Nina Lelic

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.

Rapid Intraoperative Molecular Characterization of Glioma

IMPORTANCE Conclusive intraoperative pathologic confirmation of diffuse infiltrative glioma guides the decision to pursue definitive neurosurgical resection. Establishing the intraoperative diagnosis by histologic analysis can be difficult in low-cellularity infiltrative gliomas. Therefore, we developed a rapid and sensitive genotyping assay to detect somatic single-nucleotide variants in the telomerase reverse transcriptase (TERT) promoter and isocitrate dehydrogenase 1 (IDH1). OBSERVATIONS This assay was applied to tissue samples from 190 patients with diffuse gliomas, including archived fixed and frozen specimens and tissue obtained intraoperatively. Results demonstrated 96% sensitivity (95% CI, 90%-99%) and 100% specificity (95% CI, 95%-100%) for World Health Organization grades II and III gliomas. In a series of live cases, glioma-defining mutations could be identified within 60 minutes, which could facilitate the diagnosis in an intraoperative timeframe. CONCLUSIONS AND RELEVANCE The genotyping method described herein can establish the diagnosis of low-cellularity tumors like glioma and could be adapted to the point-of-care diagnosis of other lesions that are similarly defined by highly recurrent somatic mutations.

Sporadic hemangioblastomas are characterized by cryptic VHL inactivation

Hemangioblastomas consist of 10-20% neoplastic “stromal” cells within a vascular tumor cell mass of reactive pericytes, endothelium and lymphocytes. Familial cases of central nervous system hemangioblastoma uniformly result from mutations in the Von Hippel-Lindau (VHL) gene. In contrast, inactivation of VHL has been previously observed in only a minority of sporadic hemangioblastomas, suggesting an alternative genetic etiology. We performed deep-coverage DNA sequencing on 32 sporadic hemangioblastomas (whole exome discovery cohort n = 10, validation n = 22), followed by analysis of clonality, copy number alteration, and somatic mutation. We identified somatic mutation, loss of heterozygosity and/or deletion of VHL in 8 of 10 discovery cohort tumors. VHL inactivating events were ultimately detected in 78% (25/32) of cases. No other gene was significantly mutated. Overall, deep-coverage sequence analysis techniques uncovered VHL alterations within the neoplastic fraction of these tumors at higher frequencies than previously reported. Our findings support the central role of VHL inactivation in the molecular pathogenesis of both familial and sporadic hemangioblastomas.

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.

A new patient-derived orthotopic malignant meningioma model treated with oncolytic herpes simplex virus

BACKGROUND Higher-grade meningiomas (HGMs; World Health Organization grades II and III) pose a clinical problem due to high recurrence rates and the absence of effective therapy. Preclinical development of novel therapeutics requires a disease model that recapitulates the genotype and phenotype of patient HGM. Oncolytic herpes simplex virus (oHSV) has shown efficacy and safety in cancers in preclinical and clinical studies, but its utility for HGM has not been well characterized. METHODS Tumorsphere cultures and serial orthotopic xenografting in immunodeficient mice were used to establish a patient-derived HGM model. The model was pathologically and molecularly characterized by immunohistochemistry, western blot, and genomic DNA sequencing and compared with the patient tumor. Anti-HGM effects of oHSV G47Δ were assessed using cell viability and virus replication assays in vitro and animal survival analysis following intralesional injections of G47Δ. RESULTS We established a serially transplantable orthotopic malignant meningioma model, MN3, which was lethal within 3 months after tumorsphere implantation. MN3 xenografts exhibited the pathological hallmarks of malignant meningioma such as high Ki67 and vimentin expression. Both the patient tumor and xenografts were negative for neurofibromin 2 (merlin) and had the identical NF2 mutation. Oncolytic HSV G47Δ efficiently spread and killed MN3 cells, as well as other patient-derived HGM lines in vitro. Treatment with G47Δ significantly extended the survival of mice bearing subdural MN3 tumors. CONCLUSIONS We established a new patient-derived meningioma model that will enable the study of targeted therapeutic approaches for HGM. Based on these studies, it is reasonable to consider a clinical trial of G47Δ for HGM.