Kensuke Tateishi

Extreme Vulnerability of IDH1 Mutant Cancers to NAD+ Depletion

Heterozygous mutation of IDH1 in cancers modifies IDH1 enzymatic activity, reprogramming metabolite flux and markedly elevating 2-hydroxyglutarate (2-HG). Here, we found that 2-HG depletion did not inhibit growth of several IDH1 mutant solid cancer types. To identify other metabolic therapeutic targets, we systematically profiled metabolites in endogenous IDH1 mutant cancer cells after mutant IDH1 inhibition and discovered axa0profound vulnerability to depletion of the coenzyme NAD+. Mutant IDH1 lowered NAD+ levels by downregulating the NAD+ salvage pathway enzyme nicotinate phosphoribosyltransferase (Naprt1), sensitizing to NAD+ depletion via concomitant nicotinamide phosphoribosyltransferase (NAMPT) inhibition. NAD+ depletion activated the intracellular energy sensor AMPK, triggered autophagy, and resulted in cytotoxicity. Thus, we identify NAD+ depletion as a metabolic susceptibility of IDH1 mutant cancers.

Targetable signaling pathway mutations are associated with malignant phenotype in IDH-mutant gliomas

Purpose: Isocitrate dehydrogenase (IDH) gene mutations occur in low-grade and high-grade gliomas. We sought to identify the genetic basis of malignant phenotype heterogeneity in IDH-mutant gliomas. Methods: We prospectively implanted tumor specimens from 20 consecutive IDH1-mutant glioma resections into mouse brains and genotyped all resection specimens using a CLIA-certified molecular panel. Gliomas with cancer driver mutations were tested for sensitivity to targeted inhibitors in vitro. Associations between genomic alterations and outcomes were analyzed in patients. Results: By 10 months, 8 of 20 IDH1-mutant gliomas developed intracerebral xenografts. All xenografts maintained mutant IDH1 and high levels of 2-hydroxyglutarate on serial transplantation. All xenograft-producing gliomas harbored “lineage-defining” mutations in CIC (oligodendroglioma) or TP53 (astrocytoma), and 6 of 8 additionally had activating mutations in PIK3CA or amplification of PDGFRA, MET, or N-MYC. Only IDH1 and CIC/TP53 mutations were detected in non–xenograft-forming gliomas (P = 0.0007). Targeted inhibition of the additional alterations decreased proliferation in vitro. Moreover, we detected alterations in known cancer driver genes in 13.4% of IDH-mutant glioma patients, including PIK3CA, KRAS, AKT, or PTEN mutation or PDGFRA, MET, or N-MYC amplification. IDH/CIC mutant tumors were associated with PIK3CA/KRAS mutations whereas IDH/TP53 tumors correlated with PDGFRA/MET amplification. Presence of driver alterations at progression was associated with shorter subsequent progression-free survival (median 9.0 vs. 36.1 months; P = 0.0011). Conclusion: A subset of IDH-mutant gliomas with mutations in driver oncogenes has a more malignant phenotype in patients. Identification of these alterations may provide an opportunity for use of targeted therapies in these patients. Clin Cancer Res; 20(11); 2898–909. ©2014 AACR.

Myc-driven glycolysis is a therapeutic target in glioblastoma

Purpose: Deregulated Myc drives an oncogenic metabolic state, including pseudohypoxic glycolysis, adapted for the constitutive production of biomolecular precursors to feed rapid tumor cell growth. In glioblastoma, Myc facilitates renewal of the tumor-initiating cell reservoir contributing to tumor maintenance. We investigated whether targeting the Myc-driven metabolic state could be a selectively toxic therapeutic strategy for glioblastoma. Experimental Design: The glycolytic dependency of Myc-driven glioblastoma was tested using 13C metabolic flux analysis, glucose-limiting culture assays, and glycolysis inhibitors, including inhibitors of the NAD+ salvage enzyme nicotinamide phosphoribosyl-transferase (NAMPT), in MYC and MYCN shRNA knockdown and lentivirus overexpression systems and in patient-derived glioblastoma tumorspheres with and without MYC/MYCN amplification. The in vivo efficacy of glycolyic inhibition was tested using NAMPT inhibitors in MYCN-amplified patient-derived glioblastoma orthotopic xenograft mouse models. Results: Enforced Myc overexpression increased glucose flux and expression of glycolytic enzymes in glioblastoma cells. Myc and N-Myc knockdown and Myc overexpression systems demonstrated that Myc activity determined sensitivity and resistance to inhibition of glycolysis. Small-molecule inhibitors of glycolysis, particularly NAMPT inhibitors, were selectively toxic to MYC/MYCN–amplified patient-derived glioblastoma tumorspheres. NAMPT inhibitors were potently cytotoxic, inducing apoptosis and significantly extended the survival of mice bearing MYCN-amplified patient-derived glioblastoma orthotopic xenografts. Conclusions: Myc activation in glioblastoma generates a dependency on glycolysis and an addiction to metabolites required for glycolysis. Glycolytic inhibition via NAMPT inhibition represents a novel metabolically targeted therapeutic strategy for MYC or MYCN-amplified glioblastoma and potentially other cancers genetically driven by Myc. Clin Cancer Res; 22(17); 4452–65. ©2016 AACR.

The alkylating chemotherapeutic temozolomide induces metabolic stress in IDH1-mutant cancers and potentiates NAD+ depletion-mediated cytotoxicity

IDH1-mutant gliomas are dependent upon the canonical coenzyme NAD+ for survival. It is known that PARP activation consumes NAD+ during base excision repair (BER) of chemotherapy-induced DNA damage. We therefore hypothesized that a strategy combining NAD+ biosynthesis inhibitors with the alkylating chemotherapeutic agent temozolomide could potentiate NAD+ depletion-mediated cytotoxicity in mutant IDH1 cancer cells. To investigate the impact of temozolomide on NAD+ metabolism, patient-derived xenografts and engineered mutant IDH1-expressing cell lines were exposed to temozolomide, in vitro and in vivo, both alone and in combination with nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, which block NAD+ biosynthesis. The acute time period (<3 hours) after temozolomide treatment displayed a burst of NAD+ consumption driven by PARP activation. In IDH1-mutant-expressing cells, this consumption reduced further the abnormally lowered basal steady-state levels of NAD+, introducing a window of hypervulnerability to NAD+ biosynthesis inhibitors. This effect was selective for IDH1-mutant cells and independent of methylguanine methyltransferase or mismatch repair status, which are known rate-limiting mediators of adjuvant temozolomide genotoxic sensitivity. Combined temozolomide and NAMPT inhibition in an in vivo IDH1-mutant cancer model exhibited enhanced efficacy compared with each agent alone. Thus, we find IDH1-mutant cancers have distinct metabolic stress responses to chemotherapy-induced DNA damage and that combination regimens targeting nonredundant NAD+ pathways yield potent anticancer efficacy in vivo Such targeting of convergent metabolic pathways in genetically selected cancers could minimize treatment toxicity and improve durability of response to therapy. Cancer Res; 77(15); 4102-15. ©2017 AACR.

Transaminase Inhibition by 2-Hydroxyglutarate Impairs Glutamate Biosynthesis and Redox Homeostasis in Glioma

IDH1 mutations are common in low-grade gliomas and secondary glioblastomas and cause overproduction of (R)-2HG. (R)-2HG modulates the activity of many enzymes, including some that are linked to transformation and some that are probably bystanders. Although prior work on (R)-2HG targets focused on 2OG-dependent dioxygenases, we foundxa0that (R)-2HG potently inhibits the 2OG-dependent transaminases BCAT1 and BCAT2, likely as a bystander effect, thereby decreasing glutamate levels and increasing dependence on glutaminase for the biosynthesis of glutamate and one of its products, glutathione. Inhibiting glutaminase specifically sensitized IDH mutant glioma cells to oxidative stress inxa0vitro and to radiation inxa0vitro and inxa0vivo. These findings highlight the complementary roles for BCATs and glutaminase in glutamate biosynthesis, explain the sensitivity of IDH mutant cells to glutaminase inhibitors, and suggest a strategy for maximizing the effectiveness of such inhibitors against IDH mutant gliomas.

Genotype-targeted local therapy of glioma

Significance Lower-grade gliomas are often characterized by mutations in metabolism-related genes isocitrate dehydrogenase 1 (IDH1) and IDH2. Resection of these tumors is constrained by adjacent eloquent cortex, resulting in local failures. Studies showed that IDH mutant cells are sensitive to metabolic therapeutics, but these drugs are limited by systemic toxicities. We hypothesized that application of metabolism-altering therapeutics at the surgical margin would improve tumor control and minimize toxicity. We developed an intraoperative diagnostic assay to identify IDH mutations. We show that intratumoral administration of sustained release formulations of metabolism-altering compound prolongs survival in a mouse model of IDH mutant glioma. This genotype-based paradigm introduces a workflow in surgical oncology that can be extended to other tumors characterized by targetable molecular alterations. Aggressive neurosurgical resection to achieve sustained local control is essential for prolonging survival in patients with lower-grade glioma. However, progression in many of these patients is characterized by local regrowth. Most lower-grade gliomas harbor isocitrate dehydrogenase 1 (IDH1) or IDH2 mutations, which sensitize to metabolism-altering agents. To improve local control of IDH mutant gliomas while avoiding systemic toxicity associated with metabolic therapies, we developed a precision intraoperative treatment that couples a rapid multiplexed genotyping tool with a sustained release microparticle (MP) drug delivery system containing an IDH-directed nicotinamide phosphoribosyltransferase (NAMPT) inhibitor (GMX-1778). We validated our genetic diagnostic tool on clinically annotated tumor specimens. GMX-1778 MPs showed mutant IDH genotype-specific toxicity in vitro and in vivo, inducing regression of orthotopic IDH mutant glioma murine models. Our strategy enables immediate intraoperative genotyping and local application of a genotype-specific treatment in surgical scenarios where local tumor control is paramount and systemic toxicity is therapeutically limiting.

Abstract 2675: Metabolic addiction in IDH1 mutant cancers

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.