Although considerable progress has been made toward understanding glioblastoma biology through large-scale genetic and protein expression analyses, little is known about the underlying metabolic alterations promoting their aggressive phenotype. We conducted global metabolomic profiling on patient-derived glioma specimens and identified specific metabolic programs differentiating low- and high-grade tumors, with the metabolic signature of glioblastoma reflecting accelerated anabolic metabolism. When coupled with transcriptional profiles, we identified the metabolic phenotype of the mesenchymal subtype to consist of accumulation of the glycolytic intermediate phosphoenolpyruvate and decreased pyruvate kinase activity. Unbiased hierarchical clustering of metabolomic profiles identified three subclasses, which we term energetic, anabolic, and phospholipid catabolism with prognostic relevance. These studies represent the first global metabolomic profiling of glioma, offering a previously undescribed window into their metabolic heterogeneity, and provide the requisite framework for strategies designed to target metabolism in this rapidly fatal malignancy. Cancer Res; 72(22); 5878–88. ©2012 AACR.

The relevance of cysteine metabolism in cancer has gained considerable interest in recent years, largely focusing on its role in generating the antioxidant glutathione. Through metabolomic profiling using a combination of high-throughput liquid and gas chromatography–based mass spectrometry on a total of 69 patient-derived glioma specimens, this report documents the discovery of a parallel pathway involving cysteine catabolism that results in the accumulation of cysteine sulfinic acid (CSA) in glioblastoma. These studies identified CSA to rank as one of the top metabolites differentiating glioblastoma from low-grade glioma. There was strong intratumoral concordance of CSA levels with expression of its biosynthetic enzyme cysteine dioxygenase 1 (CDO1). Studies designed to determine the biologic consequence of this metabolic pathway identified its capacity to inhibit oxidative phosphorylation in glioblastoma cells, which was determined by decreased cellular respiration, decreased ATP production, and increased mitochondrial membrane potential following pathway activation. CSA-induced attenuation of oxidative phosphorylation was attributed to inhibition of the regulatory enzyme pyruvate dehydrogenase. Studies performed in vivo abrogating the CDO1/CSA axis using a lentiviral-mediated short hairpin RNA approach resulted in significant tumor growth inhibition in a glioblastoma mouse model, supporting the potential for this metabolic pathway to serve as a therapeutic target. Collectively, we identified a novel, targetable metabolic pathway involving cysteine catabolism contributing to the growth of aggressive high-grade gliomas. These findings serve as a framework for future investigations designed to more comprehensively determine the clinical application of this metabolic pathway and its contributory role in tumorigenesis. Cancer Res; 74(3); 787–96. ©2013 AACR.

Whole brain radiotherapy (WBRT) is a mainstay of treatment in patients with both identifiable brain metastases and prophylaxis for microscopic disease. The use of WBRT has decreased somewhat in recent years due to both advances in radiation technology, allowing for a more localized delivery of radiation, and growing concerns regarding the late toxicity profile associated with WBRT. This has prompted the development of several recent and ongoing prospective studies designed to provide Level I evidence to guide optimal treatment approaches for patients with intracranial metastases. In addition to defining the role of WBRT in patients with brain metastases, identifying methods to improve WBRT is an active area of investigation, and can be classified into two general categories: Those designed to decrease the morbidity of WBRT, primarily by reducing late toxicity, and those designed to improve the efficacy of WBRT. Both of these areas of research show diversity and promise, and it seems feasible that in the near future, the efficacy/toxicity ratio may be improved, allowing for a more diverse clinical application of WBRT.

The purpose of this study was to determine the capacity of MK-1775, a potent Wee-1 inhibitor, to abrogate the radiation-induced G₂ checkpoint arrest and modulate radiosensitivity in glioblastoma cell models and normal human astrocytes. The radiation-induced checkpoint response of established glioblastoma cell lines, glioblastoma neural stem (GNS) cells, and astrocytes were determined in vitro by flow cytometry and in vivo by mitosis-specific staining using immunohistochemistry. Mechanisms underlying MK-1775 radiosensitization were determined by mitotic catastrophe and γH2AX expression. Radiosensitivity was determined in vitro by the clonogenic assay and in vivo by tumor growth delay. MK-1775 abrogated the radiation-induced G₂ checkpoint and enhanced radiosensitivity in established glioblastoma cell lines in vitro and in vivo, without modulating radiation response in normal human astrocytes. MK-1775 appeared to attenuate the early-phase of the G₂ checkpoint arrest in GNS cell lines, although the arrest was not sustained and did not lead to increased radiosensitivity. These results show that MK-1775 can selectively enhance radiosensitivity in established glioblastoma cell lines. Further work is required to determine the role Wee-1 plays in checkpoint activation of GNS cells. Mol Cancer Ther; 10(12); 2405–14. ©2011 AACR.

Purpose: Preclinical studies evaluating histone deacetylase (HDAC) inhibitor-induced radiosensitization have largely focused on the preirradiation setting based on the assumption that enhanced radiosensitivity was mediated by changes in gene expression. Our previous investigations identified maximal radiosensitization when cells were exposed to HDAC inhibitors in both the preradiation and postradiation setting. We now expand on these studies to determine whether postirradiation exposure alone affects radiosensitivity. Experimental Design: The effects of the HDAC inhibitor valproic acid (VA) on postirradiation sensitivity in human glioma cell lines were evaluated using a clonogenic assay, exposing cells to VA up to 24 h after irradiation. DNA damage repair was evaluated using γH2AX and 53BP1 foci and cell cycle phase distribution was analyzed by flow cytometry. Western blot of acetylated γH2AX was done following histone extraction on AUT gels. Results: VA enhanced radiosensitivity when delivered up to 24 h after irradiation. Cells accumulated in G₂-M following irradiation, although they returned to baseline at 24 h, mitigating the role of cell cycle redistribution in postirradiation sensitization by VA. At 12 h after irradiation, significant γH2AX and 53BP1 foci dispersal was shown in the control, although cells exposed to VA after irradiation maintained foci expression. VA alone had no effect on the acetylation or phosphorylation of H2AX, although it did acetylate radiation-induced γH2AX. Conclusions: These results indicate that VA enhances radiosensitivity at times up to 24 h after irradiation, which has direct clinical application.

A phase I study was conducted to determine the dose-limiting toxicities (DLT) and maximum tolerated dose (MTD) for the combination of vorinostat with bevacizumab and CPT-11 in recurrent glioblastoma. Vorinostat was combined with bevacizumab and CPT-11 and was escalated using a standard 3 + 3 design. Vorinostat was escalated up to 2 actively investigated doses of this compound or until the MTD was identified on the basis of DLTs. Correlative science involving proteomic profiling of serial patient plasma samples was performed. Nineteen patients were treated. The MTD of vorinostat was established at 400 mg on days 1–7 and 15–21 every 28 days when combined with bevacizumab and CPT-11. Common toxicities were fatigue and diarrhea. DLTs included fatigue, hypertension/hypotension, and central nervous system ischemia. Although the MTD was established, CPT-11 dose reductions were common early in therapy. High-dose vorinostat had an improved progression-free survival and overall survival when compared with low-dose vorinostat. Serum proteomic profiling identified IGFBP-5 and PDGF-AA as markers for improved PFS and recurrence, respectively. A MTD for the combination of vorinostat with bevacizumab and CPT-11 has been established, although it has poor long-term tolerability. With the increased toxicities associated with CPT-11 coupled with its unclear clinical significance, investigating the efficacy of vorinostat combined with bevacizumab alone may represent a more promising strategy to evaluate in the context of a phase II clinical trial.