines display a metabolic plasticity that allows them to overcome glutaminase inhibition.ConclusionsMajor metabolic adaptations involved pathways that can generate glutamate by using alternative substrates from glutamine, such as alanine or aspartate. Indeed, asparagine synthetase was upregulated both in vivo and in vitro revealing a new potential therapeutic target for a combinatory approach with CB839 against IDH1mut gliomas.

RNA-Binding Protein HuR Regulates Both Mutant and Wild-Type IDH1 in IDH1-Mutated Cancer.

Isocitrate dehydrogenase 1 (IDH1) is the most commonly mutated metabolic enzyme in human malignancy. A heterozygous genetic alteration, arginine 132, promotes the conversion of α-ketoglutarate to D-2-hydroxyglutarate (2-HG). Although pharmacologic inhibitors of mutant IDH1 are promising, resistance mechanisms to targeted therapy are not understood. Additionally, the role of wild-type IDH1 (WT.IDH1) in cancer requires further study. Recently, it was observed that the regulatory RNA-binding protein, HuR (ELAVL1), protects nutrient-deprived cancer cells without IDH1 mutations, by stabilizing WT.IDH1 transcripts. In the present study, a similar regulatory effect on both mutant (Mut.IDH1) and WT.IDH1 transcripts in heterozygous IDH1-mutant tumors is observed. In ribonucleoprotein immunoprecipitation assays of IDH1-mutant cell lines, wild-type and mutant IDH1 mRNAs each bound to HuR. Both isoforms were profoundly downregulated at the mRNA and protein levels after genetic suppression of HuR (siRNAs or CRISPR deletion) in HT1080 (R132C IDH1 mutation) and BT054 cells (R132H). Proliferation and invasion were adversely affected after HuR suppression and metabolomic studies revealed a reduction in Pentose Phosphate Pathway metabolites, nucleotide precursors, and 2-HG levels. HuR-deficient cells were especially sensitive to stress, including low glucose conditions or a mutant IDH1 inhibitor (AGI-5198). IDH1-mutant cancer cells were rescued by WT.IDH1 overexpression to a greater extent than Mut.IDH1 overexpression under these conditions. This study reveals the importance of HuR's regulation of both mutant and wild-type IDH1 in tumors harboring a heterozygous IDH1 mutation with implications for therapy. IMPLICATIONS: This study highlights the HuR-IDH1 (mutant and wild-type IDH1) regulatory axis as a critical, actionable therapeutic target in IDH1-mutated cancer, and incomplete blockade of the entire HuR-IDH1 survival axis would likely diminish the efficacy of drugs that selectively target only the mutant isoenzyme.

TMOD-36. SUCCESSFUL GENERATION OF FIVE PATIENT-DERIVED ENDOGENOUS IDH1 MUTANT GLIOMA CELL LINES PROVIDES OPPORTUNITIES FOR DEVELOPMENT OF NEW TREATMENT STRATEGIES FOR IDH MUTANT GLIOMAS

Despite the high frequency of IDH mutations in low-grade gliomas and secondary glioblastomas, in vitro models with native IDH mutations remain scarce. Development of such models is essential to study the biology of IDH1 mutations and to develop new treatment strategies. Glioma samples were obtained directly from the operating theatre as solid resection material or CUSA aspiration, were dissociated and cells were cultured under serum-free conditions. IDH1 mutations were verified with Sanger sequencing. D2HG was measured by mass spectrometry. Cell viability was measured using an ATP-based viability assay. Drug screening was performed using an FDA-approved Oncology Drugs Set from NIH as well as with specific IDH1-mutant inhibitors. Attempts to establish a cell culture were undertaken on a total of eighty IDH1 mutant gliomas. From this series, five successful IDH1 mutant cell lines were obtained (i.e. those that remain in culture >10 passages while maintaining the IDH1-mutation). All five cell lines are 1p/19q intact, WHO grade III-IV and have an IDH1-R132H mutation. Production of the mutant IDH1 derived oncometabolite D2-HG was confirmed. Addition of IDH1-mutant inhibitor AGI-5198 resulted in differential and modest effects on 3 of the IDH1 mutant cell lines. One cell line lost its IDH1 mutation around passage 16, which indicates that at least some IDH1-mutant gliomas are no longer dependent on the mutation in vitro. In contrast, a different cell line gained a copy of IDH1-R132H without compromising its proliferation potential. Drug screening with a set of FDA-approved oncology drugs (n=107) revealed several candidates that strongly decrease cell viability at clinically-feasible concentrations. An orthotopic mouse model is in development to further assess the therapeutic potential of these agents. We have established a unique set of five native IDH-mutated cell lines, therewith providing opportunities to assess new therapeutic strategies for this subtype of malignant glioma.

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.

ATPS-852-HYDROXYGLUTARATE DEPLETION IS NOT SUFFICIENT TO INHIBIT GROWTH OF SEVERAL PROGRESSIVE IDH1 MUTANT SOLID CANCER TYPES

Targeted agents that inhibit 2-hydroxyglutarate (2-HG) production by mutant IDH1 have shown considerable promise in acute leukemia. To examine the potential effects of 2-HG reduction in IDH1 mutant solid cancers, we treated endogenous IDH1 mutant tumor cells from several tissue types with a well-characterized inhibitor of IDH1 R132H and R132C (IDH1i) in vitro and in vivo. We confirmed potent inhibition of 2-HG formation in vitro in IDH1R132H glioma tumorsphere lines and a fibrosarcoma line, HT1080 (IDH1R132C). Then, we broadly tested 8 endogenous IDH1 mutant cell lines, including 6 IDH1R132H gliomas, HT1080 and a melanoma line E232-2010 (IDH1R132C). IDH1i markedly reduced 2-HG, however did not inhibit in vitro growth of any cell line. We then assessed the effect of IDH1i treatment on mice bearing patient-derived, endogenous IDH1 mutant glioma orthotopic xenografts. Continuous daily oral administration of IDH1i (200 mg BID) achieved marked 2-HG reduction within the tumor. However, we found no significant difference in tumor size or survival of IDH1i-treated versus vehicle-treated mice. Furthermore, neither short-term nor prolonged (>6mo) in vitro IDH1i exposure significantly altered the global trimethylation or dimethylation of histone 3 lysine 9 (H3K9), global trimethylation of H3K27, genomewide distribution of DNA methylation or methylation of the O-6-methylguanine-DNA methyltransferase gene promoter. Paradoxically, long-term in vitro IDH1i incubation resulted in acceleration of cell proliferation and significantly shorter animal survival upon subsequent intracerebral implantation, compared to passage-matched IDH1 mutant glioma cells grown in the absence of IDH1i. Thus, 2-HG levels can be uncoupled from cell proliferation in several IDH1 mutant solid cancer types, and the epigenetic alterations induced by mutant IDH1 are not reversed solely by depletion of 2-HG, even after many cell divisions. Alternative combination therapeutic strategies may be needed for successful growth inhibition of progressive mutant IDH1 solid cancers.

Abstract 3514: A novel inhibitor of IDH1 abrogates 2-HG production and reverses aberrant epigenetic alterations in IDH1 mutant cells

Abstract The isocitrate dehydrogenase 1 (IDH1) and IDH2 genes are mutated in acute myelogenous leukemia, low-grade glioma, intrahepatic cholangiocarcinoma, and chondrosarcomas. IDH1 and IDH2 normally function to convert isocitrate into alpha-ketoglutarate. However, when these enzymes are mutated at select residues the mutant enzymes now convert α-KG into 2-hydroxyglutarate (2-HG). In normal cells, 2-HG levels are typically extremely low, but IDH1/2 mutant cells can accumulate up to 10 mM 2-HG. In an effort to counteract the neomorphic activity of mutant IDH enzymes, we identified and developed potent inhibitors of IDH1. The compounds inhibit IDH1 catalytic activity in biochemical assays and reduce 2-HG production in IDH1-mutant cell lines. Consistent with the fact that 2-HG inhibits α-KG dependent enzymes including histone demethylases and Tet family hydroxylases, these IDH1 inhibitors induce a decrease in several histone methylation marks and also DNA methylation. These data demonstrate that small molecule inhibitors can reverse many of the epigenetic effects of mutant IDH1. Note: This abstract was not presented at the meeting. Citation Format: Cynthia Rominger, Chad Quinn, Enoch Gao, Beth Pietrak, Alan Rendina, Angela Smallwood, Arthur Groy, Susan Korenchuk, Charles McHugh, Ken Wiggall, Alexander Reif, Stanley Schmidt, Hongwei Qi, Huizhen Zhao, Nestor Concha, Christopher Carpenter, Juan Luengo, Ryan Kruger, Benjamin Schwartz, Nicholas Adams, Michael T. McCabe. A novel inhibitor of IDH1 abrogates 2-HG production and reverses aberrant epigenetic alterations in IDH1 mutant cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3514. doi:10.1158/1538-7445.AM2015-3514

Abstract 447: The IDH1 mutation in human glioblastoma and its effects on epigenetic modification and cell fate selection

Abstract Introduction: In GBM IDH mutations are associated with relatively prolonged survival compared to other subtypes. The mutations in both IDH1 result in the abnormal production of 2-hydroxyglutarate (2-HG). Although the subject of intense study, the function of 2-HG in GBM is not entirely understood. The investigation of IDH mutant tumors has been hampered by the lack of appropriate models. We have now utilized the neurosphere culture method to generate cells from 7 different tumors bearing IDH1 mutations. This study tests the validity of these lines. We utilize both these primary cultures and mouse neurospheres to test the hypotheses that mutant IDH1 is pro-proliferative and influences cell fate. Methods: We successfully propagated and obtained microarray expression data on 7 neurosphere lines bearing IDH1 mutations and 61 IDH1 wildtype cell lines for comparison. We additionally performed methylation sequencing on 3 IDH1 mutant and 3 IDH1 wildtype lines to identify sets of genes that are consistently methylated and down-regulated in these IDH1mutant cell lines. We treated 6 IDH1 mutant lines with a small molecule IDH1 mutant inhibitor that prevents 2-HG formation to to determine effects on proliferation and whether down-regulated genes were rescued by the inhibition of 2-HG formation. Finally we treated fetal murine neural progenitors with exogenous 2-HG to determine the effects on differentiation status as well as on gene expression. Results: Gene expression studies of the 7 IDH1 mutant neurosphere lines revealed 103 genes that were 4-fold down-regulated in IDH1mutant tumors. Of those 103 genes, 32 were also found to be methylated and down-regulated in IDH1mutant brain tumor samples found in public databases. Methylation sequencing revealed that 31 of these 32 genes were heavily methylated in our three IDH1mutant cell lines, while only 12 of these genes were methylated in the IDH1WT cell lines. Treatment of IDH1mutant neurosphere cultures with a small molecule inhibitor resulted in diminished growth capacity. Microarray analysis demonstrated that only one of the downregulated genes was was consistently rescued, Inhibitor of DNA binding 2 (ID2). Application of exogenous 2-HG to murine neurosphere cultures promototed the appearance of oligodendrocytes and neurons, and also induced downregulation of ID2 and upregulation of Olig 2. Discussion: Our data suggest that there is likely a conserved set of “key gateway genes” that are frequently down-regulated in these IDH1mutant tumors and that patient-derived glioma cell lines maintain these epigenetic changes. Inhibition of 2-HG formation may be an effective therapy evidenced by the fact that tumor cells grow slower without 2-HG production. However inhibition of 2-HG production did not appear to reverse the majority of epigenetic changes in gene expression. The Id2 gene is consistently rescued by 2-HG inhibition and may be an important target for future tumor therapy. Citation Format: Matthew C. Garrett, Jack Mottahedeh, Jantzen Sperry, Ascia Eskin, Giovanni Coppola, Ya-shin Shih, Albert Lai, Arthur Chou, Linda Liau, Rob Prins, Timothy Cloughesy, Hong Wu, Stanley Nelson, Harley Kornblum. The IDH1 mutation in human glioblastoma and its effects on epigenetic modification and cell fate selection. [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 447. doi:10.1158/1538-7445.AM2014-447

Abstract B159: Heterozygous IDH1 mutations modify the citric acid (TCA) cycle metabolism and sensitize cells to inhibition of mitochondrial respiration/oxidative phosphorylation.

Abstract Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) occur in a variety of tumor types. Although these mutations are loss-of-function for conversion of isocitrate to α-ketoglutarate, the mutant enzymes greatly increase the production of the proposed oncometabolite, 2-hydroxyglutarate (2-HG). However the full metabolic consequences of IDH1/2 mutation in their heterozygous cellular context have yet to be fully explored. To address this question, we utilized a panel of isogenic cell lines with wild-type IDH1/2 or clinically relevant IDH1/2 mutations and examined the metabolic consequences of IDH mutation using (13)C metabolic flux analysis (MFA). We observe a dramatic and consistent decrease in the ability of IDH1 mutant cell lines to utilize reductive glutamine metabolism via the carboxylation of α-ketoglutarate back to isocitrate. This was not seen either in IDH2 mutant cell lines or in wild-type cell lines treated with exogenous 2-HG. Consistent with these changes, the IDH1 mutant cell lines, but not IDH2 mutant or 2-HG treated cells, were deficient in the utilization of glutamine for de novo lipogenesis. Similar trends were observed in endogenous, non-engineered IDH1/2 mutant cell lines. The decrease in reductive carboxylation in the IDH1 mutant cell lines raises the hypothesis that these cells may be more reliant on mitochondrial metabolism. Indeed, IDH1 mutant cells were more sensitive to either treatment with an electron transport chain inhibitor or growth in hypoxia (which also inhibits mitochondrial metabolism). These results show heterozygous IDH1 mutation robustly impacts wild-type cellular metabolism in a different manner than IDH2 mutation. Furthermore, these results suggest that IDH1 and IDH2 mutant tumors may be differentially sensitive to inhibitors of specific metabolic pathways. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B159. Citation Format: Alexandra R. Grassian, Seth Parker, Shawn Davidson, Courtney Green, Fallon Lin, Carol Joud-Caldwell, Hong Yin, Franklin Chung, Christopher Straub, Matthew Vander Heiden, Raymond Pagliarini, Christian Metallo. Heterozygous IDH1 mutations modify the citric acid (TCA) cycle metabolism and sensitize cells to inhibition of mitochondrial respiration/oxidative phosphorylation. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B159.