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mp3 file (11 MB). In the May edition of the Cancer Discovery podcast, Science Writer Elizabeth McKenna talks with Ingo K. Mellinghoff about his paper, which suggests that the disappointing clinical activity of first-generation EGFR inhibitors in glioblastoma versus lung cancer may be attributed to the different conformational requirements of mutant EGFR in these two cancer types.

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PDF file, 80K, Supplementary Table 1: Temozolomide 150 mg/m2 Pharmacokinetic Parameters (first cycle).

PDF file, 62K, Supplementary Table 2: Grade 3 or 4 events with relationship unrelated to unlikely to combination therapy (Vorinostat + TMZ).

Purpose: A phase I, dose-finding study of vorinostat in combination with temozolomide (TMZ) was conducted to determine the maximum tolerated dose (MTD), safety, and pharmacokinetics in patients with high-grade glioma (HGG).Experimental Design: This phase I, dose-finding, investigational study was conducted in two parts. Part 1 was a dose-escalation study of vorinostat in combination with TMZ 150 mg/m2/day for 5 days every 28 days. Part 2 was a dose-escalation study of vorinostat in combination with TMZ 150 mg/m2/day for 5 days of the first cycle and 200 mg/m2/day for 5 days of the subsequent 28-day cycles.Results: In part 1, the MTD of vorinostat administered on days 1 to 7 and 15 to 21 of every 28-day cycle, in combination with TMZ, was 500 mg daily. Dose-limiting toxicities (DLT) included grade 3 anorexia, grade 3 ALT, and grade 5 hemorrhage in the setting of grade 4 thrombocytopenia. In part 2, the MTD of vorinostat on days 1 to 7 and 15 to 21 of every 28-day cycle, combined with TMZ, was 400 mg daily. No DLTs were encountered, but vorinostat dosing could not be escalated further due to thrombocytopenia. The most common serious adverse events were fatigue, lymphopenia, thrombocytopenia, and thromboembolic events. There were no apparent pharmacokinetic interactions between vorinostat and TMZ. Vorinostat treatment resulted in hyperacetylation of histones H3 and H4 in peripheral mononuclear cells.Conclusion: Vorinostat in combination with temozolomide is well tolerated in patients with HGG. A phase I/II trial of vorinostat with radiotherapy and concomitant TMZ in newly diagnosed glioblastoma is underway. Clin Cancer Res; 18(21); 6032–9. ©2012 AACR.

Background Epidermal growth factor receptor (EGFR) amplification and TP53 mutation are the two most common genetic alterations in glioblastoma multiforme (GBM). A comprehensive analysis of the TCGA GBM database revealed a subgroup with near mutual exclusivity of EGFR amplification and TP53 mutations indicative of a role of EGFR in regulating wild-type-p53 (wt-p53) function. The relationship between EGFR amplification and wt-p53 function remains undefined and this study describes the biological significance of this interaction in GBM. Methods Mass spectrometry was used to identify EGFR-dependent p53-interacting proteins. The p53 and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) interaction was detected by co-immunoprecipitation. We used CRISPR-Cas9 gene editing to knockout EGFR and DNA-PKcs and the Edit-R CRIPSR-Cas9 system for conditional knockout of EGFR. ROS activity was measured with a CM-H2DCFDA probe, and real-time PCR was used to quantify expression of p53 target genes. Results Using glioma sphere-forming cells (GSCs), we identified, DNA-PKcs as a p53 interacting protein that functionally inhibits p53 activity. We demonstrate that EGFR knockdown increased wt-p53 transcriptional activity, which was associated with decreased binding between p53 and DNA-PKcs. We further show that inhibition of DNA-PKcs either by siRNA or an inhibitor (nedisertib) increased wt-p53 transcriptional activity, which was not enhanced further by EGFR knockdown, indicating that EGFR suppressed wt-p53 activity through DNA-PKcs binding with p53. Finally, using conditional EGFR-knockout GSCs, we show that depleting EGFR increased animal survival in mice transplanted with wt-p53 GSCs. Conclusion This study demonstrates that EGFR signaling inhibits wt-p53 function in GBM by promoting an interaction between p53 and DNA-PKcs.

Imaging response assessment is a cornerstone of patient care and drug development in oncology. Clinicians/clinical researchers rely on tumor imaging to estimate the impact of new treatments and guide decision making for patients and candidate therapies. This is important in brain cancer, where associations between tumor size/growth and emerging neurological deficits are strong. Accurately measuring the impact of a new therapy on tumor growth early in clinical development, where patient numbers are small, would be valuable for decision making regarding late-stage development activation. Current attempts to measure the impact of a new therapy have limited influence on clinical development, as determination of progression, stability or response does not currently account for individual tumor growth kinetics prior to the initiation of experimental therapies. Therefore, we posit that imaging-based response assessment, often used as a tool for estimating clinical effect, is incomplete as it does not adequately account for growth trajectories or biological characteristics of tumors prior to the introduction of an investigational agent. Here, we propose modifications to the existing framework for evaluating imaging assessment in primary brain tumors that will provide a more reliable understanding of treatment effects. Measuring tumor growth trajectories prior to a given intervention may allow us to more confidently conclude whether there is an anti-tumor effect. This updated approach to imaging-based tumor response assessment is intended to improve our ability to select candidate therapies for later-stage development, including those that may not meet currently sought thresholds for “response” and ultimately lead to identification of effective treatments.

Determination of therapeutic benefit in intracranial tumors is intimately dependent on serial assessment of radiographic images. The Response Assessment in Neuro-Oncology (RANO) criteria were established in 2010 to provide an updated framework to better characterize tumor response to contemporary treatments. Since this initial update a number of RANO criteria have provided some basic principles for the interpretation of changes on MR images; however, the details of how to operationalize RANO and other criteria for use in clinical trials are ambiguous and not standardized. In this review article designed for the neuro-oncologist or treating clinician, we outline essential steps for performing radiographic assessments by highlighting primary features of the Imaging Charter (referred to as the Charter for the remainder of this article), a document that describes the clinical trial imaging methodology and methods to ensure operationalization of the Charter into the workings of a clinical trial. Lastly, we provide recommendations for specific changes to optimize this methodology for neuro-oncology, including image registration, requirement of growing tumor for eligibility in trials of recurrent tumor, standardized image acquisition guidelines, and hybrid reader paradigms that allow for both unbiased measurements and more comprehensive interpretation.

Background Temozolomide (TMZ) resistance in glioblastoma multiforme (GBM) is mediated by the DNA repair protein O6-methylguanine DNA methyltransferase (MGMT). MGMT promoter methylation (occurs in about 40% of patients) is associated with loss of MGMT expression (MGMT−) that compromises DNA repair, leading to a favorable response to TMZ therapy. The 60% of patients with unmethylated MGMT (MGMT+) GBM experience resistance to TMZ; in these patients, understanding the mechanism of MGMT-mediated repair and modulating MGMT activity may lead to enhanced TMZ activity. Here, we report a novel mode of regulation of MGMT protein activity by poly(ADP-ribose) polymerase (PARP). Methods MGMT-PARP interaction was detected by co-immunoprecipitation. PARylation of MGMT and PARP was detected by co-immunoprecipitation with anti-PAR antibody. O6-methylguanine (O6-MetG) adducts were quantified by immunofluorescence assay. In vivo studies were conducted in mice to determine the effectiveness of PARP inhibition in sensitizing GBM to TMZ. Results We demonstrated that PARP physically binds with MGMT and PARylates MGMT in response to TMZ treatment. In addition, PARylation of MGMT by PARP is required for MGMT binding to chromatin to enhance the removal of O6-MetG adducts from DNA after TMZ treatment. PARP inhibitors reduced PARP-MGMT binding and MGMT PARylation, silencing MGMT activity to repair O6-MetG. PARP inhibition restored TMZ sensitivity in vivo in MGMT-expressing GBM. Conclusion This study demonstrated that PARylation of MGMT by PARP is critical for repairing TMZ-induced O6-MetG, and inhibition of PARylation by PARP inhibitor reduces MGMT function rendering sensitization to TMZ, providing a rationale for combining PARP inhibitors to sensitize TMZ in MGMT-unmethylated GBM.

Purpose:Exploration of novel strategies to extend the benefit of PARP inhibitors beyond BRCA-mutant cancers is of great interest in personalized medicine. Here, we identified EGFR amplification as a potential biomarker to predict sensitivity to PARP inhibition, providing selection for the glioblastoma (GBM) patient population who will benefit from PARP inhibition therapy.Experimental Design:Selective sensitivity to the PARP inhibitor talazoparib was screened and validated in two sets [test set (n = 14) and validation set (n = 13)] of well-characterized patient-derived glioma sphere-forming cells (GSC). FISH was used to detect EGFR copy number. DNA damage response following talazoparib treatment was evaluated by γH2AX and 53BP1 staining and neutral comet assay. PARP–DNA trapping was analyzed by subcellular fractionation. The selective monotherapy of talazoparib was confirmed using in vivo glioma models.Results:EGFR-amplified GSCs showed remarkable sensitivity to talazoparib treatment. EGFR amplification was associated with increased reactive oxygen species (ROS) and subsequent increased basal expression of DNA-repair pathways to counterelevated oxidative stress, and thus rendered vulnerability to PARP inhibition. Following talazoparib treatment, EGFR-amplified GSCs showed enhanced DNA damage and increased PARP–DNA trapping, which augmented the cytotoxicity. EGFR amplification–associated selective sensitivity was further supported by the in vivo experimental results showing that talazoparib significantly suppressed tumor growth in EGFR-amplified subcutaneous models but not in nonamplified models.Conclusions:EGFR-amplified cells are highly sensitive to talazoparib. Our data provide insight into the potential of using EGFR amplification as a selection biomarker for the development of personalized therapy.

Background We sought to ascertain the immune effector function of pembrolizumab within the glioblastoma (GBM) microenvironment during the therapeutic window. Methods In an open-label, single-center, single-arm phase II “window-of-opportunity” trial in 15 patients with recurrent (operable) GBM receiving up to 2 pembrolizumab doses before surgery and every 3 weeks afterward until disease progression or unacceptable toxicities occurred, immune responses were evaluated within the tumor. Results No treatment-related deaths occurred. Overall median follow-up time was 50 months. Of 14 patients monitored, 10 had progressive disease, 3 had a partial response, and 1 had stable disease. Median progression-free survival (PFS) was 4.5 months (95% CI: 2.27, 6.83), and the 6-month PFS rate was 40%. Median overall survival (OS) was 20 months, with an estimated 1-year OS rate of 63%. GBM patients’ recurrent tumors contained few T cells that demonstrated a paucity of immune activation markers, but the tumor microenvironment was markedly enriched for CD68+ macrophages. Conclusions Immune analyses indicated that pembrolizumab anti–programmed cell death 1 (PD-1) monotherapy alone can’t induce effector immunologic response in most GBM patients, probably owing to a scarcity of T cells within the tumor microenvironment and a CD68+ macrophage preponderance.

PI3K-targeting therapy represents one of the most sought-after therapies for glioblastoma (GBM). Several small-molecule inhibitors have been evaluated in clinical trials, however, the emergence of resistance limits treatment potential. Here, we generated a patient-derived glioma sphere–forming cell (GSC) xenograft model resistant to the PI3K-specific inhibitor BKM-120. Integrated RNA sequencing and high-throughput drug screening revealed that the Aurora A kinase (Aurora A)/Polo-like kinase 1 (PLK1)/cyclin-dependent kinase 1 (CDK1) signaling pathway was the main driver of PI3K inhibitor resistance in the resistant xenografts. Aurora kinase was upregulated and pCDK1 was downregulated in resistant tumors from both xenografts and tumor tissues from patients treated with the PI3K inhibitor. Mechanistically, the tyrosine kinase receptor Tie2 physically interacted with FGFR1, promoting STAT3 phosphorylation and binding to the AURKA promoter, which increased Aurora A expression in resistant GSCs. Concurrent inhibition of Aurora A and PI3K signaling overcame PI3K inhibitor–induced resistance. This study offers a proof of concept to target PI3K and the collateral-activated pathway to improve GBM therapy.Significance:These findings provide novel insights into the mechanisms of PI3K inhibitor resistance in glioblastoma.

Glioblastoma is the most common malignant primary brain tumor in adults and is almost invariably fatal. Despite our growing understanding of the various mechanisms underlying treatment failure, the standard-of-care therapy has not changed over the last two decades, signifying a great unmet need. The challenges of treating glioblastoma are many and include inadequate drug or agent delivery across the blood–brain barrier, abundant intra- and intertumoral heterogeneity, redundant signaling pathways, and an immunosuppressive microenvironment. Here, we review the innate and adaptive molecular mechanisms underlying glioblastoma’s treatment resistance, emphasizing the intrinsic challenges therapeutic interventions must overcome—namely, the blood–brain barrier, tumoral heterogeneity, and microenvironment—and the mechanisms of resistance to conventional treatments, targeted therapy, and immunotherapy.

Diffuse midline gliomas driven by lysine27-to-methionine mutations in histone 3 (H3-K27M DMGs) are among the most fatal brain tumors. Molecular studies including single cell RNA-sequencing (scRNA-seq) of pediatric and predominantly pontine H3-K27M DMGs have shown that the H3-K27M oncohistone keeps glioma cells locked in a stem-like oligodendrocyte precursor cell (OPC) state that is capable of self-renewal and tumor-initiation. However, a comprehensive dissection of the cellular architecture of H3-K27M DMGs across different midline regions and age groups is required to better understand the cell-intrinsic and contextual regulation of H3-K27M DMG cell identities. In particular, the more recently described group of adult H3-K27M DMGs remains understudied. Here, we have collected and characterized 45 H3-K27M mutant patient tumors, spanning pontine (n=26), thalamic (n=17), and spinal (n=2) locations. Median age at surgery was 12 (2-68) years, encompassing 21 early childhood (0-10 years), 12 adolescent (11-20 years), and 12 adult (≥ 21 years) tumors. The majority of samples were obtained pre-treatment (n=28), as opposed to post-treatment or at autopsy (n=17). We profiled all 45 tumors by single cell/single nucleus RNA-seq and selected tumors were further characterized by the single cell assay for transposase-accessible chromatin (scATAC-seq). Our integrated analyses highlight the predominance of transcriptionally and epigenetically defined OPC-like tumor cells as the main cell population of H3-K27M DMGs across all age groups and locations. We further identify distinct age- and location-specific OPC-like cell subpopulations. Comparison of pediatric and adult tumors further demonstrates a significant increase of mesenchymal cell states in adult H3-K27M DMGs, which we link to differences in glioma-associated immune cell compartments between age groups. Together, this study sheds light on the effects of age- and region-dependent microenvironments in shaping cellular identities in H3-K27M DMGs.

Histone 3 lysine27-to-methionine (H3-K27M) mutations most frequently occur in diffuse midline gliomas (DMGs) of the childhood pons but are also increasingly recognized in adults. Their potential heterogeneity at different ages and midline locations is vastly understudied. Here, through dissecting the single-cell transcriptomic, epigenomic and spatial architectures of a comprehensive cohort of patient H3-K27M DMGs, we delineate how age and anatomical location shape glioma cell-intrinsic and -extrinsic features in light of the shared driver mutation. We show that stem-like oligodendroglial precursor-like cells, present across all clinico-anatomical groups, display varying levels of maturation dependent on location. We reveal a previously underappreciated relationship between mesenchymal cancer cell states and age, linked to age-dependent differences in the immune microenvironment. Further, we resolve the spatial organization of H3-K27M DMG cell populations and identify a mitotic oligodendroglial-lineage niche. Collectively, our study provides a powerful framework for rational modeling and therapeutic interventions.

Histone 3 lysine27-to-methionine mutant diffuse midline gliomas (H3-K27M DMGs) are among the most lethal brain tumors. Their putative cellular hierarchy has been shown to be driven by self-renewing stem-like cells arrested in an oligodendrocyte precursor-like (OPC-like) state, of which few cells are able to differentiate towards more mature astrocyte (AC)-like and oligodendrocyte (OC)-like cells. However, the spatial organization underlying this tumor cell architecture and its microenvironmental interactions in intact H3-K27M DMG tissues remain unknown. Here, we profiled the single cell transcriptomes of 45 patient H3-K27M DMGs and derived cell population-specific marker gene combinations to characterize the single cell spatial organization of 16 tumors using targeted in situ sequencing. We thereby resolved different malignant and non-malignant cell populations including cycling, OPC-like, AC-like, OC-like, mesenchymal tumor cells, and non-malignant oligodendrocytes, astrocytes, neurons, myeloid cells, T cells, and vascular cells directly in situ. Global neighborhood analyses indicate a higher tendency of cycling OPC-like cells, vascular cells, and neurons to localize within a more restricted homogeneous compartment, whereas AC-like cells, non-malignant astrocytes and myeloid cells tend to intermingle with different cell populations in a more diffuse manner. Among malignant cells, we observed cycling OPC-like and OC-like cells to co-localize within a niche-like structure that is surrounded by more differentiated AC-like cells. We further validated this stem-like niche at the protein level using multiplexed immunofluorescence via the CODEX system. Finally, we characterized relationships between malignant and non-malignant cells, consistently identifying preferred neighborhoods of mesenchymal tumor cells with vascular and myeloid cells. Together, this study resolves the spatial architecture of H3-K27M DMG malignant and non-malignant cells at single cell resolution and identifies a local niche of the oligodendroglial lineage containing the OPC-like cancer stem-like cells, thus providing novel insights into the cancer stem-like compartment in H3-K27M DMGs and suggesting potential avenues for its perturbation.

The most common cancer therapies including chemo- and radiation therapy are designed to induce cell death by DNA damage. However, DNA damage response (DDR) repair pathways initiated by tumor cells can resist the effect of anticancer drugs thus providing the rationale for combining DNA repair pathway inhibitors with radiation and DNA damaging anticancer drugs. The reliance of different cancer to different DDR mechanism can guide for targeted use of specific DDR inhibitor to maximize tumor cell killing. Here, we show the differential activities of two classes of DDR pathway inhibitors, Ataxia Telangiectasia- and Rad3-related kinase (ATRi) and poly(ADP-ribose) polymerases inhibitors (PARPi) across 20 glioma stem cell (GSC) lines. Using a panel of 3 ATRi, we identified a subgroup of GSCs, showing significant synergism with TMZ combination but resistant to ATRi single agent monotherapy. Interestingly, majority of the synergistic cell lines are associated with MGMT promoter methylation, suggesting that only tumor cells with no MGMT repair will benefit from ATR inhibition. Moreover, we also observe that PARPi + TMZ showed synergism only in MGMT unmethylated GSC cells suggesting that this synergism is a result of the dual function of PARPi in blocking BER repair and blocking MGMT PARylation thus abolishing MGMT mediated repair. Altogether, we believe MGMT methylation status in primary GBM could function as a strong biomarker for patient selection for ATRi or PARPi combination with TMZ in the clinic. We are currently exploring the activity of the triple combination of TMZ + PARPi + ATRi for the small subgroup of GSCs that do not show response to either TMZ + ATRi or PARPi + TMZ combinations.

Multiple genomic aberrations and distinct molecular subtypes of glioblastoma (GBM) have been identified through multi-omics analyses including single-cell genomic and epigenomic analysis. Epidermal Growth Factor Receptor (EGFR) amplification is found in nearly 50% of GBM tumors. Several EGFR inhibitors have been tested in GBM but failed to demonstrate long-term therapeutic benefit, presumably due to target heterogeneity, escape signal pathways, and acquired resistance. Therefore, we investigated downstream signaling, mitogen-activated protein kinase (MEK1/2) as a potential therapeutic target for GBM. We utilized an unbiased high-throughput screening with a panel of glioma stem-like cells (GSCs) and identified that GBM cells harboring EGFR amplification showed anti-proliferative and apoptotic cell death (sensitive) to multiple MEK inhibitors. RNA-seq analysis of cells pre- and post-treatment with trametinib, a potent MEK inhibitor, revealed an upregulation of neurogenesis and neuronal differentiation genes such as β-III tubulin, ASCL1, DLL1, DLL3, NeuroD4, PAX6 and NCAM1. In addition, downregulation of the several MEK targets including erythroblast transformation specific (ETS) family genes, particularly ETV5 was observed. We demonstrated that MEK inhibition increased neuronal differentiation demonstrated by immunofluorescence, western blot, and RT-qPCR experiments using selected differentiation markers (β-III tubulin, ASCL1, DLL3, and NeuroD4). In addition, ETV-5 knockout in GSCs by CRISPR/Cas-9 promoted the expression of neuronal differentiation genes. Oral treatment of trametinib in an orthotopic GSC xenograft model significantly improved animal survival, in which 25-30% of mice are long-term survivors. This was accompanied by decreased MEK/ERK signal and cell proliferation in tumor tissues. Thus, we demonstrated here that MEK1/2 inhibition by trametinib induces neuronal differentiation mediated through downregulation of ETV-5 in GBM, a potential novel mechanism of action of MEK1/2 signal inhibition.

On July 24, 2020, a workshop sponsored by the National Brain Tumor Society was held on innovating brain tumor clinical trials based on lessons learned from the COVID-19 experience. Various stakeholders from the brain tumor community participated including the US Food and Drug Administration (FDA), academic and community clinicians, researchers, industry, clinical research organizations, patients and patient advocates, and representatives from the Society for Neuro-Oncology and the National Cancer Institute. This report summarizes the workshop and proposes ways to incorporate lessons learned from COVID-19 to brain tumor clinical trials including the increased use of telemedicine and decentralized trial models as opportunities for practical innovation with potential long-term impact on clinical trial design and implementation.

Malignant gliomas are a group of intracranial cancers associated with disproportionately high mortality and morbidity. Here, we report ultradeep targeted sequencing of a prospective cohort of 237 tumors from 234 patients consisting of both glioblastoma (GBM) and lower-grade glioma (LGG) using our customized gene panels. We identified 2,485 somatic mutations, including single-nucleotide substitutions and small indels, using a validated in-house protocol. Sixty-one percent of the mutations were contributed by 12 hypermutators. The hypermutators were enriched for recurrent tumors and had comparable outcome, and most were associated with temozolomide exposure. TP53 was the most frequently mutated gene in our cohort, followed by IDH1 and EGFR. We detected at least one EGFR mutation in 23% of LGGs, which was significantly higher than 6% seen in The Cancer Genome Atlas, a pattern that can be partially explained by the different patient composition and sequencing depth. IDH hotspot mutations were found with higher frequencies in LGG (83%) and secondary GBM (77%) than primary GBM (9%). Multivariate analyses controlling for age, histology, and tumor grade confirm the prognostic value of IDH mutation. We predicted 1p/19q status using the panel sequencing data and received only modest performance by benchmarking the prediction to FISH results of 50 tumors. Targeted therapy based on the sequencing data resulted in three responders out of 14 participants. In conclusion, our study suggests ultradeep targeted sequencing can recapitulate previous findings and can be a useful approach in the clinical setting.

Background It is estimated only 8–11% of patients with glioblastoma (GBM) enrol in clinical trials, limiting treatment development. We analysed the clinical and demographic features of patients with GBM enroled in clinical trials at the University of Texas MD Anderson Cancer Center (MDACC). Methods We reviewed the records of adult patients treated for primary GBM between 2007 and 2012 at the MDACC. A total of 755 patients were identified: 133 were deemed non-eligible, 111 were deemed trial eligible but received standard care and 511 participated in a clinical trial (311 for newly diagnosed glioblastoma [nGBM] and 200 for recurrent glioblastoma [rGBM]). Population characteristics were analysed using descriptive statistics, and survival end-points were evaluated with the Kaplan–Meier method. Results The median age of clinical trial participants and trial eligible patients was 53.2 years (standard deviation 12.1). Most patients (49.4%) were enroled in a clinical trial protocol for nGBM. The majority of nGBM trial participants were male patients (65.1%), white (86.3%), married (84.4%) and in state (59.9%). Employment status, education, symptoms, tumour location, performance status, extent of resection and treatment facility differed between nGBM trial participants and non-participants. Patients who were eligible but did not enrol tended to be older, have worse performance status and live farther away from the MDACC. Conclusion Numerous disease and demographic barriers exist in trial enrolment in patients with GBM. This study highlights some of these obstacles, which require attention to improve patient enrolment to clinical trials. Patient and physician engagement in novel therapeutic strategies is essential to improving outcomes in this disease.

The evolutionary processes that drive universal therapeutic resistance in adult patients with diffuse glioma remain unclear1,2. Here we analysed temporally separated DNA-sequencing data and matched clinical annotation from 222 adult patients with glioma. By analysing mutations and copy numbers across the three major subtypes of diffuse glioma, we found that driver genes detected at the initial stage of disease were retained at recurrence, whereas there was little evidence of recurrence-specific gene alterations. Treatment with alkylating agents resulted in a hypermutator phenotype at different rates across the glioma subtypes, and hypermutation was not associated with differences in overall survival. Acquired aneuploidy was frequently detected in recurrent gliomas and was characterized by IDH mutation but without co-deletion of chromosome arms 1p/19q, and further converged with acquired alterations in the cell cycle and poor outcomes. The clonal architecture of each tumour remained similar over time, but the presence of subclonal selection was associated with decreased survival. Finally, there were no differences in the levels of immunoediting between initial and recurrent gliomas. Collectively, our results suggest that the strongest selective pressures occur during early glioma development and that current therapies shape this evolution in a largely stochastic manner.

2006 Background: Checkpoint inhibitor (CPI) therapy has demonstrated overall limited efficacy in the treatment of GBM. Sixty newly diagnosed GBM patients unselected for MGMT status underwent treatment with concurrent atezolizumab with radiation therapy and TMZ followed by adjuvant atezolizumab and TMZ (NCT03174197). Clinical data has been reported previously. Methods: Genomic (WES with somatic mutation and SCNA determination N = total 42 samples, 33 baseline, 9 TP-2), transcriptomic (RNA seq N = total 72 samples, 54 baseline, 18 TP-2), and metagenomic sequencing of fecal samples (N = total 45 samples, 26 pre samples, 13 post RT samples, six 6m samples) analyses were conducted on pre-treatment samples. Findings were correlated with clinical outcome including OS and PFS. Twenty of the 60 patients underwent re-resection for suspected recurrent disease of which nine patients had WES and RNA seq performed successfully on paired pre and post treatment samples. Results: Somatic mutation, copy number and ploidy profiles were consistent with known aberrations in GBM. An unsupervised molecular network-based stratification of pre-treatment tumor mutations resulted in patients being grouped in 3 clusters with survival difference. Patients with GBM harboring an EGFR aberrancy were associated with a relatively worse mOS following treatment compared to patients with tumors enriched with PTEN alterations, while patients with IDH1 mutations had the longest mOS. Gene set enrichment analysis of gene expression in tumors from patients ( < mOS vs ≥mOS) identified genes associated with lymphocyte activation and immune response in patients with longer survival (p < 0.01) Unsupervised hierarchical clustering of bacterial taxa demonstrated two distinct clusters with significant difference by OS. Survival analysis and Analysis of Compositions of Microbiomes with Bias Correction (ANCOM-BC) revealed distinct taxa associated with OS ( Ruminococcus spp.) and response to treatment ( Eubacterium spp.), respectively. Conclusions: In this small CPI-treated GBM cohort, WES, SCNA and RNA seq identified pre-treatment tumor features that separated patients by survival. The fecal microbiome observations in our GBM cohort warrants further investigation. Clinical trial information: NCT03174197.

BACKGROUND: Repurposed memantine, mefloquine, and metformin have putative anticancer activity. The objective of this phase 1 study was to determine the maximum tolerated doses (MTDs) of combinations of these agents with temozolomide (TMZ). METHODS: Adults with newly diagnosed glioblastoma who completed chemoradiation were eligible. The patients were assgined to receive doublet, triplet, or quadruplet therapy with TMZ combined with mefloquine, memantine, and/or metformin. Dose-limiting toxicities (DLTs) were determined, using a 3 + 3 study design. RESULTS: Of 85 enrolled patients, 4 did not complete cycle 1 (the DLT observation period) for nontoxicity reasons, and 81 were evaluable for DLT. The MTDs for doublet therapy were memantine 20 mg twice daily, mefloquine 250 mg 3 times weekly, and metformin 850 mg twice daily. For triplet therapy, the MTDs were memantie 10 mg twice daily, mefloquine 250 mg 3 times weekly, and metformin 850 mg twice daily. For triplet therapy, the MTDs were memantine 10 mg twice daily, mefloquine 250 mg 3 times weekly, and metformin 850 mg twice daily. For quadruplet therapy, the MTDs were memantine 10 mg twice daily, mefloquine 250 mg 3 times weekly, and metformin 500 mg twice daily. DLTs included dizziness (memantine) and gastrointestinal effects (metformin). Lymphopenia was the most common adverse event (66%). From study entry, the median survival was 21 months, and the 2-year survival rate was 43%. CONCLUSIONS: Memantine, mefloquine, and metformin can be combined safely with TMZ in patients with newly diagnosed glioblastoma.

Glioblastoma (GBM), the most aggressive primary brain tumor, has a dismal prognosis. Despite our growing knowledge of genomic and epigenomic alterations in GBM, standard therapies and outcomes have not changed significantly in the past two decades. There is therefore an urgent unmet need to develop novel therapies for GBM. The inter- and intratumoral heterogeneity of GBM, inadequate drug concentrations in the tumor owing to the blood–brain barrier, redundant signaling pathways contributing to resistance to conventional therapies, and an immunosuppressive tumor microenvironment, have all hindered the development of novel therapies for GBM. Given the high frequency of DNA damage pathway alterations in GBM, researchers have focused their efforts on pharmacologically targeting key enzymes, including poly(ADP-ribose) polymerase (PARP), DNA-dependent protein kinase, ataxia telangiectasia-mutated, and ataxia telangiectasia and Rad3-related. The mainstays of GBM treatment, ionizing radiation and alkylating chemotherapy, generate DNA damage that is repaired through the upregulation and activation of DNA damage response (DDR) enzymes. Therefore, the use of PARP and other DDR inhibitors to render GBM cells more vulnerable to conventional treatments is an area of intense investigation. In this review, we highlight the growing body of data behind DDR inhibitors in GBM, with a focus on putative predictive biomarkers of response. We also discuss the challenges involved in the successful development of DDR inhibitors for GBM, including the intracranial location and predicted overlapping toxicities of DDR agents with current standards of care, and propose promising strategies to overcome these hurdles.

Background There is an urgent need for additional therapies to treat recurrent glioblastoma (GBM). Preclinical studies suggest that high dose macitentan, an oral dual endothelin receptor antagonist, enhances the cytotoxic effects of temozolomide (TMZ) in GBM, improving survival. This phase I trial investigated the maximum tolerated dose of macitentan combined with TMZ in patients with recurrent GBM and assessed the safety and tolerability of high dose macitentan in these patients (NCT01499251). Methods Adults with recurrent GBM received ascending doses of macitentan from 30 mg once daily concomitantly with TMZ. Safety and tolerability were assessed in addition to exploratory efficacy and pharmacokinetic endpoints. An ancillary study examined biomarker expression following macitentan treatment prior to surgical resection of recurrent GBM. Results Thirty-eight patients with recurrent GBM were administered macitentan doses up to 300 mg once daily; no dose-limiting toxicities were observed, and a maximum tolerated dose was not determined. All patients experienced at least one treatment-emergent adverse event (TEAE), the majority associated with GBM or TMZ treatment. TEAEs related to macitentan and TMZ were reported for 16 (42.1%) and 26 (68.4%) patients, respectively, with no serious macitentan-related TEAEs. Macitentan concentrations increased with dose, with no plateau in exposure. Substantial heterogeneity was observed in the expression of efficacy biomarkers within tumors. The Kaplan-Meier estimate of median overall survival across all dose groups was 9.4 (95% CI 8.5, 13.4) months. Conclusion High-dose macitentan was well tolerated in recurrent GBM patients concomitantly receiving TMZ. TEAEs were consistent with those seen in patients receiving either drug individually.

BACKGROUND WHO grade II and III IDH wild-type astrocytomas behave more aggressively than their IDH mutated counterparts. A subpopulation shares molecular features with the novel entity proposed in cIMPACT-NOW Update 3 “Diffuse astrocytic glioma, IDH wild-type, with molecular features of glioblastoma (GBM), WHO IV”. METHODS We performed a retrospective analysis of clinical and molecular features, management and survival of 134 adult patients treated for grade II and III IDH wild-type astrocytoma between 06/2012 and 12/2018 at MD Anderson Cancer Center (MDACC - 112) and UT Health Science Center at Houston (UTHSC - 22). All patients had IDH1 sequenced, all but 2 had IDH2 sequenced, and 73 had further next generation sequencing. RESULTS Median age at diagnosis was 53 (interquartile range 18-83). 82 patients (61%) were male. 31 patients were histologically diagnosed with grade II astrocytoma, 102 with grade III astrocytoma, and one with diffuse glioma (insufficient tissue to render histologic grade or perform sequencing). EGFR alterations were found in 31 patients and TERT promoter mutations in 22. 84 (63%) received concurrent chemoradiation and adjuvant temozolomide (grade II, n=9; grade III, n=74; NOS, n=1). PFS overall was 12.0 months (grade II = 17.9; grade III = 10.7). OS in patients treated with concurrent chemoradiation and adjuvant temozolomide was 17.1 months versus 17.7 in patients treated with sequential radiation and temozolomide (p = NS), and 10.6 in patients treated with RT alone or surveillance (p< 0.016). The highest 2-year OS was seen in grade II patients treated with concurrent chemoradiation and adjuvant temozolomide (60%). CONCLUSIONS WHO grade II and III IDHwt astrocytoma survival is similar to historical GBM cohorts. The proportion meeting molecular criteria for GBM is yet undefined. Groups who received chemotherapy may perform better than those who do not. Further analysis of MGMT methylation and other molecular factors is ongoing.

Developing new therapies for patients with glioblastoma (GBM) requires focused interaction between industry, academia, nonprofits, patient advocacy, and health authorities, and novel approaches to clinical trials. GBM Adaptive Global Innovative Learning Environment (GBM AGILE) Trial was designed by over 130 global key opinion leaders in consultation with health authorities to provide an optimal mechanism for phase 2/3 development in GBM. The Sponsor of GBM AGILE is the Global Coalition for Adaptive Research, whose mission is to accelerate the development of treatments rare and deadly diseases by serving as sponsor of innovative trials. GBM AGILE is an international platform trial designed to evaluate multiple therapies in newly diagnosed and recurrent GBM. Its goals are to identify effective therapies for GBM and match effective therapies with patient subtypes, with data generated to support regulatory filing for new drug applications. Bayesian response adaptive randomization is used within subtypes of the disease to assign participants to investigational arms based on their performance. The primary endpoint is overall survival. The trial is being conducted under a master Investigational New Drug Application/Clinical Trial Agreement and Master Protocol, allowing multiple drugs from different companies to be evaluated simultaneously and/or over time. The plan is to add experimental therapies as new information is identified and remove therapies as they complete their individual evaluation against a common control. GBM AGILE received IND approval from the FDA in April 2019, screening its first patient in June 2019. As of June 2020 over 200 patients have been screened. Expansion to Canada, Europe, China, and Australia is also underway. There is currently one investigational arm under evaluation in the trial, with two additional arms to be added in Q4 2020/ Q1 2021. Clinical trial information: NCT03970447.

Current standard-of-care for glioblastoma (GBM) includes surgery followed by concurrent therapy with radiation and temozolomide (TMZ) followed by adjuvant TMZ. Almost all GBM patients experience recurrent/progressive disease despite upfront standard of care treatment, with a median survival after recurrence of 3–9 months. Unmethylated promoter for O6-methylguanine-DNA-methyltransferase (MGMT) is a validated biomarker for TMZ-resistance and is correlated with poor patient prognosis. VAL-083 is a bi-functional DNA-targeting agent which rapidly induces inter-strand cross-links at N7-guanine inducing double-strand breaks causing cell death and acts independent of MGMT DNA repair. This trial is an open-label two-arm biomarker-driven phase 2 clinical trial in MGMT-unmethylated bevacizumab-naïve GBM patients with either recurrent (Group 1) or newly diagnosed GBM requiring adjuvant therapy after chemo-radiation with temozolomide (Group 2). Patients receive VAL-083 IV at 30 or 40 mg/m2/d on days 1, 2, and 3 of a 21-day cycle. The primary objective of this study is to determine the effect of VAL-083 on median overall survival (mOS) in MGMT-unmethylated recurrent GBM patients (Group 1); and progression-free survival (PFS) in newly diagnosed GBM patients requiring adjuvant therapy after chemo-irradiation with temozolomide (Group 2), compared to historical controls in both groups. Tumor response will be assessed by MRI every 42 days, using RANO criteria. The initial starting dose in this study was 40 mg/m2/d on days 1, 2, and 3 of a 21-day cycle, which was subsequently reduced to 30 mg/m2/d to improve tolerance due to myelosuppression. As of June 2-2020, 35 patients with recurrent GBM (Group 1) have received 40 mg/m2/d and 39 patients have received 30 mg/m2/d VAL-083. In the adjuvant setting (Group 2), 25 patients have been enrolled (30 mg/m2/day). Enrollment, safety data and study updates will be presented at the meeting. Clinicaltrials.gov identifier: NCT02717962.

Diffuse infiltrating gliomas are a clinically and molecularly heterogeneous group of tumors that are uniformly incurable. Despite our growing knowledge of genomic and epigenomic alterations in gliomas, standard treatments have not changed in the past 2 decades and remain limited to surgical resection, ionizing radiation, and alkylating chemotherapeutic agents. Development of novel therapeutics for diffuse gliomas has been challenging due to inter- and intra-tumoral heterogeneity, diffuse infiltrative nature of gliomas, inadequate tumor/drug concentration due to blood–brain barrier, and an immunosuppressive tumor microenvironment. Given the high frequency of DNA damage pathway alterations in gliomas, researchers have focused their efforts in targeting the DNA damage pathways for the treatment of gliomas. A growing body of data has shed light on the role of poly(ADP-ribose) polymerase (PARP) in combination with radiation and temozolomide in high-grade gliomas. Furthermore, a novel therapeutic strategy in low-grade glioma is the recent elucidation for a potential role of PARP inhibitors in gliomas with IDH1/2 mutations. This review highlights the concepts behind targeting PARP in gliomas with a focus on putative predictive biomarkers of response. We further discuss the challenges involved in the successful development of PARP inhibitors in gliomas, including the intracranial location of the tumor and overlapping toxicities with current standards of care, and promising strategies to overcome these hurdles.

Active telomerase is essential for stem cells and most cancers to maintain telomeres. The enzymatic activity of telomerase is related but not equivalent to the expression of TERT, the catalytic subunit of the complex. Here we show that telomerase enzymatic activity can be robustly estimated from the expression of a 13-gene signature. We demonstrate the validity of the expression-based approach, named EXTEND, using cell lines, cancer samples, and non-neoplastic samples. When applied to over 9,000 tumors and single cells, we find a strong correlation between telomerase activity and cancer stemness. This correlation is largely driven by a small population of proliferating cancer cells that exhibits both high telomerase activity and cancer stemness. This study establishes a computational framework for quantifying telomerase enzymatic activity and provides new insights into the relationships among telomerase, cancer proliferation, and stemness., Telomerase activity correlates with distinct cell states, but can be challenging to quantify. Here the authors quantify telomerase activity across a range of biological samples using the expression of 13 genes, and show it correlates with cancer cell proliferation and stemness.

Background Receptor tyrosine kinases such as epidermal growth factor receptors (EGFRs) and their downstream signaling pathways such as the Ras-Raf-mitogen-activated protein kinase (MAPK) pathway play important roles in glioblastoma (GBM). This study investigated the safety, pharmacokinetics, and efficacy of sorafenib (Ras/Raf/MAPK inhibitor) in combination with erlotinib (EGFR inhibitor) for treatment of recurrent GBMs. Methods Patients with recurrent GBM were eligible. A novel sequential accrual trial design was used, where patients were sequentially accrued into separate treatment arms in phase I and phase II investigations to optimize recruitment efficiency. In phase I, a standard 3 + 3 format was used to identify dose-limiting toxicities (DLTs), determine maximum tolerated dose (MTD), and investigate pharmacokinetics. Phase II followed a 2-stage design with the primary endpoint being 6-month progression-free survival (PFS6). Results Sixteen patients were recruited for phase I, and the MTD was determined to be sorafenib 200 mg twice daily and erlotinib 100 mg once daily. DLTs include Grade 3 hypertension, Grade 3 elevated liver transaminases, and Grade 4 elevated lipase. While erlotinib did not affect sorafenib levels, sorafenib reduced erlotinib levels. In phase II, 3 of 19 stage 1 participants were progression free at 6 months. This did not meet the predetermined efficacy endpoint, and the trial was terminated. Conclusion This study identified the MTD and DLTs for sorafenib and erlotinib combination therapy for recurrent GBMs; however, efficacy data did not meet the primary endpoint. This study also demonstrates the feasibility of a novel sequential accrual clinical trial design that optimizes patient recruitment for multiarm studies, which is particularly effective for multicenter clinical trials.

Purpose To estimate the maximum tolerated dose (MTD) and/or identify the recommended Phase II dose (RP2D) for combined INC280 and buparlisib in patients with recurrent glioblastoma with homozygous phosphatase and tensin homolog (PTEN) deletion, mutation or protein loss. Methods This multicenter, open-label, Phase Ib/II study included adult patients with glioblastoma with mesenchymal-epithelial transcription factor (c-Met) amplification. In Phase Ib, patients received INC280 as capsules or tablets in combination with buparlisib. In Phase II, patients received INC280 only. Response was assessed centrally using Response Assessment in Neuro-Oncology response criteria for high-grade gliomas. All adverse events (AEs) were recorded and graded. Results 33 patients entered Phase Ib, 32 with altered PTEN. RP2D was not declared due to potential drug–drug interactions, which may have resulted in lack of efficacy; thus, Phase II, including 10 patients, was continued with INC280 monotherapy only. Best response was stable disease in 30% of patients. In the selected patient population, enrollment was halted due to limited activity with INC280 monotherapy. In Phase Ib, the most common treatment-related AEs were fatigue (36.4%), nausea (30.3%) and increased alanine aminotransferase (30.3%). MTD was identified at INC280 Tab 300 mg twice daily + buparlisib 80 mg once daily. In Phase II, the most common AEs were headache (40.0%), constipation (30.0%), fatigue (30.0%) and increased lipase (30.0%). Conclusion The combination of INC280/buparlisib resulted in no clear activity in patients with recurrent PTEN-deficient glioblastoma. More stringent molecular selection strategies might produce better outcomes. Trial registration: NCT01870726.

PURPOSE: From 1990 to 1994, patients with newly diagnosed malignant gliomas were enrolled and randomized between hyperfractionated radiation (HFX) of 72.0 Gy in 60 fractions given twice daily and 60.0 Gy in 30 fractions given once daily. All patients received 80 mg/m(2) of 1,3 bis (2 chloroethyl)-1 nitrosourea on days 1–3 q8 weeks for 1 year. METHODS: Patients were stratified by age, KPS, and histology. The primary endpoint was overall survival (OS), with secondary endpoints including progression-free survival (PFS) and toxicity. RESULTS: Out of the 712 patients accrued, 694 (97.5%) were analyzable cases (350 HFX, 344 standard arm). There was no significant difference between the arms on overall acute or late treatment-related toxicity. No statistically significant effect for HFX, as compared to standard therapy, was found on either OS, with a median survival time (MST) of 11.3 mo vs. 13.1 mo (p=0.20) or PFS, with a median PFS time of 5.7 mo vs. 6.9 mo (p=0.18). The treatment effect on OS remained insignificant based on the multivariate analysis (hazard ratio=1.16; p=0.0682). When OS was analyzed by histology subgroup there was also no significant difference between the two arms for patients with glioblastoma multiforme (MST: 10.3 mo vs. 11.2 mo; p=0.34), anaplastic astrocytoma (MST: 69.8 mo vs 50.0 mo; p=0.91) or anaplastic oligodendroglioma (MST: 92.1 mo vs. 66.5 mo; p=0.33). CONCLUSION: Though this trial provided many invaluable secondary analyses, there was no trend or indication of a benefit to HFX radiation to 72.0 Gy in any subset of malignant glioma patients.

Current standard-of-care for glioblastoma (GBM) includes surgery followed by concurrent therapy with radiation and temozolomide (TMZ) followed by adjuvant TMZ. Almost all GBM patients experience recurrent/progressive disease despite upfront standard-of-care treatment, with a median survival of 3-9 months after recurrence. Unmethylated promoter for O6-methylguanine-DNA-methyltransferase (MGMT) is a validated biomarker for TMZ-resistance and is correlated with poor patient prognosis. VAL-083 is a bi-functional DNA-targeting agent which rapidly induces interstrand DNA cross-links at N7-guanine inducing double-strand breaks causing cell death and acts independently of MGMT DNA repair. This trial is an open-label two-arm biomarker-driven phase 2 clinical trial in MGMT-unmethylated, bevacizumab-naïve GBM patients with either recurrent (Group 1) or newly diagnosed GBM requiring adjuvant therapy after chemo-irradiation with temozolomide (Group 2). Patients receive VAL-083 IV at 30 or 40 mg/m2/d on days 1, 2, and 3 of a 21-day cycle. The primary objective of this study is to determine the effect of VAL-083 on median overall survival (mOS) in MGMT-unmethylated recurrent GBM patients (Group 1); and progression-free survival (PFS) in newly diagnosed GBM patients requiring adjuvant therapy after chemo-irradiation with temozolomide (Group 2), compared to historical controls in both groups. Tumor response is assessed by MRI every 42 days, using RANO criteria. The initial starting dose in this study was 40 mg/m2/d on days 1, 2, and 3 of a 21-day cycle, which was subsequently reduced to 30 mg/m2/d to improve tolerance due to myelosuppression. As of May 2021, Group 1 (Recurrent GBM) is fully enrolled: 35 evaluable patients have received 40 mg/m2/d and 48 evaluable patients have received 30 mg/m2/d VAL-083. In the adjuvant setting (Group 2), 35 evaluable patients have been enrolled (30 mg/m2/day). Enrollment, safety data and efficacy updates will be presented at the meeting. Clinicaltrials.gov identifier: NCT02717962.

Glioblastoma (GBM) is an aggressive brain tumor and has an extremely poor prognosis despite the use of multiple treatment modalities. DNA damage response (DDR) signaling plays an important role in inducing radiation and temozolomide (TMZ) resistance and hence has emerged as a molecular target for therapeutic development. The Ataxia Telangiectasia and Rad3-related protein (ATR) kinase is a key regulator of the DDR machinery, activated by DNA damage. Here, we show that three clinical-grade ATR inhibitors (Bay1895344, AZD6738 and Berzosertib) had similar selective sensitivity pattern across 16 glioma-like stem cell (GSC) lines tested. ATR inhibitors inhibited the growth of GSCs at low nanomolar range concentrations. Interestingly, all three ATR inhibitors showed a significant synergism with TMZ in a selective group of GSCs (Combination index and Bliss model). Importantly, we demonstrate that MGMT promoter methylation status was associated with cellular response to combination treatment with preferential inhibition of cell growth in MGMT promotor methylated (MGMT deficient) cell lines. Further, we compared the RNA-seq data from GSCs in the synergism and non-synergism group and used multiple complementary approaches to identify the response marker that confer sensitivity to combination therapy. Our preliminary data analysis identified several genes that confer sensitivity to combination treatment and studies are underway to validate the data. We also investigated the efficacy of BBB penetrant ATR inhibitor BAY 1895344 in orthotropic xenografts and administration of BAY 1895344 and TMZ combination significantly reduced tumor size and extended survival in an intracranial animal model. Combining ATR inhibitor with TMZ was well tolerated and did not confer additional toxicity as the body weights of TMZ and combination groups were comparable. The findings from this study provides a rationale to use ATR inhibitors in combination with TMZ in MGMT methylated tumors to improve therapeutic efficacy of standard of care for GBM patients.

BACKGROUND Checkpoint inhibitor therapy has demonstrated overall limited efficacy in the treatment of GBM. Mechanisms of resistance to checkpoint blockade need to be better elucidated. Analysis of the tumor microenvironment is critical to identify correlates of response to immune checkpoint blockade. 60 newly diagnosed GBM patients unselected for MGMT status underwent treatment with concurrent atezolizumab with radiation therapy and TMZ followed by adjuvant atezolizumab and TMZ (NCT03174197). Clinical data has been reported previously. METHODS Tissue image immunoprofiling was conducted using 2 multiplex immunofluorescence (mIF) panels against; CD3, CD8, PD-1, PD-L1, Granzyme B, FOXP3, CD45RO, CD68, and GFAP antibodies. PDL-1 (Clone SP263) malignant cells expression was assessed by immunohistochemistry. Correlations between mIF biomarkers co-expressions, IHC PD-L1, and clinical outcome including OS, radiographic response, and PFS were evaluated. RESULTS Of 60 patients enrolled, image immunoprofiling was performed successfully on pre-treatment tissue in 48 patients. 20 of 60 patients underwent re-resection for suspected recurrent disease of which 10 patients had immunoprofiling performed successfully on pre and post treatment samples. An analysis of CD3CD8+ cytotoxic T lymphocytes was consistent with prior work, showing no or relatively low levels at baseline, and no association with clinical outcome. PDL-1 expression by IHC, at thresholds of >1% or >5%, was not associated with clinical outcome. Tumors with a higher number of GFAP-expressing cancer cells had a significantly lower tumor response (p< 0.05) and median OS (430 vs. 799 days, p< 0.01). CONCLUSIONS For newly diagnosed GBM patients treated with standard of care radiation and temozolomide in combination with atezolizumab, T-cell levels and PDL-1 expression were not predictive of outcome. GFAP may represent a novel predictive biomarker of overall survival. Ongoing studies to evaluate the gut microbiome and tumor genomic (WES, CNA) and transcriptomic (RNAseq) features of these and matched tumors are underway.

Recurrent glioblastoma (GBM) has a very low 6-month progression free survival (PFS) with currently available treatments. Combination chemotherapy to target multiple cell signaling pathways is currently being investigated in order to improve prognosis for recurrent disease. The purpose of this phase I study was to determine the maximum tolerated dose (MTD) for the combination of tipifarnib and sorafenib for the treatment of recurrent GBM. Patients with pathologically proven WHO grade IV GBM and radiographically proven tumor recurrence were eligible for this study. Treatments included sorafenib at twice daily and escalating dosages of tipifarnib. Dose-limiting toxicity (DLT) was determined over the first 28-days of treatments, and the MTD was determined in a 3 + 3 study design. We enrolled 24 patients, and 21 patients completed the MTD period. The study was stopped early with no MTD determination for excessive toxicities. The last dose level reached was sorafenib at 200mg twice a day and tipifarnib 100mg twice a day on an alternating week schedule. The DLTs included diarrhea, lipase elevation, hypophosphatemia, and arthralgia. The combination of sorafenib and tipifarnib has excessive toxicities and full single agent dosages could not be achieved in combination.

Genomic, transcriptional, and proteomic analyses of brain tumors reveal that subtypes differ in their pathway activity, progression, and response to therapy. We performed an expression profiling of Glioma Initiating Cells (GICs) and comparative analysis between different groups of GICs indicates major variations in gene expression. Hierarchical clustering analysis revealed groups of GICs reflecting their heterogeneity, and among some of the genes as major regulators of mesenchymal phenotype, we identified ABOBEC3G as one of the most discriminating genes in mesenchymal group. ABOBEC3G revealed a strong correlation with overall survival in TCGA GBM patient cohorts. APOBEC3G regulates cell invasion and silencing of this gene in GICs inhibits cell invasion and also glioma sphere initiation. APOBEC3G controls invasion through TGFβ/Smad2 pathway by regulating Smad2 target genes Thrombospondin 1, matrix metallopeptidase 2 and TIMP metallopeptidase inhibitor 1. We also show that targeting APOBEC3G can sensitize cancer cells to radiation induced cell death by attenuating activation of the DNA repair pathway. This response is mainly shown by decreased pChk2 expression in knockdown APOBEC3G cells. Taken together, we show that APOBEC3G gene is a mesenchymal enriched gene that controls invasion and knockdown of APOBEC3G sensitizes cells to radiation induced cell death, suggesting that APOBEC3G can be considered for use in stratifying patients with GBM for prognostic considerations.

Glioblastoma (GBM) is the most common and lethal primary intracranial tumor. Aggressive surgical resection plus radiotherapy and temozolomide have prolonged patients’ median survival to only 14.6 months. Therefore, there is a critical need to develop novel therapeutic strategies for GBM. In this study, we evaluated the effect of NOTCH signaling intervention by gamma-secretase inhibitors (GSIs) on glioma sphere-forming cells (GSCs). GSI sensitivity exhibited remarkable selectivity among wild-type TP53 (wt-p53) GSCs. GSIs significantly impaired the sphere formation of GSCs harboring wt-p53. We also identified a concurrence between GSI sensitivity, NOTCH1 expression, and wt-p53 activity in GSCs. Through a series of gene editing and drug treatment experiments, we found that wt-p53 did not modulate NOTCH1 pathway, whereas NOTCH1 signaling positively regulated wt-p53 expression and activity in GSCs. Finally, GSIs (targeting NOTCH signaling) synergized with doxorubicin (activating wt-p53) to inhibit proliferation and induce apoptosis in wt-p53 GSCs. Taken together, we identified wt-p53 as a potential marker for GSI sensitivity in GSCs. Combining GSI with doxorubicin synergistically inhibited the proliferation and survival of GSCs harboring wt-p53.

PURPOSE Phosphatidylinositol 3-kinase (PI3K) signaling is highly active in glioblastomas. We assessed pharmacokinetics, pharmacodynamics, and efficacy of the pan-PI3K inhibitor buparlisib in patients with recurrent glioblastoma with PI3K pathway activation. METHODS This study was a multicenter, open-label, multi-arm, phase II trial in patients with PI3K pathway–activated glioblastoma at first or second recurrence. In cohort 1, patients scheduled for re-operation after progression received buparlisib for 7 to 13 days before surgery to evaluate brain penetration and modulation of the PI3K pathway in resected tumor tissue. In cohort 2, patients not eligible for re-operation received buparlisib until progression or unacceptable toxicity. Once daily oral buparlisib 100 mg was administered on a continuous 28-day schedule. Primary end points were PI3K pathway inhibition in tumor tissue and buparlisib pharmacokinetics in cohort 1 and 6-month progression-free survival (PFS6) in cohort 2. RESULTS Sixty-five patients were treated (cohort 1, n = 15; cohort 2, n = 50). In cohort 1, reduction of phosphorylated AKTS473 immunohistochemistry score was achieved in six (42.8%) of 14 patients, but effects on phosphoribosomal protein S6S235/236 and proliferation were not significant. Tumor-to-plasma drug level was 1.0. In cohort 2, four (8%) of 50 patients reached 6-month PFS6, and the median PFS was 1.7 months (95% CI, 1.4 to 1.8 months). The most common grade 3 or greater adverse events related to treatment were lipase elevation (n = 7 [10.8%]), fatigue (n = 4 [6.2%]), hyperglycemia (n = 3 [4.6%]), and elevated ALT (n = 3 [4.6%]). CONCLUSION Buparlisib had minimal single-agent efficacy in patients with PI3K-activated recurrent glioblastoma. Although buparlisib achieved significant brain penetration, the lack of clinical efficacy was explained by incomplete blockade of the PI3K pathway in tumor tissue. Integrative results suggest that additional study of PI3K inhibitors that achieve more-complete pathway inhibition may still be warranted.

Poly-ADP-ribose polymerase (PARP) is an enzyme critical for regulating a variety of DNA damage repair mechanisms. In this study, we report that PARP inhibitor, talazoparib, showed strong single-agent cytotoxicity and remarkable selective activity in glioma stem-like cells (GSCs). This single agent activity was strongly correlated with EGFR amplification as shown by genomic analysis. GSCs withEGFR amplification (which occurs in about 45% of GBMs) exhibited higher oxidative base damage, DNA breaks, and genomic instability than non-amplified GSCs. To sustain the elevated basal oxidative stress, EGFR amplified GSCs harbored increased basal expression of DNA repair proteins. As a result, DNA damage and PARP-DNA complexes increased in the amplified GSCs following talazoparib treatment, which may explain the sensitivity of these GSCs to talazoparib. Further, we show that EGFR kinase activity is important for talazoparib sensitivity, as kinase-inactive EGFR mutant and EGFR knockout cell lines were resistant to talazoparib. Intriguingly, another PAPR inhibitor Olaparib, with similar PARP enzymatic inhibition potential but less PARP-trapping ability compared with talazoparib, did not show selective sensitivity in EGFR amplified GSCs. Our data provide insight into the anti-cancer activity of talazoparib through PARP inhibition and by trapping PARP-DNA complexes. In conclusion we propose the potential of EGFR amplification as a biomarker for selection of the development of personalized therapy.

A comprehensive characterization of the somatic alterations and molecular subtypes of glioma at diagnosis has been established. However, gliomas undergo significant molecular changes over time, some of these causing malignant progression or associated with therapy. Understanding this molecular evolution may uncover therapeutic vulnerabilities and facilitate development of more effective systemic therapies. The Glioma Longitudinal Analysis (GLASS) Consortium is an international effort to systemically catalogue the longitudinal changes in gliomas through multi-platform characterization. GLASS has developed computational and clinical infrastructure to enable its mission of establishing a well annotated longitudinal molecular dataset of 1500 gliomas including 500 gliomas per each of the IDHwt/IDH-mut-non-codel/IDH-mut-codel subtypes. An initial dataset comprising 150 cases with exome sequencing at multiple timepoints has been constructed, including cases diagnosed as IDHwt (55%), IDH-mut-non-codel (38%) and IDH-mut-codel (6%). Treatment regimens followed the expected combinations of chemo- and radiation therapy while survival was more favorable than reported in literature, suggested a bias in our dataset towards better performing cases. This is possibly explained by the need for two consecutive surgical procedures to be included in the analysis. While more than half of mutations in IDHwt primary tumors were recovered in their matching recurrences, this fraction was less than 30% in the other two subtypes. The disparity in mutations between primary and recurrences may reflect intratumoral heterogeneity in both primary and recurrence, and clonal selection patterns. We observed temozolomide treatment-associated hypermutation in up to 13 of our cohort. A more detailed analysis of mutational and DNA copy number data is underway. A cohort of 75 additional patients with multi-timepoint exome data is being processed and, through several funded projects, multi-platform characterization of 250 gliomas is in progress. In summary, GLASS will provide a rich resource to the glioma community with the potential for paradigm shifting discoveries.