Disha Patel

Current Therapeutic Paradigms in Glioblastoma

Glioblastoma (GBM), a WHO grade IV malignant glioma, is the most common and lethal adult primary brain tumor. Median survival rates range from 12-15 months. The current standard of care for GBM has evolved from resection followed by adjuvant radiotherapy to resection, concurrent adjuvant chemotherapy (temozolomide) and radiation, and additional adjuvant chemotherapy. The expression of specific molecular biomarkers, especially O-6-methylguanine methyltransferase (MGMT) status, may determine the response of the tumor to treatment, and helps in identifying the magnitude of benefit from this regimen. By identifying further biological subtypes of GBM at the molecular level, specific targeted therapies could be developed and used in the future for more individualized therapeutic regimens. This article will review the current therapies for GBM and the investigation of new molecular and targeted therapies, such as EGFR inhibitors, mTOR/PI3Kinase inhibitors, and anti-angiogenesis agents.

Molecular Advances of Brain Tumors in Radiation Oncology

Glioblastoma, grade IV malignant glioma based on the World Health Organization classification, is the most common primary brain tumor in adults. The average survival time of less than 1 year has not improved notably over the last 3 decades. Surgery and radiotherapy, the traditional cornerstones of therapy, provide palliative benefit, whereas the value of chemotherapy has been marginal and controversial. The dismal prognosis of glioblastoma patients is largely caused by the striking radioresistance of these tumors. A better understanding of the molecular mechanisms that underlie the malignant phenotype of glioblastomas and plausible mechanisms of radiation resistance can provide new possibilities in terms of targeted therapeutic strategies. Despite the genetic heterogeneity of malignant gliomas, common aberrations in the signaling elements of the growth and survival pathways are found. New treatments have emerged to target molecules in these signaling pathways with the goal to increase specific efficacy and minimize toxicity. Monoclonal antibodies and low molecular-weight kinase inhibitors are the most common classes of agents in targeted cancer treatment. This review introduces these new targeted therapies in the context of current treatment options for patients with glioblastoma. It is hoped that this combined approach will overcome the current limitations in the treatment of patients with glioblastoma and result in a better prognosis for these patients.

Technological Advances in Radiation Oncology for Central Nervous System Tumors

Advances in computer software technology have led to enormous progress that has enabled increasing levels of complexity to be incorporated into radiotherapy treatment planning systems. Because of these changes, the delivery of radiotherapy evolved from therapy designed primarily on plain 2-dimensional X-ray images and hand calculations to therapy based on 3-dimensional images incorporating increasingly complex computer algorithms in the planning process. In addition, challenges in treatment planning and radiation delivery, such as problems with setup error and organ movement, have begun to be systematically addressed, ushering in an era of so-called 4-dimensional radiotherapy. This review article discusses how these advances have changed the way in which many common neoplasms of the central nervous system are being treated at present.

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.

Biomarkers of clinical responsiveness in brain tumor patients : progress and potential.

Gliomas are the most common primary brain tumors in adults. Anaplastic astrocytoma and glioblastoma multiforme represent malignant astrocytomas, which are the most common type of malignant gliomas. Despite research efforts in cancer therapy, the prognosis of patients with malignant gliomas remains poor. Research efforts in recent years have focused on investigating the cellular, molecular, and genetic pathways involved in the progression of malignant gliomas. As a result, biomarkers have emerged as diagnostic, predictive, and prognostic tools that have the potential to transform the field of brain tumor diagnostics. An increased understanding of the important molecular pathways that have been implicated in the progression of malignant gliomas has led to the identification of potential diagnostic, prognostic, and predictive biomarkers, some bearing clinical implications for targeted therapy. Some of the most promising biomarkers to date include loss of chromosomes 1p/19q in oligodendrogliomas and expression of O-6-methylguanine-DNA methyltransferase (MGMT) or epidermal growth factor receptor (EGFR) status in glioblastomas. Other promising biomarkers in glioma research include glial fibrillary acidic protein, galectins, Kir potassium channel proteins, angiogenesis, and apoptosis pathway markers. Research into the clinical relevance and applicability of such biomarkers has the potential to revolutionize our approach to the diagnosis and treatment of patients with malignant gliomas.

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 AACR. See 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.

Abstract 522: Role and regulation of Aurora A in radiation resistance mechanisms in glioblastoma

Glioblastoma (GBM) is the most aggressive tumor of the human brain, which inevitably escapes the standard treatment modalities that include surgery, radiotherapy and chemotherapy. Consequently, the median survival of GBM patients remains 12-15 months despite these aggressive treatments. Therefore, to develop effective therapies, it is important to understand how GBM cells develop therapeutic resistance. Aurora A is a ubiquitously expressed serine/threonine kinase, which plays essential roles in mitotic regulation. This protein kinase, which phosphorylates p53, PP1, CEBP, and histone H3 among other regulators of cell division, is overexpressed in various neoplasms including GBM and invariably associated with poor prognosis. Recent studies have demonstrated that Aurora A contributes to radio-resistance in various solid neoplasm including GBM. Importantly, Aurora A inhibitors are currently in phase I/II clinical trials for solid cancer. Notably, Aurora A inhibitor MLN8237 that crosses the blood brain barrier specifically inhibits Aurora A at concentrations ≤ maximally tolerated dose in animal models and in phase I clinical trials. We found that Aurora A protein level is upregulated by ionizing radiation in GBM cells. This was, in part, mediated by radiation-induced inhibition of 26S-proteosomal degradation of Aurora A. Aurora A was silenced using both lentiviral mediated shRNA expression and a specific Aurora A inhibitor in three different established cell lines, and were treated with 6 gy radiation. We found that pharmacological inhibition and RNAi-mediated knockdown of Aurora A sensitized the GBM cells to radiation therapy resulting in reduced cell proliferation and increased apoptosis. Interestingly, we observed that RNAi-mediated down-regulation of Aurora A was associated with a decrease in X-linked inhibitor of apoptosis (XIAP) expression in GBM cells. We also found that Aurora A and XIAP were co-localized in GBM cells. Taken, together our results reveal a mechanism by which Aurora A confers radio-resistance in GBM cells and further rationalize the use of Aurora A inhibitors as radio-sensitizer in solid neoplasms including GBM. Funding sources: This work was supported by R01CA108633 (To AC), 1RC2CA148190 (To AC) U10CA180850-01 (To AC), 1R01CA169368 (To AC) from the National Cancer Institute (NCI), Brain Tumor Funders Collaborative Grant (To AC), Ohio State University Comprehensive Cancer Center Award (To AC) Citation Format: Brinda Ramasubramanian, Kamalakannan Palanichamy, Disha Patel, Saikh Jaharul Haque, Arnab Chakravarti. Role and regulation of Aurora A in radiation resistance mechanisms in glioblastoma. [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 522.

Abstract LB-6: Exploring nicotinamide-N-methyltransferase kinetics through targeted metabolomic profiling for its prognostic value in glioblastoma

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Purpose: Glioblastoma (GBM) is the most aggressive form of glioma. With average patient survival of about 14 months, largely due to its resistance to conventional therapies, there is opportunity for significant improvements to care. Nicotinamide-N-methyltransferase (NNMT) is an enzyme responsible for the methylation of Nicotinamide and other xenobiotic compounds. Although NNMTs exact role in the malignant phenotype remains poorly understood, it has been investigated for its potential use as a prognostic marker or therapeutic target in other cancers. It is detected at very low levels in normal brain tissue but at significantly higher levels in GBM. Additionally, Kaplan-Meier plots from publically available data sets suggest that higher NNMT expression is correlated with adverse patient outcome. In addition to gene interference and various functional assays, we employed a targeted metabolomic approach, using LC-MS Quadropole Time of Flight (Q-TOF) and LC-MS Triple Quad (QQQ) instruments to study intracellular levels of Nicotinamide and N-methylnicotinamide to gain a better understanding of NNMTs role in GBM. Methods: We determined the expression profile of NNMT in established (ATCC) and in our panel of patient derived, primary GBM cell lines. By generating an NNMT isogenic model in U87 cells, we were able to study the functional consequences of differential NNMT expression using in vitro assays such as MTS Assay, Clonogenic Survival Assay, Annexin V, ATP Assay, Comet Assay, and Western Blotting. We intracranially injected NOD-SCID mice with U87 NNMT cells to assess differences in tumorgenicity in vivo. We have also used LC-MS Quadropole Time of Flight (Q-TOF) and LC-MS Triple Quad (QQQ) instruments to measure intracellular levels of the metabolites relevant to NNMT enzymatic function. Results and Conclusions: Results indicate that NNMT protein is highly expressed in primary cell lines. Encouragingly, U87 NNMT Knockdown cells are less proliferative, more sensitive to radiation in vitro, and are less tumorgenic in vivo. Recapitulating the clinical observations, these results could justify why clinically patients with lower expression of NNMT enjoyed increased overall survival. These results also suggest that finding ways to decrease NNMT in patients tumors may improve response to radiation. Additional preliminary results indicate that intracellular N-methylnicotinamide levels correlate directly with NNMT protein expression and could be used a surrogate biomarker for intracellular NNMT levels, potentially predicting not only overall survival but perhaps response to radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-6. doi:1538-7445.AM2012-LB-6

Abstract 2478: Silencing Aurora Kinase A radio-sensitizes glioma cells

Purpose: It has been reported that Aurora Kinase A (AurKA) is overexpressed in many cancer types, including breast, ovarian, pancreatic, head and neck, esophageal, renal, and lung. Upregulation of AurKA can lead to oncogenic transformation and aneuploidy. AurKA is a member of the serine/threonine family of protein kinases that is localized to the mitotic spindle during cell division. AurKA is involved in spindle assembly and regulated by phosphorylation-dependent proteosomal degradation. Cells that overexpress AurKA surpass the G2/M checkpoint prematurely, resulting in chromosomal abnormalities. Silencing AurKA will prevent premature progression through the G2/M checkpoint, allowing for cells to be targeted in the radiosensitive G2/M phase. Materials and Methods: We first looked at the expression of AurKA in established (ATCC) and primary glioblastoma (GBM) cell lines. Stable silencing of AurKA was done via a lentiviral transfection with shRNA in U87, LN18, and LN229 cell lines. We performed clonogenic survival and MTS assays to measure the cell death after radiation of these AurKA knockdown (AurKA KD) cells versus cells transfected with non-targeting (NT) shRNA. Western blot was performed using these same cells post-radiation treatment to investigate the expression of proteins in both apoptotic and pro-survival pathways. To investigate the effect of AurKA KD on the formation of polyploidy cells and cell cycle regulation. Cell cycle analysis on AurKA KD and NT cells was conducted using flow cytometry. AurKA is also known to phosphorylate tumor suppressor protein p53 at Ser315, which leads to degradation of p53 via ubiquitination by Mdm2. We carried out experiments where AurKA KD cells, with either intact or mutant p53, were treated with a p53 inhibitor Pifithrin α and subsequently measured apoptosis using Annexin V and Western blot. Initial sequencing results of AurKA in our GBM cell lines did not lead to any conclusive results. Furthermore, we assessed the copy number variation of AurKA to correlate with radiosensitivity of our GBM cell lines. Results and Conclusions: Radiation treatment increases AurKA expression in our established and primary GBM cell lines. Next we silenced AurKA using lentiviral mediated shRNA transduction. AurKA KD cells exhibit greater radiosensitivity and reduced proliferation when compared to control cells, as shown by the clonogenic survival and MTS assays, respectively. Increased expression of apoptotic proteins, such as cleaved PARP and cleaved caspase-3, is seen in AurKA KD cells after treatment with radiation when compared to controls. Moreover, silencing AurKA prevented the accumulation of polyploid cells after radiation treatment. Our data suggest that AurKA could be a promising therapeutic target for radiosensitizing of GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2478. doi:10.1158/1538-7445.AM2011-2478