Abstract A049: Age-Associated Transcriptomic Alterations in Glioblastoma Following Temozolomide Treatment

Simple SummaryThere is a critical need to identify readily translatable adjuncts to potentiate the dismal median survivals of only 15–20 months in glioblastoma (GBM) patients after standard of care, i.e., concurrent Temozolomide (TMZ) and radiation (XRT) therapy. Here we demonstrated that the Polo-like kinase 1 (PLK1) inhibitor volasertib, which has been employed in cancer clinical trials, has activity against GBM in the contexts of both as monotherapy and as an adjunct to standard of care (SOC). In addition to corroborating the known effects of volasertib, we found novel impacts of volasertib on mitochondrial membrane potential, ROS generation, persistent DNA damage and signaling pathways such as ERK/MAPK, AMPK and glucocorticoid receptor. Together these studies support the potential importance of PLK1 inhibitors as an adjunct to GBM SOC therapy that warrants further preclinical investigation.New strategies that improve median survivals of only ~15–20 months for glioblastoma (GBM) with the current standard of care (SOC) which is concurrent temozolomide (TMZ) and radiation (XRT) treatment are urgently needed. Inhibition of polo-like kinase 1 (PLK1), a multifunctional cell cycle regulator, overexpressed in GBM has shown therapeutic promise but has never been tested in the context of SOC. Therefore, we examined the mechanistic and therapeutic impact of PLK1 specific inhibitor (volasertib) alone and in combination with TMZ and/or XRT on GBM cells. We quantified the effects of volasertib alone and in combination with TMZ and/or XRT on GBM cell cytotoxicity/apoptosis, mitochondrial membrane potential (MtMP), reactive oxygen species (ROS), cell cycle, stemness, DNA damage, DNA repair genes, cellular signaling and in-vivo tumor growth. Volasertib alone and in combination with TMZ and/or XRT promoted apoptotic cell death, altered MtMP, increased ROS and G2/M cell cycle arrest. Combined volasertib and TMZ treatment reduced side population (SP) indicating activity against GBM stem-like cells. Volasertib combinatorial treatment also significantly increased DNA damage and reduced cell survival by inhibition of DNA repair gene expression and modulation of ERK/MAPK, AMPK and glucocorticoid receptor signaling. Finally, as observed in-vitro, combined volasertib and TMZ treatment resulted in synergistic inhibition of tumor growth in-vivo. Together these results identify new mechanisms of action for volasertib that provide a strong rationale for further investigation of PLK1 inhibition as an adjunct to current GBM SOC therapy.

High-grade glioma: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up

Malignant primary spinal cord gliomas (PSCGs) are rare, and the optimal treatment for these lesions remains controversial. We report herein treatment outcomes of six malignant PSCGs managed with temozolomide (TMZ)-based multidisciplinary treatment. TMZ was administered concomitantly with fractionated radiotherapy for two newly diagnosed primary spinal cord glioblastoma multiforme (GBM), followed by adjuvant chemotherapy with TMZ. For one anaplastic astrocytoma (AA) and one anaplastic ependymoma (AEPN), TMZ was given as adjuvant therapy at first recurrence. One malignantly transformed ependymoma (EPN) and one malignantly transformed diffuse astrocytoma (DA) were treated with TMZ after radiotherapy at second recurrence. Two patients with newly diagnosed GBM died, 12 and 16 months, respectively, after being treated with TMZ, during and after radiation therapy. One patient with AA and one with malignantly transformed EPN showed good response to salvage therapy with TMZ and had stable disease 21 and 20 months, respectively, after TMZ treatment. One patient with recurrent AEPN and one with malignantly transformed DA died from uncontrolled progression of the lesions despite TMZ chemotherapy. Three patients developed grade 1 or 2 neutropenia, anemia, and infection. Nonhematologic toxicities occurred in all patients; however, they were below grade 3 in severity. TMZ treatment may have a positive effect on control of malignant PSCGs and survival for some patients. Specifically, treatment with TMZ during and after radiation therapy might provide survival benefit to patients with primary spinal cord GBM. A multicenter cooperative investigation for a large-scale study on malignant PSCGs may be required.

Background: Upregulation of cellular signaling pathways for proliferation is one of the characteristic features of high-grade glioblastomas (GBM), the most common and lethal type of adult brain tumors. Common oncogenic alterations in GBM include a characteristic gain-of-function of growth-promoting EGFR gene, a functionally relevant activation of p21-RAS pathway or a loss-of-expression of growth-attenuating tumor suppressor PTEN gene. Here we tested the role of genetic backgrounds (RAS pathway upregulation and PTEN nullness) on the sensitivity of ABT888 in the presence of either temozolomide (TMZ) or carboplatin (C). Methods: We examined the anti-tumor effects of ABT888 using PTEN null U87MG cell line and a transgenic mouse model (12V-Ha-Ras transgenic mice) of human glioma expressing V12 RAS (Can. Res. 2001). LacZ, GFAP and PCNA-positive primary astrocytes (derivative astrocytes, passage 100) were used for the study. The effect of the drugs were tested by (1) soft agar colony growth (2) 3D- On-Top assay (3) cell cycle analyses (4) vascular mimicry (real time) and (5) Western blots in a dose and time dependent manner. Results: Expression of the transgene in 12V-Ha-Ras mice (tail biopsy of mice from which derivative glioma cells cultures were established) and in the derivative glioma cells (established from the transgene-expressing mice) at different passages were confirmed by genotyping. Testing for astrocytic marker GFAP by ICC (>95% GFAP positive at passage 10 and onwards). Both U87MG glioma cells and astrocytoma cells from 12V-Ha-Ras mice were positive for GFAP and PCNA. Our results show that (1) the combination of ABT888 plus TMZ is additive in reducing soft agar colony growth in both U87MG and V12RAS astrocytes (2) ABT888 sensitized both U87MG and V12RAS astrocytes to TMZ in 3D On-Top assay, (3) in both U87MG and V12RAS astrocytes the largest amount of G0 was observed following the combination of ABT888 plus C at 72 hours (4) ABT888 plus C as well as ABT888 plus TMZ blocked vascular mimicry in both U87MG and V12RAS astrocytes and (5) at 72 hours PTEN null U87MG cells exhibited an increase in pro-apoptotic cl-PARP and a decrease in anti-apoptotic MCL-1 following both combination treatments, an effect not observed in V12RAS astrocytes. At 48 hours the TMZ induced increase in PAR is blocked by ABT888, an effect more pronounced in U87MG cells. Conclusion: The data demonstrate that ABT888 sensitizes the effect of TMZ in PTEN null glioma cells. The functional link between tumor suppressor, PTEN and the sensitization of PARP inhibitor is being currently worked out utilizing glioma cells expressing PTEN to specifically address the role of PTEN nullness in the context of ABT888 in combination with TMZ, the results of which will be presented at the meeting. Citation Format: Jennifer H. Carlson, Brian Leyland-Jones, David Martin, Pradip De, Nandini Dey. Does genetic background in glioma influence the sensitivity of tumor cells to a PARP inhibitor in the presence of different DNA damaging agents. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5126. doi:10.1158/1538-7445.AM2014-5126

Nek1-inhibitor and temozolomide-loaded microfibers as a co-therapy strategy for glioblastoma treatment

Simple SummaryTemozolomide (TMZ) is the first-line drug for chemotherapy of GBM, the most aggressive and incurable brain tumor. Acquired chemoresistance is a hallmark that causes the poor prognosis of GBM. Therefore, understanding the underlying mechanisms by using a proper model becomes emergent. Previous models usually take weeks/months and are often not fully representative of characteristics of TMZ resistance. We established an acute acquired TMZ resistance model using GBM cell lines with different genomic backgrounds. In response to TMZ, the resistant cells showed less susceptibility and sustained regrowth, high clonogenicity, reduced DNA damage accompanied by attenuated MMR, shortened G2/M arrest, uncontrolled DNA replication, and evasion of apoptosis. Moreover, these TMZ resistant cells presented stem cell properties that are critical for chemoresistance. Thus, our model recapitulates all key features of TMZ resistance and is believed to be a promising model to study the underlying mechanisms and define therapeutics for GBM in the future.Temozolomide (TMZ) is the first line of standard therapy in glioblastoma (GBM). However, relapse occurs due to TMZ resistance. We attempted to establish an acquired TMZ resistance model that recapitulates the TMZ resistance phenotype and the relevant gene signature. Two GBM cell lines received two cycles of TMZ (150 µM) treatment for 72 h each. Regrown cells (RG2) were defined as TMZ resistant cells. MTT assay revealed significantly less susceptibility and sustained growth of RG2 compared with parental cells after TMZ challenge. TMZ-induced DNA damage significantly decreased in 53BP1-foci reporter transduced-RG2 cells compared with parental cells, associated with downregulation of MSH2 and MSH6. Flow cytometry revealed reduced G2/M arrest, increased EdU incorporation and suppressed apoptosis in RG2 cells after TMZ treatment. Colony formation and neurosphere assay demonstrated enhanced clonogenicity and neurosphere formation capacity in RG2 cells, accompanied by upregulation of stem markers. Collectively, we established an acute TMZ resistance model that recapitulated key features of TMZ resistance involving impaired mismatch repair, redistribution of cell cycle phases, increased DNA replication, reduced apoptosis and enhanced self-renewal. Therefore, this model may serve as a promising research tool for studying mechanisms of TMZ resistance and for defining therapeutic approaches to GBM in the future.

Temozolomide (TMZ) is widely used for treating glioblastoma multiforme (GBM), however, the treatment of such brain tumors remains a challenge due to the development of resistance. Increasing studies have found that TMZ treatment could induce autophagy that may link to therapeutic resistance in GBM, but, the precise mechanisms are not fully understood. Understanding the molecular mechanisms underlying the response of GBM to chemotherapy is paramount for developing improved cancer therapeutics. In this study, we demonstrated that the loss of DOC-2/DAB2 interacting protein (DAB2IP) is responsible for TMZ-resistance in GBM through ATG9B. DAB2IP sensitized GBM to TMZ and suppressed TMZ-induced autophagy by negatively regulating ATG9B expression. A higher level of ATG9B expression was associated with GBM compared to low-grade glioma. The knockdown of ATG9B expression in GBM cells suppressed TMZ-induced autophagy as well as TMZ-resistance. Furthermore, we showed that DAB2IP negatively regulated ATG9B expression by blocking the Wnt/β-catenin pathway. To enhance the benefit of TMZ and avoid therapeutic resistance, effective combination strategies were tested using a small molecule inhibitor blocking the Wnt/β-catenin pathway in addition to TMZ. The combination treatment synergistically enhanced the efficacy of TMZ in GBM cells. In conclusion, the present study identified the mechanisms of TMZ-resistance of GBM mediated by DAB2IP and ATG9B which provides insight into a potential strategy to overcome TMZ chemo-resistance.

Temozolomide (TMZ) is the preferred chemotherapeutic drug approved for the Glioblastoma multiforme (GBM) treatment. However, resistance to TMZ is the most intractable challenge for treatment of GBM. Screening of miRNAs is becoming a novel strategy to reveal underlying mechanism of drug-resistance of human tumors. We conducted RNA sequencing (RNA-seq) for GBM cells treated continuously with TMZ 1 or 2 week or not. Bioinformatic analysis was used to predict targets of these altered miRNAs. Subsequently, we studied the potential role of miR-1268a in TMZ-resistance of GBM cells. Expression levels of 55 miRNAs were identified altering both after 1 and 2 weeks TMZ treatment. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were conducted to illuminate the biological implication and related pathways of predicted target genes. We showed that miR-1268a was downregulated after TMZ treatment and targeted ABCC1/MRP1, a membrane transporter contributing to drug resistance, using dual-luciferase assay. Furthermore, we confirmed overexpression of miR-1268a inhibited protein translation of ABCC1 and restored upregulated expression of ABCC1 due to TMZ. Inversely, knockdown of miR-1268a increased ABCC1 at protein level and enhanced upregulation of ABCC1 with TMZ treatment. In addition, our data indicated that miR-1268a enhanced TMZ sensitivity in GBM cells. Through RNA-seq analysis, we discovered miR-1268a and elucidated its role in modulating TMZ-resistance of GBM cells by targeting ABCC1.

Temozolomide (TMZ) is the standard of care for patients with newly diagnosed glioblastoma (GBM) as well as those with recurrent anaplastic glioma (AG) and GBM. It has become common practice to re-expose patients to TMZ who had been previously treated with TMZ, or to switch patients to alternative dosing regimens of TMZ when there are signs of relapse or progress on standard TMZ therapeutic regimens. To date, however, there is a scarcity of data on the efficacy of this therapeutic strategy, currently referred to as TMZ rechallenge. We have conducted a retrospective review of patients with recurrent glioma rechallenged with TMZ. Patients experiencing progressive disease (PD) during TMZ therapy who were rechallenged with alternative TMZ regimens and patients rechallenged after stable disease in a TMZ-free interval were evaluated separately. A total of 90 rechallenges were identified in 80 patients. The progression-free survival at 6 months (PFS-6) was 48% in patients with AG (12/25) and 27.7% in those with GBM (14/47). The PFS-6 was 16.7% in AG and 26.3% in GBM for patients switched during TMZ and 57.9 and 28.6% in patients rechallenged after a TMZ-free interval of at least 8 weeks. Relevant hematological toxicity (NCI-CTC grade 3-5) was observed in 22 of 90 rechallenges, and relevant non-hematological in ten of 90 rechallenges. Temozolomide was well tolerated and generated promising PFS-6 in patients who had previously failed TMZ, regardless if they progressed during TMZ treatment, or if they were rechallenged after a TMZ-free interval. These results suggest that the TMZ rechallenge strategy warrants further investigation in a prospective randomized trial.

Glioblastoma multiforme (GBM) is a highly invasive and chemoradioresistant brain malignancy. Temozolomide (TMZ), a DNA-alkylating agent, is effective against GBM and has become the standard first-line drug. However, the mechanism by which TMZ regulates the progression of GBM remains elusive. Here, we demonstrate that TMZ targets TAp63, a p53 family member, inducing its expression to suppress the progression of human GBM. High levels of TAp63 expression in GBM tissues after TMZ treatment was an indicator of favourable prognosis. In human GBM cells, TMZ-induced TAp63 directly repressed MYC transcription. Activation of this TAp63-MYC pathway by TMZ inhibited human GBM progression both in vitro and in vivo. Furthermore, downregulation of MYC mRNA levels in recurrent GBMs after TMZ treatment correlated with better patient survival. Therefore, our results suggest that the TAp63-mediated transcriptional repression of MYC is a novel pathway regulating TMZ efficacy in GBM.

e14547 Background: Glioblastoma (GBM) is the most aggressive brain malignancy with a heterogeneity molecular profile and accounts for no more than 3-5% of Central Nervous System tumors in children. Despite its rarity, pediatric GBM pertain different molecular genetics, outcome and effectiveness to therapies and remains an equally lethal tumor in children. Identification of genetic alterations in GBM of childhood and adolescence is important to refine molecular classification, define prognosis and therapeutic strategies. We aimed to detect and investigate molecular changes with potential prognostic marker and therapeutic target in GBM, using next generation sequencing (NGS) strategy. Methods: We selected 41 GBM samples from patients treated at Pediatric Oncology Institute/GRAACC. NGS was performed to identified genetic alterations in tumor samples using Oncomine Childhood Cancer Research Assay (OCCRA) panel, specific genetic panel for childhood and adolescence neoplasms. Results: We selected 41 GBM samples and identified 33 mutated genes. The most commonly detected genetic alterations were involving TP53, PDGFRA, PIK3CA, NF1, MYC, MET and genes with histone-related functions, including H3F3A and ATRX. Mutations in H3F3A K28M, ATRX and TP53 were most recurrent. ATRX alterations, either nonsense substitution or frameshift deletion, were exclusively found in patients with H3F3A K28M mutations. TP53 loss-of-function was exclusive in 9 of 41 (22%) tumors. In 7 of 9 cases (78%) harboring both H3F3A and TP53 mutations, patients died due to disease progression . In these patients, H3F3A-TP53 mutation is suggested to be unfavorable prognostic factor. Copy number alterations were found in 30% of GBM cases and PDGFRA-amplifications were the most frequent. PDGFRA-amplification was exclusively found in patients with H3F3A K28M mutations and 75% of these patients did not survived longer than two years. Conclusions: Molecular profiling based on NGS genetic panel, specific for pediatric tumors, can provide information about potential prognostic biomarkers for GBM of childhood and adolescence. Thus, wider understanding about GBM biologic heterogeneity may lead to personalized therapeutic strategies for pediatric patients.

Temozolomide (TMZ) is a chemotherapy agent used to treat Grade IV astrocytoma, also known as glioblastoma (GBM). TMZ treatment causes DNA damage that results in tumor cell apoptosis and increases the survival rate of GBM patients. However, chemoresistance as a result of TMZ-induced autophagy significantly reduces this anticancer effects over time. Statins are competitive inhibitors of HMG-CoA reductase, the rate-limiting enzyme of the mevalonate (MEV) cascade. Statins are best known for their cholesterol (CH)-lowering effect. Long-term consumption of statins, prior to and in parallel with other cancer therapeutic approaches, has been reported to increase the survival rate of patients with various forms of cancers. In this study, we investigated the potentiation of TMZ-induced apoptosis by simvastatin (Simva) in human GBM cell lines and patient GBM cells, using cell monolayers and three-dimensional cell culture systems. The incubation of cells with a combination of Simva and TMZ resulted in a significant increase in apoptotic cells compared to cells treated with TMZ alone. Incubation of cells with CH or MEV cascade intermediates failed to compensate the decrease in cell viability induced by the combined Simva and TMZ treatment. Simva treatment inhibited the autophagy flux induced by TMZ by blocking autophago-lysosome formation. Our results suggest that Simva sensitizes GBM cells to TMZ-induced cell death in a MEV cascade-independent manner and identifies the inhibition of autophagosome-lysosome fusion as a promising therapeutic strategy in the treatment of GBM.

Despite advances in therapies, glioblastoma (GBM) recurrence is almost inevitable due to the aggressive growth behavior of GBM cells and drug resistance. Temozolomide (TMZ) is the preferred drug for GBM chemotherapy, however, development of TMZ resistance is over 50% cases in GBM patients. To investigate the mechanism of TMZ resistance and invasive characteristics of GBM, analysis of combined RNA-seq and ChIP-seq was performed in GBM cells in response to TMZ treatment. We found that the PERK/eIF2α/ATF4 signaling was significantly upregulated in the GBM cells with TMZ treatment, while blockage of ATF4 effectively inhibited cell migration and invasion. SPHK1 expression was transcriptionally upregulated by ATF4 in GBM cells in response to TMZ treatment. Blockage of ATF4-SPHK1 signaling attenuated the cellular and molecular events in terms of invasive characteristics and TMZ resistance. In conclusion, GBM cells acquired chemoresistance in response to TMZ treatment via constant ER stress. ATF4 transcriptionally upregulated SPHK1 expression to promote GBM cell aggression and TMZ resistance. The ATF4-SPHK1 signaling in the regulation of the transcription factors of EMT-related genes could be the underlying mechanism contributing to the invasion ability of GBM cells and TMZ resistance. ATF4-SPHK1-targeted therapy could be a potential strategy against TMZ resistance in GBM patients.

Objectives Temozolomide (TMZ) is an effective drug for glioblastoma multiforme (GBM), but the mechanism underlying TMZ resistance is poorly understood. New evidence has revealed that the release of heat shock proteins (Hsps) derived from extracellular vesicles (EVs) play an important role in cancer progression by modulating tumor microenvironment and cellular cross-talk. This study aims to evaluate the effects of TMZ on the expression of EV-derived and cellular Hsps and cell motility in U87MG human glioblastoma cell line. Methods Glial-EVs were isolated from the culture medium and characterized by SEM and immunoblotting. The effect of TMZ treatments (25, 200 and 750 µM) on cell proliferation (MTT assay), migration (scratch assay), and Hsp60 and Hsp70 levels (immunoblotting) were evaluated. Results TMZ treatments led to an increase in intracellular Hsp70 while decreasing EV-derived Hsp70. Cellular Hsp60 level was elevated at the low dose of TMZ, but it reduced at higher TMZ concentrations. Hsp60 was also decreased in EVs secreted from TMZ-treated cells. Besides, TMZ treatment reduced the proliferation and migration of glioma cells in a dose-dependent manner. Conclusions Our results suggest that TMZ has the potential to target both EV-derived and cellular Hsps for GBM treatment, thus it may reduce cell motility.

e13006 Background: The 5-year survival for patients (pts) with glioblastoma (GBM) is low at approximately 3%. Radiotherapy plus concomitant and adjuvant temozolomide (TMZ) remain the standard of care. The optimal duration of therapy with TMZ is unknown, though treatment periods of 6 months (mo), 12 mo and longer have been utilized. Whether or not there is a benefit with longer treatment duration is controversial. Methods: A retrospective chart review of all pts diagnosed with GBM who were treated at a regional referral center was conducted with data obtained from their electronic medical records. These pts were treated with TMZ for up to 2 years between January 1, 2002 and December 31, 2011. Survival was calculated as the time from initial surgical diagnosis until death. The Kaplan-Meier method with log-rank test was used to estimate the progression-free survival (PFS) as well as the overall survival (OS) distribution of pts after treatment. The results were compared to historical controls and data from previous clinical trials of pts treated up to 1 year. Results: Data from 56 pts were evaluated, the majority of whom had gross total resection and had external pathology review confirming the diagnosis of GBM. The OS probability was 55.4% (SE = 0.068) at 1 year, 26.9% (SE = 0.067) at 2 years and 20.1% (SE = 0.065) at 3 years. The median PFS time in this study group was 8 mo (95% CI = 4.0 – 9.0 mo). The probability of no progression at 2 years was 8.6% (SE = 0.05). Seven pts (12.5%) were treated with TMZ for 2 years. The probability of disease progression at 2 years among these pts was 33.3% with a median time-to-progression of 20 mo (95% CI = 5.0-28.0). These patients showed an increased survival probability at 3 years compared to pts who did not receive the 2 year treatment of TMZ (log-rank test Chi-square = 12.4, p = 0.0004). Conclusions: This analysis suggests that there may be an advantage for a longer duration of TMZ therapy in pts with GBM. In this review, treatment with TMZ for 2 years was associated with an increased survival benefit. While we consider the sample size to be too small for generalization, a prospective/multicenter study with a larger sample size might better evaluate the question of duration of TMZ therapy, particularly if both clinical and basic science data are paired.