Lack of Constitutively Active DNA Repair Sensitizes Glioblastomas to Akt Inhibition and Induces Synthetic Lethality with Radiation Treatment in a p53-Dependent Manner.

Treatment refractory glioblastoma (GBM) remains a major clinical problem globally, and targeted therapies in GBM have not been promising to date. The Cancer Genome Atlas integrative analysis of GBM reported the striking finding of genetic alterations in the p53 and PI3K pathways in more than 80% of GBMs. Given the role of these pathways in making cell-fate decisions and responding to genotoxic stress, we investigated the reliance of these two pathways in mediating radiation resistance. We selected a panel of GBM cell lines and glioma stem cells (GSC) with wild-type TP53 (p53-wt) and mutant TP53, mutations known to interfere with p53 functionality (p53-mt). Cell lines were treated with a brain permeable inhibitor of P-Akt (ser473), phosphatidylinositol ether lipid analogue (PIA), with and without radiation treatment. Sensitivity to treatment was measured using Annexin-V/PI flow cytometry and Western blot analysis for the markers of apoptotic signaling, alkaline COMET assay. All results were verified in p53 isogenic cell lines. p53-mt cell lines were selectively radiosensitized by PIA. This radiosensitization effect corresponded with an increase in DNA damage and a decrease in DNA-PKcs levels. TP53 silencing in p53-wt cells showed a similar response as the p53-mt cells. In addition, the radiosensitization effects of Akt inhibition were not observed in normal human astrocytes, suggesting that this treatment strategy could have limited off-target effects. We demonstrate that the inhibition of the PI3K/Akt pathway by PIA radiosensitizes p53-mt cells by antagonizing DNA repair. In principle, this strategy could provide a large therapeutic window for the treatment of TP53-mutant tumors. Mol Cancer Ther; 17(2); 336-46. ©2017 AACRSee all articles in this MCT Focus section, "Developmental Therapeutics in Radiation Oncology."

NNMT Silencing Activates Tumor Suppressor PP2A, Inactivates Oncogenic STKs, and Inhibits Tumor Forming Ability.

Purpose: To identify potential molecular hubs that regulate oncogenic kinases and target them to improve treatment outcomes for glioblastoma patients.Experimental Design: Data mining of The Cancer Genome Atlas datasets identified nicotinamide-N-methyl transferase (NNMT) as a prognostic marker for glioblastoma, an enzyme linked to the reorganization of the methylome. We tested our hypothesis that NNMT plays a crucial role by modulating protein methylation, leading to inactivation of tumor suppressors and activation of oncogenes. Further experiments were performed to understand the underlying biochemical mechanisms using glioblastoma patient samples, established, primary, and isogenic cells.Results: We demonstrate that NNMT outcompetes leucine carboxyl methyl transferase 1 (LCMT1) for methyl transfer from principal methyl donor SAM in biological systems. Inhibiting NNMT increased the availability of methyl groups for LCMT1 to methylate PP2A, resulting in the inhibition of oncogenic serine/threonine kinases (STK). Further, NNMT inhibition retained the radiosensitizer nicotinamide and enhanced radiation sensitivity. We have provided the biochemical rationale of how NNMT plays a vital role in inhibiting tumor suppressor PP2A while concomitantly activating STKs.Conclusions: We report the intricate novel mechanism in which NNMT inhibits tumor suppressor PP2A by reorganizing the methylome both at epigenome and proteome levels and concomitantly activating prosurvival STKs. In glioblastoma tumors with NNMT expression, activation of PP2A can be accomplished by FDA approved perphenazine (PPZ), which is currently used to treat mood disorders such as schizophrenia, bipolar disorder, etc. This study forms a foundation for further glioblastoma clinical trials using PPZ with standard of care treatment. Clin Cancer Res; 23(9); 2325-34. ©2016 AACR.

Methionine and Kynurenine Activate Oncogenic Kinases in Glioblastoma, and Methionine Deprivation Compromises Proliferation.

PURPOSE: We employed a metabolomics-based approach with the goal to better understand the molecular signatures of glioblastoma cells and tissues, with an aim toward identifying potential targetable biomarkers for developing more effective and novel therapies. EXPERIMENTAL DESIGN: We used liquid chromatography coupled with mass spectrometry (LC-MS/Q-TOF and LC-MS/QQQ) for the discovery and validation of metabolites from primary and established glioblastoma cells, glioblastoma tissues, and normal human astrocytes. RESULTS: We identified tryptophan, methionine, kynurenine, and 5-methylthioadenosine as differentially regulated metabolites (DRM) in glioblastoma cells compared with normal human astrocytes (NHAs). Unlike NHAs, glioblastoma cells depend on dietary methionine for proliferation, colony formation, survival, and to maintain a deregulated methylome (SAM:SAH ratio). In methylthioadenosine phosphorylase (MTAP)-deficient glioblastoma cells, expression of MTAP transgene did not alter methionine dependency, but compromised tumor growth in vivo We discovered that a lack of the kynurenine-metabolizing enzymes kynurenine monooxygenase and/or kynureninase promotes the accumulation of kynurenine, which triggers immune evasion in glioblastoma cells. In silico analysis of the identified DRMs mapped the activation of key oncogenic kinases that promotes tumorigenesis in glioblastoma. We validated this result by demonstrating that the exogenous addition of DRMs to glioblastoma cells in vitro results in oncogene activation as well as the simultaneous downregulation of Ser/Thr phosphatase PP2A. CONCLUSIONS: We have connected a four-metabolite signature, implicated in the methionine and kynurenine pathways, to the promotion and maintenance of glioblastoma. Together, our data suggest that these metabolites and their respective metabolic pathways serve as potential therapeutic targets for glioblastoma. Clin Cancer Res; 22(14); 3513-23. ©2016 AACR.