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Glioblastoma is an aggressive cancer of the brain and spine. While analysis of glioblastoma ‘omics data has somewhat improved our understanding of the disease, it has not led to direct improvement in patient survival. Cancer survival is often characterized by differences in gene expression, but the mechanisms that drive these differences are generally unknown. We therefore set out to model the regulatory mechanisms associated with glioblastoma survival. We inferred individual patient gene regulatory networks using data from two different expression platforms from The Cancer Genome Atlas. We performed comparative network analysis between patients with long- and short-term survival. Seven pathways were identified as associated with survival, all of them involved in immune signaling; differential regulation of PD1 signaling was validated to correspond with outcome in an independent dataset from the German Glioma Network. In this pathway, transcriptional repression of genes for which treatment options are available was lost in short-term survivors; this was independent of mutational burden and only weakly associated with T-cell infiltration. Collectively, these results provide a new way to stratify patients with glioblastoma that uses network features as biomarkers to predict survival. They also identify new potential therapeutic interventions, underscoring the value of analyzing gene regulatory networks in individual patients with cancer. Significance: Genome-wide network modeling of individual glioblastomas identifies dysregulation of PD1 signaling in patients with poor prognosis, indicating this approach can be used to understand how gene regulation influences cancer progression.

Germline POT1 mutations are found in a spectrum of cancers and confer increased risk. Recently, we identified a series of novel germline POT1 mutations that predispose carrier families to the development of glioma. Despite these strong associations, how these glioma-associated POT1 mutations contribute to glioma tumorigenesis remains undefined. Here we show that POT1-G95C increases proliferation in glioma-initiating cells in vitro and in progenitor populations in the developing brain. In a native mouse model of glioma, loss of Pot1a/b resulted in decreased survival in females compared with males. These findings were corroborated in human glioma, where low POT1 expression correlated with decreased survival in females. Transcriptomic and IHC profiling of Pot1a/b-deficient glioma revealed that tumors in females exhibited decreased expression of immune markers and increased expression of cell-cycle signatures. Similar sex-dependent trends were observed in human gliomas that had low expression of POT1. Together, our studies demonstrate context-dependent functions for POT1 mutation or loss in driving progenitor proliferation in the developing brain and sexual dimorphism in glioma. Significance: This study shows that manipulation of POT1 expression in glioma has sex-specific effects on tumorigenesis and associated immune signatures.

EGFR is frequently amplified, mutated, and overexpressed in malignant gliomas. Yet the EGFR-targeted therapies have thus far produced only marginal clinical responses, and the underlying mechanism remains poorly understood. Using an inducible oncogenic EGFR-driven glioma mouse model system, our current study reveals that a small population of glioma cells can evade therapy-initiated apoptosis and potentiate relapse development by adopting a mesenchymal-like phenotypic state that no longer depends on oncogenic EGFR signaling. Transcriptome analyses of proximal and distal treatment responses identified TGFβ/YAP/Slug signaling cascade activation as a major regulatory mechanism that promotes therapy-induced glioma mesenchymal lineage transdifferentiation. Following anti-EGFR treatment, TGFβ secreted from stressed glioma cells acted to promote YAP nuclear translocation that stimulated upregulation of the pro-mesenchymal transcriptional factor SLUG and subsequent glioma lineage transdifferentiation toward a stable therapy-refractory state. Blockade of this adaptive response through suppression of TGFβ-mediated YAP activation significantly delayed anti-EGFR relapse and prolonged animal survival. Together, our findings shed new insight into EGFR-targeted therapy resistance and suggest that combinatorial therapies of targeting both EGFR and mechanisms underlying glioma lineage transdifferentiation could ultimately lead to deeper and more durable responses. Significance: This study demonstrates that molecular reprogramming and lineage transdifferentiation underlie anti-EGFR therapy resistance and are clinically relevant to the development of new combinatorial targeting strategies against malignant gliomas with aberrant EGFR signaling.

The tumor microenvironment plays an essential role in supporting glioma stemness and radioresistance. Following radiotherapy, recurrent gliomas form in an irradiated microenvironment. Here we report that astrocytes, when pre-irradiated, increase stemness and survival of cocultured glioma cells. Tumor-naïve brains increased reactive astrocytes in response to radiation, and mice subjected to radiation prior to implantation of glioma cells developed more aggressive tumors. Extracellular matrix derived from irradiated astrocytes were found to be a major driver of this phenotype and astrocyte-derived transglutaminase 2 (TGM2) was identified as a promoter of glioma stemness and radioresistance. TGM2 levels increased after radiation in vivo and in recurrent human glioma, and TGM2 inhibitors abrogated glioma stemness and survival. These data suggest that irradiation of the brain results in the formation of a tumor-supportive microenvironment. Therapeutic targeting of radiation-induced, astrocyte-derived extracellular matrix proteins may enhance the efficacy of standard-of-care radiotherapy by reducing stemness in glioma. Significance: These findings presented here indicate that radiotherapy can result in a tumor-supportive microenvironment, the targeting of which may be necessary to overcome tumor cell therapeutic resistance and recurrence.

Glioma stem cells (GSC) are a subpopulation of tumor cells with special abilities to proliferate and differentiate in gliomas. They are one of the main causes of tumor recurrence, especially under hypoxic conditions. Although long noncoding RNAs (lncRNA) are known to be involved in numerous biological processes and are implied in the occurrence of certain diseases, their role in tumor development and progression remains poorly understood. Here we explored the mechanisms by which lncRNA derived from hypoxic GSCs (H-GSC) cause glioma progression. Isolation and identification of the Linc01060 gene, the exosomes containing them, and the proteins from tumor cells regulating the gene allowed for studying the effects of Linc01060 on proliferation and glycometabolism. H-GSC exerted their effects by transferring exosomes to glioma cells, resulting in a significant increase in Linc01060 levels. Mechanistically, Linc01060 directly interacted with the transcription factor myeloid zinc finger 1 (MZF1) and enhanced its stability. Linc01060 facilitated nuclear translocation of MZF1 and promoted MZF1-mediated c-Myc transcriptional activities. In addition, c-Myc enhanced the accumulation of the hypoxia-inducible factor-1 alpha (HIF1α) at the posttranscriptional level. HIF1α bound the hormone response elements of the Linc01060 promoter, upregulating the transcription of Linc01060 gene. Clinically, Linc01060 was upregulated in glioma and was significantly correlated with tumor grade and poor clinical prognosis. Overall, these data show that secretion of Linc01060-containing exosomes from H-GSCs activates prooncogenic signaling pathways in glioma cells to promote disease progression. Significance: These findings suggest that inhibition of Linc01060-containing exosomes or targeting the Linc01060/MZF1/c-Myc/HIF1α axis may be an effective therapeutic strategy in glioma.

Malignant glioma constitutes one of the fatal primary brain tumors in adults. Such poor prognosis calls for a better understanding of cancer-related signaling pathways of this disease. Here we elucidate a MYC-miRNA-MXI1 feedback loop that regulates proliferation and tumorigenesis in glioma. MYC suppressed MXI1 expression via microRNA-155 (miR-155) and the microRNA-23a∼27a∼24-2 cluster (miR-23a cluster), whereas MXI1, in turn, inhibited MYC expression by binding to its promoter. Overexpression of miR-155 and the miR-23a cluster promoted tumorigenesis in U87 glioma cells. Furthermore, fat mass and obesity-associated protein (FTO), an N6-methyladenosine (m6A) RNA demethylase, regulated the loop by targeting MYC. The ethyl ester form of meclofenamic acid (MA2) inhibited FTO and enhanced the effect of the chemotherapy drug temozolomide on suppressing proliferation of glioma cells and negatively regulated the loop. These data collectively highlight a key regulatory circuit in glioma and provide potential targets for clinical treatment. Significance: These findings elucidate a novel feedback loop that regulates proliferation in glioma and can be targeted via inhibition of FTO to enhance the efficacy of temozolomide.

Anti-VEGF therapy prolongs recurrence-free survival in patients with glioblastoma but does not improve overall survival. To address this discrepancy, we investigated immunologic resistance mechanisms to anti-VEGF therapy in glioma models. A screening of immune-associated alterations in tumors after anti-VEGF treatment revealed a dose-dependent upregulation of regulatory T-cell (Treg) signature genes. Enhanced numbers of Tregs were observed in spleens of tumor-bearing mice and later in tumors after anti-VEGF treatment. Elimination of Tregs with CD25 blockade before anti-VEGF treatment restored IFNγ production from CD8+ T cells and improved antitumor response from anti-VEGF therapy. The treated tumors overexpressed the glutamate/cystine antiporter SLC7A11/xCT that led to elevated extracellular glutamate in these tumors. Glutamate promoted Treg proliferation, activation, suppressive function, and metabotropic glutamate receptor 1 (mGlutR1) expression. We propose that VEGF blockade coupled with glioma-derived glutamate induces systemic and intratumoral immunosuppression by promoting Treg overrepresentation and function, which can be pre-emptively overcome through Treg depletion for enhanced antitumor effects. Significance: Resistance to VEGF therapy in glioblastoma is driven by upregulation of Tregs, combined blockade of VEGF, and Tregs may provide an additive antitumor effect for treating glioblastoma.

The development of potent and selective therapeutic approaches to glioblastoma (GBM), one of the most aggressive primary brain tumors, requires identification of molecular pathways that critically regulate the survival and proliferation of GBM. Previous studies have reported that deregulated expression of N-myc downstream regulated gene 1 (NDRG1) affects tumor growth and clinical outcomes of patients with various types of cancer including glioma. Here, we show that high level expression of NDRG1 in tumors significantly correlated with better prognosis of patients with GBM. Loss of NDRG1 in GBM cells upregulated GSK3β levels and promoted cell proliferation, which was reversed by selective inhibitors of GSK3β. In contrast, NDRG1 overexpression suppressed growth of GBM cells by decreasing GSK3β levels via proteasomal degradation and by suppressing AKT and S6 cell growth signaling, as well as cell-cycle signaling pathways. Conversely, GSK3β phosphorylated serine and threonine sites in the C-terminal domain of NDRG1 and limited the protein stability of NDRG1. Furthermore, treatment with differentiation inducing factor-1, a small molecule derived from Dictyostelium discoideum, enhanced NDRG1 expression, decreased GSK3β expression, and exerted marked NDRG1-dependent antitumor effects in vitro and in vivo. Taken together, this study revealed a novel molecular mechanism by which NDRG1 inhibits GBM proliferation and progression. Our study thus identifies the NDRG1/GSK3β signaling pathway as a key growth regulatory program in GBM, and suggests enhancing NDRG1 expression in GBM as a potent strategy toward the development of anti-GBM therapeutics. Significance: This study identifies NDRG1 as a potent and endogenous suppressor of glioblastoma cell growth, suggesting the clinical benefits of NDRG1-targeted therapeutics against glioblastoma.

Chaperone-mediated autophagy (CMA) is a homeostatic process essential for the lysosomal degradation of a selected subset of the proteome. CMA activity directly depends on the levels of LAMP2A, a critical receptor for CMA substrate proteins at the lysosomal membrane. In glioblastoma (GBM), the most common and aggressive brain cancer in adulthood, high levels of LAMP2A in the tumor and tumor-associated pericytes have been linked to temozolomide resistance and tumor progression. However, the role of LAMP2A, and hence CMA, in any cancer stem cell type or in glioblastoma stem cells (GSC) remains unknown. In this work, we show that LAMP2A expression is enriched in patient-derived GSCs, and its depletion diminishes GSC-mediated tumorigenic activities. Conversely, overexpression of LAMP2A facilitates the acquisition of GSC properties. Proteomic and transcriptomic analysis of LAMP2A-depleted GSCs revealed reduced extracellular matrix interaction effectors in both analyses. Moreover, pathways related to mitochondrial metabolism and the immune system were differentially deregulated at the proteome level. Furthermore, clinical samples of GBM tissue presented overexpression of LAMP2, which correlated with advanced glioma grade and poor overall survival. In conclusion, we identified a novel role of CMA in directly regulating GSCs activity via multiple pathways at the proteome and transcriptome levels. Significance: A receptor of chaperone-mediated autophagy regulates glioblastoma stem cells and may serve as a potential biomarker for advanced tumor grade and poor survival in this disease.

Cancer cells rely on mitochondrial functions to regulate key survival and death signals. How cancer cells regulate mitochondrial autophagy (mitophagy) in the tumor microenvironment as well as utilize mitophagy as a survival signal is still not well understood. Here, we elucidate a key survival mechanism of mitochondrial NIX-mediated mitophagy within the hypoxic region of glioblastoma, the most malignant brain tumor. NIX was overexpressed in the pseudopalisading cells that envelop the hypoxic–necrotic regions, and mitochondrial NIX expression was robust in patient-derived glioblastoma tumor tissues and glioblastoma stem cells. NIX was required for hypoxia and oxidative stress–induced mitophagy through NFE2L2/NRF2 transactivation. Silencing NIX impaired mitochondrial reactive oxygen species clearance, cancer stem cell maintenance, and HIF/mTOR/RHEB signaling pathways under hypoxia, resulting in suppression of glioblastoma survival in vitro and in vivo. Clinical significance of these findings was validated by the compelling association between NIX expression and poor outcome for patients with glioblastoma. Taken together, our findings indicate that the NIX-mediated mitophagic pathway may represent a key therapeutic target for solid tumors, including glioblastoma. Significance: NIX-mediated mitophagy regulates tumor survival in the hypoxic niche of glioblastoma microenvironment, providing a potential therapeutic target for glioblastoma.

Human astrocytomas and oligodendrogliomas are defined by mutations of the metabolic enzymes isocitrate dehydrogenase (IDH) 1 or 2, resulting in the production of the abnormal metabolite D-2 hydroxyglutarate. Here, we studied the effect of mutant IDH on cell proliferation and apoptosis in a glioma mouse model. Tumors were generated by inactivating Pten and p53 in forebrain progenitors and compared with tumors additionally expressing the Idh1 R132H mutation. Idh-mutant cells proliferated less in vitro and mice with Idh-mutant tumors survived significantly longer compared with Idh-wildtype mice. Comparison of miRNA and RNA expression profiles of Idh-wildtype and Idh-mutant cells and tumors revealed miR-183 was significantly upregulated in IDH-mutant cells. Idh-mutant cells were more sensitive to endoplasmic reticulum (ER) stress, resulting in increased apoptosis and thus reduced cell proliferation and survival. This was mediated by the interaction of miR-183 with the 5′ untranslated region of semaphorin 3E, downregulating its function as an apoptosis suppressor. In conclusion, we show that mutant Idh1 delays tumorigenesis and sensitizes tumor cells to ER stress and apoptosis. This may open opportunities for drug treatments targeting the miR-183–semaphorin axis. Significance: The pathologic metabolite 2-hydroxyglutarate, generated by IDH-mutant astrocytomas, sensitizes tumor cells to ER stress and delays tumorigenesis.

Gliomas are classified by combining histopathologic and molecular features, including isocitrate dehydrogenase (IDH) status. Although IDH-wild-type diffuse astrocytic glioma (DAG) shows a more aggressive phenotype than IDH-mutant type, lack of knowledge regarding relevant molecular drivers for this type of tumor has hindered the development of therapeutic agents. Here, we examined human IDH-wild-type DAGs and a glioma mouse model with a mosaic analysis with double markers (MADM) system, which concurrently lacks p53 and NF1 and spontaneously develops tumors highly comparable with human IDH-wild-type DAG without characteristic molecular features of glioblastoma (DAG-nonMF). During tumor formation, enhancer of zeste homolog (EZH2) and the other polycomb repressive complex 2 (PRC2) components were upregulated even at an early stage of tumorigenesis, together with an increased number of genes with H3K27me3 or H3K27me3 and H3K4me3 bivalent modifications. Among the epigenetically dysregulated genes, frizzled-8 (Fzd8), which is known to be a cancer- and stem cell reprogramming–related gene, was gradually silenced during tumorigenesis. Genetic and pharmacologic inhibition of EZH2 in MADM mice showed reactivation of aberrant H3K27me3 target genes, including Fzd8, together with significant reduction of tumor size. Our study clarifies a pathogenic molecular pathway of IDH-wild-type DAG-nonMF that depends on EZH2 activity and provides a strong rationale for targeting EZH2 as a promising therapeutic approach for this type of glioma. Significance: EZH2 is involved in the generation of IDH-wild-type diffuse astrocytic gliomas and is a potential therapeutic target for this type of glioma.

O6-methylguanine-DNA methyltransferase (MGMT) is an enzyme that removes alkyl groups at the O6-position of guanine in DNA. MGMT expression is reduced or absent in many tumor types derived from a diverse range of tissues, most notably in glioma. Low MGMT expression confers significant sensitivity to DNA alkylating agents such as temozolomide, providing a natural therapeutic index over normal tissue. In this study, we sought to identify novel approaches that could maximally exploit the therapeutic index between tumor cells and normal tissues based on MGMT expression, as a means to enhance selective tumor cell killing. Temozolomide, unlike other alkylators, activated the ataxia telangiectasia and Rad3–related (ATR)–checkpoint kinase 1 (Chk1) axis in a manner that was highly dependent on MGMT status. Temozolomide induced growth delay, DNA double-strand breaks, and G2–M cell-cycle arrest, which led to ATR-dependent phosphorylation of Chk1; this effect was dependent on reduced MGMT expression. Treatment of MGMT-deficient cells with temozolomide increased sensitivity to ATR inhibitors both in vitro and in vivo across numerous tumor cell types. Taken together, this study reveals a novel approach for selectively targeting MGMT-deficient cells with ATR inhibitors and temozolomide. As ATR inhibitors are currently being tested in clinical trials, and temozolomide is a commonly used chemotherapeutic, this approach is clinically actionable. Furthermore, this interaction potently exploits a DNA-repair defect found in many cancers. Significance: Monofunctional alkylating agents sensitize MGMT-deficient tumor cells to ATR inhibitors.

Homozygous deletion of methylthioadenosine phosphorylase (MTAP) is one of the most frequent genetic alterations in glioblastoma (GBM), but its pathologic consequences remain unclear. In this study, we report that loss of MTAP results in profound epigenetic reprogramming characterized by hypomethylation of PROM1/CD133–associated stem cell regulatory pathways. MTAP deficiency promotes glioma stem-like cell (GSC) formation with increased expression of PROM1/CD133 and enhanced tumorigenicity of GBM cells and is associated with poor prognosis in patients with GBM. As a combined consequence of purine production deficiency in MTAP-null GBM and the critical dependence of GSCs on purines, the enriched subset of CD133+ cells in MTAP-null GBM can be effectively depleted by inhibition of de novo purine synthesis. These findings suggest that MTAP loss promotes the pathogenesis of GBM by shaping the epigenetic landscape and stemness of GBM cells while simultaneously providing a unique opportunity for GBM therapeutics. Significance: This study links the frequently mutated metabolic enzyme MTAP to dysregulated epigenetics and cancer cell stemness and establishes MTAP status as a factor for consideration in characterizing GBM and developing therapeutic strategies.

Circulating tumor cells (CTC) are known to be present in the blood of patients with glioblastoma (GBM). Here we report that GBM-derived CTC possess a cancer stem cell (CSC)-like phenotype and contribute to local tumorigenesis and recurrence by the process of self-seeding. Genetic probes showed that mouse GBM-derived CTC exhibited Sox2/ETn transcriptional activation and expressed glioma CSC markers, consistent with robust expression of stemness-associated genes including SOX2, OCT4, and NANOG in human GBM patient-derived samples containing CTC. A transgenic mouse model demonstrated that CTC returned to the primary tumor and generated new tumors with enhanced tumorigenic capacity. These CTCs were resistant to radiotherapy and chemotherapy and to circulation stress-induced cell apoptosis. Single-cell RNA-seq analysis revealed that Wnt activation induced stemness and chemoresistance in CTC. Collectively, these findings identify GBM-derived CTC as CSC-like cells and suggest that targeting Wnt may offer therapeutic opportunities for eliminating these treatment-refractory cells in GBM. Significance: These findings identify CTCs as an alternative source for in situ tumor invasion and recurrence through local micrometastasis, warranting eradication of systemic "out-of-tumor" CTCs as a promising new therapeutic opportunity for GBM.

Isocitrate dehydrogenase-mutant low-grade gliomas (IDHmut-LGG) grow slowly but frequently undergo malignant transformation, which eventually leads to premature death. Chemotherapy and radiotherapy treatments prolong survival, but can also induce genetic (or epigenetic) alterations involved in transformation. Here, we developed a mathematical model of tumor progression based on serial tumor volume data and treatment history of 276 IDHmut-LGGs classified by chromosome 1p/19q codeletion (IDHmut/1p19qcodel and IDHmut/1p19qnoncodel) and performed genome-wide mutational analyses, including targeted sequencing and longitudinal whole-exome sequencing data. These analyses showed that tumor mutational burden correlated positively with malignant transformation rate, and chemotherapy and radiotherapy significantly suppressed tumor growth but increased malignant transformation rate per cell by 1.8 to 2.8 times compared with before treatment. This model revealed that prompt adjuvant chemoradiotherapy prolonged malignant transformation-free survival in small IDHmut-LGGs (≤ 50 cm3). Furthermore, optimal treatment differed according to genetic alterations for large IDHmut-LGGs (> 50 cm3); adjuvant therapies delayed malignant transformation in IDHmut/1p19qnoncodel but often accelerated it in IDHmut/1p19qcodel. Notably, PI3K mutation was not associated with malignant transformation but increased net postoperative proliferation rate and decreased malignant transformation-free survival, prompting the need for adjuvant therapy in IDHmut/1p19qcodel. Overall, this model uncovered therapeutic strategies that could prevent malignant transformation and, consequently, improve overall survival in patients with IDHmut-LGGs. Significance: A mathematical model successfully estimates malignant transformation-free survival and reveals a link between genetic alterations and progression, identifying precision medicine approaches for optimal treatment of IDH-mutant low-grade gliomas.

F-box and WD repeat domain containing 7 (FBXW7) is a substrate receptor of the ubiquitin ligase SKP1-Cullin1-F-box complex and a potent tumor suppressor that prevents unregulated cell growth and tumorigenesis. However, little is known about FBXW7-mediated control of cell metabolism and related functions in cancer therapy. Here, we report that FBXW7 expression inversely correlates with the expression levels of the key metabolic enzyme isocitrate dehydrogenase 1 (IDH1) in patients with glioma and public glioma datasets. Deletion of FBXW7 significantly increased both wild-type (WT) and mutant IDH1 expression, which was mediated by blocking degradation of sterol regulatory element binding protein 1 (SREBP1). The upregulation of neomorphic mutant IDH1 by FBXW7 deletion stimulated production of the oncometabolite 2-hydroxyglutarate at the expense of increasing pentose phosphate pathway activity and NADPH consumption, limiting the buffering ability against radiation-induced oxidative stress. In addition, FBXW7 knockout and IDH1 mutations induced nonhomologous end joining and homologous recombination defects, respectively. In vitro and in vivo, loss of FBXW7 dramatically enhanced the efficacy of radiation treatment in IDH1-mutant cancer cells. Taken together, this work identifies FBXW7 deficiency as a potential biomarker representing both DNA repair and metabolic vulnerabilities that sensitizes IDH1-mutant cancers to radiotherapy. Significance: Deficiency of FBXW7 causes defects in DNA repair and disrupts NADPH homeostasis in IDH1-mutant glioma cells, conferring high sensitivity to radiotherapy.

Glioma stem-like cells (GSC) in glioblastoma (GBM) structure tumor cells into a hierarchical organization and are postulated to be recalcitrant to conventional treatments, resulting in fatal relapse of the disease. A better understanding of these cells would be essential for meaningful and lasting treatments. In this issue of Cancer Research, Virolle and colleagues report a fascinating phenotype whereby the extracellular signal-regulated kinase (ERK) pathway regulates a mechanism of dedifferentiation of GBM cells into a stem-like state expressing markers of pluripotency through an EGFR-ERK-EGR1–dependent axis. This aptly termed “toggle switch” may contribute to maintenance of GSCs, promote intratumor heterogeneity, and potentially provide innovative treatment options. See related article by Almairac et al., p. 3236

Cancer evolves from premalignant clones that adopt unusual cell states to achieve transformation. We previously pinpointed the oligodendrocyte precursor cell (OPC) as a cell of origin for glioma, but the early changes of mutant OPCs during premalignancy remained unknown. Using mice engineered for inducible Nf1-Trp53 loss in OPCs, we acutely isolated labeled mutant OPCs by laser-capture microdissection, determined global gene-expression changes by bulk RNA sequencing, and compared with cell-state fluctuations at the single-cell level by stochastic profiling, which uses RNA-sequencing measurements from random pools of 10 mutant cells. At 12 days after Nf1-Trp53 deletion, bulk differences were mostly limited to mitotic hallmarks and genes for ribosome biosynthesis, and stochastic profiling revealed a spectrum of stem-progenitor (Axl, Aldh1a1), proneural, and mesenchymal states as potential starting points for gliomagenesis. At 90 days, bulk sequencing detected few differentially expressed transcripts, whereas stochastic profiling revealed cell states for neurons and mural cells that do not give rise to glial tumors, suggesting cellular dead-ends for gliomagenesis. Importantly, mutant OPCs that strongly expressed key effectors of nonsense-mediated decay (Upf3b) and homology-dependent DNA repair (Rad51c, Slx1b, Ercc4) were identified along with DNA-damage markers, suggesting transcription-associated replication stress. Analysis of 10-cell transcriptomes at 90 days identified a locus of elevated gene expression containing an additional repair endonuclease (Mus81) and Rin1, a Ras-Raf antagonist and possible counterbalance to Nf1 loss, which was microdeleted or downregulated in gliomas at 150 days. These hidden cell-state variations uncover replication stress as a potential bottleneck that must be resolved for glioma initiation. Significance: Profiling premalignant cell states in a mouse model of glioma uncovers regulatory heterogeneity in glioma cells-of-origin and defines a state of replication stress that precedes tumor initiation. See related articles by Singh and colleagues, p. 1840 and Schaff and colleagues, p. 1853

13C MRI of hyperpolarized [1-13C]pyruvate metabolism has been used in oncology to detect disease, investigate disease progression, and monitor response to treatment with a view to guiding treatment in individual patients. This technique has translated to the clinic with initial studies in prostate cancer. Here, we use the technique to investigate its potential uses in patients with glioblastoma (GB). We assessed the metabolism of hyperpolarized [1-13C]pyruvate in an orthotopically implanted cell line model (U87) of GB and in patient-derived tumors, where these were produced by orthotopic implantation of cells derived from different patients. Lactate labeling was higher in the U87 tumor when compared with patient-derived tumors, which displayed intertumoral heterogeneity, reflecting the intra- and intertumoral heterogeneity in the patients' tumors from which they were derived. Labeling in some patient-derived tumors could be observed before their appearance in morphologic images, whereas in other tumors it was not significantly greater than the surrounding brain. Increased lactate labeling in tumors correlated with c-Myc–driven expression of hexokinase 2, lactate dehydrogenase A, and the monocarboxylate transporters and was accompanied by increased radioresistance. Because c-Myc expression correlates with glioma grade, this study demonstrates that imaging with hyperpolarized [1-13C]pyruvate could be used clinically with patients with GB to determine disease prognosis, to detect early responses to drugs that modulate c-Myc expression, and to select tumors, and regions of tumors for increased radiotherapy dose. Significance: Metabolic imaging with hyperpolarized [1-13C]pyruvate detects low levels of c-Myc–driven glycolysis in patient-derived glioblastoma models, which, when translated to the clinic, could be used to detect occult disease, determine disease prognosis, and target radiotherapy. Cancer Res; 78(18); 5408–18. ©2018 AACR.

Tumor metabolism is reprogrammed to meet the demands of proliferating cancer cells. In particular, cancer cells upregulate synthesis of the membrane phospholipids phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdE) in order to allow for rapid membrane turnover. Nonetheless, we show here that, in mutant isocitrate dehydrogenase 1 (IDHmut) gliomas, which produce the oncometabolite 2-hydroxyglutarate (2-HG), PtdCho and PtdE biosynthesis is downregulated and results in lower levels of both phospholipids when compared with wild-type IDH1 cells. 2-HG inhibited collagen-4-prolyl hydroxylase activity, leading to accumulation of misfolded procollagen-IV in the endoplasmic reticulum (ER) of both genetically engineered and patient-derived IDHmut glioma models. The resulting ER stress triggered increased expression of FAM134b, which mediated autophagic degradation of the ER (ER-phagy) and a reduction in the ER area. Because the ER is the site of phospholipid synthesis, ER-phagy led to reduced PtdCho and PtdE biosynthesis. Inhibition of ER-phagy via pharmacological or molecular approaches restored phospholipid biosynthesis in IDHmut glioma cells, triggered apoptotic cell death, inhibited tumor growth, and prolonged the survival of orthotopic IDHmut glioma-bearing mice, pointing to a potential therapeutic opportunity. Glioma patient biopsies also exhibited increased ER-phagy and downregulation of PtdCho and PtdE levels in IDHmut samples compared with wild-type, clinically validating our observations. Collectively, this study provides detailed and clinically relevant insights into the functional link between oncometabolite-driven ER-phagy and phospholipid biosynthesis in IDHmut gliomas. Significance: Downregulation of phospholipid biosynthesis via ER-phagy is essential for proliferation and clonogenicity of mutant IDH1 gliomas, a finding with immediate therapeutic implications. Cancer Res; 78(9); 2290–304. ©2018 AACR.

Chimeric antigen receptor (CAR) T-cell therapy is an emerging immunotherapy against several malignancies including glioblastoma, the most common and most aggressive malignant primary brain tumor in adults. The challenges in solid tumor immunotherapy comprise heterogenously expressed tumor target antigens and restricted trafficking of CAR T cells to and impaired long-term persistence at the tumor site, as well as the unaddressed integration of CAR T-cell therapy into conventional anticancer treatments. We addressed these questions using a NKG2D-based chimeric antigen receptor construct (chNKG2D) in fully immunocompetent orthotopic glioblastoma mouse models. ChNKG2D T cells demonstrated high IFNγ production and cytolytic activity in vitro. Upon systemic administration in vivo, chNKG2D T cells migrated to the tumor site in the brain, did not induce adverse events, prolonged survival, and cured a fraction of glioma-bearing mice. Surviving mice were protected long-term against tumor rechallenge. Mechanistically, this was not solely the result of a classical immune memory response, but rather involved local persistence of chNKG2D T cells. A subtherapeutic dose of local radiotherapy in combination with chNKG2D T-cell treatment resulted in synergistic activity in two independent syngeneic mouse glioma models by promoting migration of CAR T cells to the tumor site and increased effector functions. We thus provide preclinical proof-of-concept of NKG2D CAR T-cell activity in mouse glioma models and demonstrate efficacy, long-term persistence, and synergistic activity in combination with radiotherapy, providing a rationale to translate this immunotherapeutic strategy to human glioma patients. Significance: These findings provide evidence for synergy of conventional anticancer therapy and CAR T cells and heralds future studies for other treatment combinations. Cancer Res; 78(4); 1031–43. ©2017 AACR.

Glioma-associated microglia and macrophages (GAM), which infiltrate high-grade gilomas, constitute a major cellular component of these lesions. GAM behavior is influenced by tumor-derived cytokines that suppress initial antitumorigenic properties, causing them to support tumor growth and to convert and suppress adaptive immune responses to the tumor. Mice that lack the transmembrane receptor neuropilin-1 (Nrp1), which modulates GAM immune polarization, exhibit a decrease in glioma volumes and neoangiogenesis and an increase in antitumorigenic GAM infiltrate. Here we show that replacing the peripheral macrophage populations of wild-type mice with Nrp1-depleted bone marrow-derived macrophages (BMDM) confers resistance to the development of glioma. This resistance occurred in a similar fashion seen in mice in which all macrophages lacked Nrp1 expression. Tumors had decreased volumes, decreased vascularity, increased CTL infiltrate, and Nrp1-depleted BMDM adopted a more antitumorigenic phenotype relative to wild-type GAMs within the tumors. Mice with Nrp1-deficient microglia and wild-type peripheral macrophages showed resistance to glioma development and had higher microglial infiltrate than mice with wild-type GAMs. Our findings show how manipulating Nrp1 in either peripheral macrophages or microglia reprograms their phenotype and their pathogenic roles in tumor neovascularization and immunosuppression. Significance: This study highlights the proangiogenic receptor neuropilin 1 in macrophages and microglial cells in gliomas as a pivotal modifier of tumor neovascularization and immunosuppression, strengthening emerging evidence of the functional coordination of these two fundamental traits of cancer. Cancer Res; 78(3); 685–94. ©2017 AACR.

Despite considerable promise and emerging clinical success, several challenges impede the broader implementation of novel immunotherapies such as oncolytic virus(OV)-based gene therapy, including for patients with pancreatic cancer (PDAC). One of such challenge is inter-patient variability that may impact on OV selectivity and killing efficacy for tumor cells. For this study, we selected the rat parvovirus H-1 (H-1PV) that is nonpathogenic in humans and has a natural oncolytic activity in several cancer models. The safety and tolerability of H-1PV was recently demonstrated in early clinical trials for glioma and PDAC. However, we report here that H-1PV infection, oncolytic and pro-apoptotic activity are limited in PDAC cells, including patient-derived primary cells. To address this concern, we applied a directed evolution strategy to generate H-1PV variants with specific activity towards PDAC cells. Following selection using patient primary cells, we managed to isolate clonal, PDAC-adapted H-1PVs that induce PDAC cells lysis as compared to parental H-1PV while infection of normal pancreatic cells remained negligeable. Genome sequencing of the tumor adapted virus reveals mutations in promoting and viral capsid sequences. In vivo, the tumor-adapted H1PV demonstrates greater anti-tumor effect than parental H-1PV, following intravenous administration in an experimental model of orthotopic pancreatic tumors engrafted in immunodeficient mice. To our knowledge, we report here for the first time the production of highly selective and potent OV using directed evolution to override PDAC resistance to virotherapy. While the molecular mechanisms involved are still under investigation, this project is a first step towards precision medicine strategies based on OV. Citation Format: Pierre Garcin, Monireh Kazemimanesh, Hubert Lulka, Nelson Dusetti, Guillaume Labrousse, Emilie Benuzzi, Louis Buscail, Pierre Cordelier. Directed evolution generates novel oncolytic H-1 parvoviruses with improved therapeutic efficacy in virus-resistant pancreatic cancer cells [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-016.

Introduction: Olfactory ensheathing glia (OEG) is a fully differentiated cell that promotes the continuous regeneration of odor receptors from the nasal mucosa to the olfactory bulb during adult lifespan. OEG secretes several factors making the environment more permissive to regeneration, and promotes differentitation of progenitor/neural stem cells. Recently, our group showed for the first time that OEG can target glioblastoma (GBM) cells and GBM stem-like cells (GSC) in the mice brain via intranasal delivery, the natural route of OEG to the brain. Here, we test the potential of OEG in inhibiting the proliferation and stemness of GSC, leading to their differentiation (similar to their neural stem cells counterpart), making them susceptible to conventional therapy. Methods: OEG derived from mice OB (1.5e+5 cells) were co-cultured with GSC at 1:1 ratio. We used a dual secreted luciferase reporter that can be multiplexed together to monitor both OEG and GSCs viability and proliferation in the same well. For in vivo analysis, mice-bearing patient-derived xenograft GSCs were treated with a single injection of 1e+5 OEG intranasally or control. One week later, each group was subdivided into two groups which either received ionizing radiation (IR, one dose of 4Gy) or control. Results: GSC or their corresponding differentiated glioma cells (cultured in serum for 10 days) displayed a decrease in cell viability when co-cultured with OEG compared to control. Analysis of mRNA levels and immunofluorescence images from these co-cultures (using a Transwell) showed a significant decrease in markers associated with stemness and aggressiveness, and an increase in both neuronal and astrocytic markers compared to control. When assessed tumor formation, GSC cultured with OEG conditioned media (OEGCM) failed to form tumors and became more susceptible to IR and Temozolomide. In mice bearing PDX, intranasal injection of non-modified OEG lead to inhibition of tumor growth prolonged mouse survival (48 days vs 34 days for control; P Conclusion: Our findings show that OEG inhibits GBM stemness and malignancy, induces differentiation of GSCs, making them prone to conventional therapy. OEG have the advantage over other typical cell therapies since they are terminally differentiated and can be easily obtained from the olfactory mucosa, a simple procedure typically done for patients with spinal cord injury allowing autologous transplantation. Citation Format: Litia Carvalho, Renata Fleming, Gulsah Erel-Akbaba, Ghazal Lashgari, Daniel Ryan, Elie Tabet, Max Zinter, Jian Teng, Bakhos Tannous. Olfactory ensheathing glia as a cell-based therapy for glioblastomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3114.

Adducin 3 (ADD3) is a major cytoskeleton protein in glioma cells. Our previous studies demonstrated a significant downregulation of ADD3 in GBM when compared with lower-grade gliomas, and that ADD3 knockdown promoted malignant phenotypes. GBMs are usually characterized by high levels of genomic instability, including frequent loss of heterozygosity (LOH) of chromosome 10q, whilst ADD3 is also located in chromosome 10q25.1-25.2 where it is known to be a tumor-suppressor region. However, it is not known whether ADD3 downregulation in malignant gliomas is associated with allelic loss in 10q. To determine 10q LOH on different polymorphic DNA loci (covering ADD3 locus) on glioma tissues, specimens of Normal, Grade II, Grade III, Grade IV GBM and recurrent GBM tissue were included. DNA fragments were evaluated by using a PCR-based microsatellite capillary electrophoresis and analyzed by a fragment analyzer. LOH status were correlated with patient survival for potential prognostic and diagnostic implications. LOH on ADD3 locus was detected in high grade and recurrent GBMs. It is revealed that downregulation of ADD3 level in GBM is associated with genetic instability of LOH in 10q and associated with recurrence disease. This study would provide significant implications, particularly for understanding pathogenesis of GBM and add valuable credits on the diagnostic as well as prognostic implications. Citation Format: Mei-Yee Kiang, Stella Sun, Gilberto Ka-Kit Leung. Detection of loss of heterozygosity in GBM revealed the role of a tumor suppressive gene in regulating glioma progression and tumor recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2484.

Background: Gliomas are the most common malignant brain tumor, have a very aggressive behavior, and invariably relapse and progress. Despite the recent advances, only a few drugs are approved and they present limited success. Currently, there are numerous clinical trials evaluating the efficacy of immunotherapy for gliomas, which are not completed yet. Deciphering the composition of the tumor microenvironment (TME) can have an important and immediate impact on therapeutic interventions and on the development of prognostic and predictive biomarkers for gliomas immunotherapy. To investigate the molecular dynamics over time and in response to therapeutic pressures, the Glioma Longitudinal AnalySiS (GLASS) Consortium, a multinational collaboration, is investigating epigenome-wide molecular data from primary and recurrent matched pairs. Objective: Our aim is to evaluate glioma TME using the deconvolution method methylCIBERSORT applied to DNA methylation data from GLASS. Methods: We generated and validated a customized reference signature defining 10 cell types to predict the relative proportions of immune cell type in the TME of 370 glioma specimens, including 132 longitudinal pairs (initial and recurrent tumors) in association with clinical features (recurrence, survival etc). Results: We found that the TME differs across gliomas of different subtypes. In general, IDHmut subtypes (Codel, GCIMP-high, and GCIMP-low) presented less immune infiltration than IDHwt (Classic-like, Mesenchymal-like, and PA-like). The most abundant estimated infiltrated cell types in IDHmut and IDHwt gliomas were TCD4 cells and macrophages, respectively. Post-treatment (chemo+radiotherapy), we found a decrease of TCD4 and an increase of TCD8 cells in recurrent Codel and G-CIMP-high subtypes; and an increase of macrophages in classic recurrent tumors. High frequency of macrophages and TCD8 cells were associated with poorer overall survival in the IDHwt patients (log-rank p=0.040, hazard ratio (HR) = 1.38; log-rank p=0.046, HR = 2.37, respectively). Conclusions: Using a DNA methylation-based deconvolution approach, we have described the TME of longitudinal gliomas. We found a TME diversity across glioma molecular subtypes and an association with IDH mutation and overall survival. Our findings indicate that the epigenomic deconvolution of TME has a potential therapeutic and prognostic implication to guide the management of patients with gliomas. Citation Format: Tathiane Maistro Malta, Indrani Datta, Thais Sabedot, Ruicong She, AnaValeria Castro, GLASS Consortium GLASS Consortium, Antonio Iavarone, Laila M. Poisson, Houtan Noushmehr. Glioma immune microenvironment change during tumor recurrence [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2717.

Pediatric low-grade gliomas (pLGG) are the most common central nervous system (CNS) tumor in children. Although the majority of pLGG has unregulated MAPK pathway activity, and clinical MAPK pathway inhibitors are available, pLGG treatment remains challenging. Tumor progression, rebound, and therapy resistance limit the success of MAPK pathway inhibitors, especially in high risk subgroups, such as BRAF V600E mutant CDKN2A deleted gliomas.To define mechanisms for progression and overcome resistance, we generated BRAF V600E mutant, CDKN2A deleted murine glioma models of different grades and drug sensitivities. Here, we use genetically engineered mice to target expression of BRAF V600E and deletion of CDKN2A to various germinal and non-germinal areas, assessing neuro-anatomical aspects of oncogene sensitivity. Using neural stem cell (NSC)- and astrocyte-, and oligodendrocyte progenitor cell (OPC)-selective driver line, we investigate the likely origin for pLGG and therapy resistant glioma stem cells, developing models that faithfully recapitulate human tumor development. Our numerous immunocompetent, orthotopic BRAF V600E mutant glioma model derived from endogenous tumors exhibit a range of sensitivities to MAPK inhibitor therapies, allowing us to define mechanisms for therapy resistance. In syngeneic, orthotopic BRAF V600E mutant CDKN2A deleted gliomas, we define MAPK pathway inhibitor-induced-changes in the tumor microenvironment, including the immune infiltrate, and investigate immune modulatory therapy as novel treatment strategy for children with BRAF mutant gliomas. Our numerous models for BRAF mutant glioma will facilitate research on tumor development, progression and the tumor-immune interactions. Collectively, our results describe genetic alteration- and microenvironment-based mechanisms for BRAF mutant tumor initiation and progression, and suggest therapeutic combination therapies for glioma. Citation Format: Claudia K. Petritsch, Stefan Grossauer, Wei Wang, Anne Marie Barrette, Jongwhi Park. Clinically relevant MAPK pathway inhibition reverses stem cell fate defects and sensitize BRAF mutant glioma to immune modulatory therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3072.

Background: Glioblastoma Multiforme (GBM-Grade IV) is the most aggressive form of glioma in the USA carrying only a 15 month median survival time. IDH1R132H is an early onset mutation present in 70-80% of low grade gliomas (LGG-Grade I and II) and secondary GBM patients that causes intracellular oxidative stress and impairment of post-radiation DNA damage repair (DDR) via Homologous Recombination (HR). Although IDH1R132H is associated with improved patient survival and clinical response to chemotherapy, astrocytoma (LGG) patients specifically also harbor ATRXLoss, a genetic alteration that inactivates the ATRX chromatin remodeling protein and impairs DDR via non-homologous end joining (NHEJ). Importantly, the recent discovery of glioma stem cells (GSCs), a subpopulation of chemoradioresistant, self-renewable, tumor initiating cells known to promote tumor recurrence, presents a new target for therapeutic intervention. However, the mechanisms by which IDH1R132H and ATRXLoss contribute to GSC sensitivity to proton radiotherapy (PRT), a more tumor specific and cytotoxic form of radiation therapy than conventional X-rays (XRT), is not well understood. We hypothesize that both IDH1R132H and ATRXLoss contribute to the impairment of GSC self-renewal after PRT, but not after XRT. Methods: Isogenic MGG18-IDH1WT, MGG18-IDH1R132H, TS543-ATRXWT, and TS543-ATRXLoss patient derived glioma cells were cultured as neurospheres and treated with 3-6 Gy of XRT or PRT. GSC self-renewal, based on neurosphere formation frequency, was quantified using extreme limiting dilution analysis (ELDA). Results: TS543-ATRXLoss significantly inhibited GSC self-renewal over TS543-ATRXWT when subject to PRT (Pairwise Testing Pr(>χ2)χ2)χ2)χ2) Conclusions: We conclude that ATRXLoss and IDH1R132H both substantially contribute to PRT sensitivity in GSCs, thereby supporting the benefits of PRT for astrocytoma patients. Our future experiments will further elucidate the biological mechanisms by which ATRXLoss and IDH1R132H confer PRT sensitivity, especially via dsDNA damage and apoptosis. Citation Format: Ángel Adrian Garcés, Lawrence Bronk, Krishna P. Bhat, David R. Grosshans. ATRXLoss and IDH1R132H impair self-renewal of glioma stem cells after proton radiotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3077.

Background: Glioma is the most common malignant tumor of the central nervous system, often behaving very aggressively. Recently, The Cancer Genome Atlas (TCGA) and others have shown that epigenomic alterations in primary glioma tumors have prognostic and predictive roles, but there is a gap in knowledge of the molecular alterations after glioma treatment. In order to fill this gap, the Glioma Longitudinal AnalySiS (GLASS) Consortium, a multi-national collaboration from 13 institutions, is investigating genome-wide molecular data from primary and recurrent matched pairs. The current data freeze has DNA methylation data for 266 primary-recurrent pairs fromIllumina 450K and EPIC array platforms. Methods: We hypothesize that there will be evidence of aggressivity in the DNA methylation profiles of recurrent tumors relative to their matched primary, and we explored this through tumor subtyping, patterns of differentially methylated CpGs (DMPs), and epigenomic aging. Results: The subtype classification in the primary tumors was as follows for IDH wildtype tumors - 43.8%Classical, 47.3% Mesenchymal, 8.7% PA-like, none LGm6-GBM and for IDH mutant tumors 19.1% Codel, 79.4% G-CIMP-high, 1.3% G-CIMP-low. We observed that among IDH wildtype tumors 29.8% changed subtype, 47.1% of which shifted to the more aggressive Mesenchymal-like subtype. In IDH mutant tumors, 26.0% changed at recurrence, of which 57.9% shifted to the aggressive G-CIMP-low subtype. Patterns of DMPs in IDH mutant-code tumors (15 pairs) showed a loss of methylation upon recurrence, with 651 DMPs identified (paired Wilcoxon test, FDR &lt0.05). In unsupervised clustering, recurrent Codel tumors thus move away from IDH mutant tumors and align more closely to IDH wildtype tumors. We do not see the same increases in hypomethylation upon recurrence among IDHwt tumors, with only 17 DMPs at FDR &lt0.05 in the classical subtype (24 pairs). It has been shown that biological age estimates of the tumor using DNA methylation can predict aggressivity of certain tumor types. We examined epigenetic aging changes between primary and recurrent tumors, relative to the patients chronological age, with Horvath (tissue-based) and epiTOC (mitotic-based) DNA methylation clocks. Both clocks showed shifts in age acceleration, that is increased biological age in relation to chronological age. For the Horvath clock, we found increased age acceleration in Classical-like tumors (primary=39.5±8.9, recurrent 41.7± 19.7) and Codel tumors (primary= 57.0±22.3, recurrent= 64.2±31.8). Conclusions: Collectively, we observed glioma tumor epigenetic changes from the primary to recurrent state and these differences tend to reflect a shift to a more aggressive phenotype. Future work will explore the relationship of these findings with clinical treatments received between primary and recurrent states. Citation Format: Indrani Datta, Tathi Malta, Thais Sabedot, Ruicong She, The GLASS Consortium, Antonio Iavarone, Houtan Noushmehr, Laila M. Poisson. The evolutionary trajectory of epigenomics in adult glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2085.

ONC201, a first-in-class, small molecule, imipridone therapy, is known to induce apoptosis via upregulation of TNF-related apoptosis inducing ligand (TRAIL) and its proapoptotic death receptor (DR5), independent of p53. Current trials have been exploring ONC201 as a monotherapy or in conjunction with radiotherapy, in treatment of patients with newly diagnosed and refractory diffuse intrinsic pontine gliomas (DIPG), a disease which remains incurable at present. We sought to identify synergy between ONC201 or second generation impridones (ONC206 and ONC212), and other chemotherapeutics with known promising pre-clinical activity against DMGs. Seven patient derived H3K27M mutant DIPG cell lines (SU-DIPG-IV, SU-DIPG-13, SU-DIPG-25, SUDIPG-27, SU-DIPG-29, SU-DIPG-36, SF8628) were grown in culture and exposed to ONC201, ONC206, or ONC212 either as monotherapy or in combination with other FDA approved chemotherapeutics (histone de-acetylase inhibitors [HDACi], marizomib, etoposide, and temozolomide). Dose dependent response to imipridone therapy was noted in all cell lines, with mean half maximal inhibitory concentration (IC50) of 1.46 µM in ONC201, 0.11 µM in ONC206, and 0.03 µM in ONC212. Synergy was identified via combination studies, with combination indices of Citation Format: Robyn Borsuk, Lanlan Zhou, Yiqun Zhang, Varun V. Prabhu, Joshua E. Allen, Nikos Tapinos, Rishi Lulla, Wafik S. El-Deiry. Response to novel imipridone combination therapies targeting H3K27M mutant diffuse midline glioma (DMG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 635.

INTRODUCTION: Glioblastoma tumors harbor an immunosuppressive microenvironment which is sustained by key players such as TGF-β1/2 and IL-10 cytokines; and mediators of the PD-1/PD-L1/2 pathway. Previously, our lab has shown the role of a new gene, FAT1, in promoting gliomagenesis by supporting the production of pro-inflammatory cytokines (IL-1β, IL-6, etc.) in glioma cells. Here, we show a potent role of FAT1 gene in regulation of anti-inflammatory genes in human glioma tumors, cell lines and primary cultures. Our findings indicate an additional way in which FAT1 might contribute to gliomagenesis. METHODS: q-PCR was used to analyse transcript levels of FAT1 and genes of the TGF-β1/2, IL-10 and PD-L1/2 pathways in fresh-frozen glioblastoma samples relative to normal brain RNA. Gene expression correlation was analysed using Spearman's test and semi-supervised clustering. TCGA expression data was extracted to analyse the correlation of FAT1 with anti-inflammatory genes. Primary tumor cultures and cell lines were transfected with FAT1-specific siRNA to check the effect on expression of anti-inflammatory cytokines, as well as effect on migratory properties of monocytes towards FAT1-knockdown tumor cells (supernatants). RESULTS: Significant positive correlation was found between upregulation of FAT1 and that of anti-inflammatory genes in fresh-frozen glioblastoma samples as well as TCGA datasets. Upon siRNA-mediated knockdown of FAT1 in in-vitro glioma cell cultures, we observed decreased anti-inflammatory genes' expression. We also found increased transmigration of monocytes towards the supernatants from siFAT1-transfected cells. CONCLUSION: FAT1 favors the immunosuppressive milieu in glioblastoma by supporting expression of anti-inflammatory genes. This is an emerging role of the new oncogene FAT1 and crucial to our understanding of the contributions of FAT1 to hallmarks of cancer. FUNDING: SERB Start-Up Grant (DST, India); DHR Young Scientist Scheme (ICMR, India) Citation Format: Khushboo Irshad, Chitrangda Srivastava, Nargis Malik, Yakhlesh Gupta, Vaishali Suri, Swati Mahajan, Deepak Gupta, Ashish Suri, Parthaprasad Chattopadhyay, Subrata Sinha, Kunzang Chosdol. FAT1 and the immunosuppressive milieu in glioblastoma tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3175.

Development of an electrochemical diagnostic test would provide quantitative detection of biomarkers characteristic of clinical serum samples that may facilitate early detection and triage of glioma patients in the clinical setting. Today, 32 individuals will receive a confirmatory diagnosis of a brain tumor in the UK, equivalent to 3% of all patients diagnosed with cancer annually. Implications for patients and families are tragic, brain cancer is responsible for >5,000 mortalities annually in the UK, with 20.1 mean years of life lost to the disease per patient, greater than any other cancer. Presently, ~22% of brain cancer patients receive diagnosis through GP referral, with over a third of patients attending their health practice on five occasions prior to diagnosis. Brain tumor symptoms are diverse and often non-specific in the absence of epileptic seizures, with an extremely poor positive predictive value. Consequently, suspected patients experience significant diagnostic delays, with significant time spent in primary care settings. Heightened symptoms often translates to emergency presentation, where >60% of brain cancer patients currently receive diagnosis, associated with reduced survival compared to non-emergency pathways. Hence, earlier detection of brain cancer patients is a clinical need within primary care settings to prevent significant diagnostic delays and improve patient outcomes. Introduction of a widespread screening program with medical imaging modalities is not economically viable for early detection of brain cancer given high test costs and low incidence in the general population. However, a recent health economic study suggested a blood-based test may prove cost saving to NHS services if employed to triage brain tumor patients for medical imaging in primary and secondary care. Blood is fundamental to physiological function of cells and their perpetual interaction provides a rich source of endogenous molecules that may reflect biochemical events of gliomagenesis. Our current work towards development of a blood-based electrochemical platform for brain cancer focuses on screening of clinical serum samples for a large panel of cytokines from confirmed patients with glioma tumor types. Thereafter, our work demonstrates electrochemical detection of elevated cytokine biomarkers identified within our serum samples. Previously, we demonstrated the potential of this platform technology for early detection of Hodgkin lymphoma, with successful discrimination between cancerous and non-cancerous patient samples, which we now hope to extend to early detection of brain cancer. Implementation of a low-cost, point-of-care triage blood test for brain cancer would have significant clinical and economical benefits, ultimately facilitating the promotion of early detection strategies in primary care to enable timely cancer diagnosis and improve patient outcomes. Citation Format: Christopher Rinaldi, Ashton G. Theakstone, Damion Corrigan, Paul Brennan, Matthew J. Baker. Towards an electrochemical serum-based platform for early diagnosis and stratification of glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2598.

Background: More effective treatments are needed to improve outcomes in HGG and LGG. Activating BRAF mutations occur in ~3% of glioblastomas and 15% of LGGs. BRAF inhibitor dabrafenib + MEK inhibitor trametinib combination is FDA-approved in BRAF V600-positive melanoma, NSCLC, and anaplastic thyroid cancer. Methods: We conducted a nonrandomized, open-label, phase 2 basket study (NCT02034110) of dabrafenib + trametinib in pts with BRAF V600E mutation-positive rare cancers; here we report results for the HGG and LGG cohorts. Adult pts with histologically confirmed recurrent/progressive HGG (Grade III, IV) or LGG (Grade I, II) per WHO 2007 classification received oral dabrafenib, 150 mg twice daily, and oral trametinib, 2 mg once daily, until unacceptable toxicity, disease progression, or death. The primary endpoint was investigator-assessed objective response rate (ORR) using RANO criteria. Secondary endpoints included progression-free survival (PFS), duration of response (DOR), overall survival (OS), and safety; molecular characterization of baseline tumor samples was an exploratory endpoint. Results: As of Sept 14, 2020, 45 pts (23 male) were enrolled in the HGG cohort; 35 discontinued, 6 remained on treatment, 4 were in follow up. The majority had glioblastoma (69%), followed by anaplastic pleomorphic xanthoastrocytoma and anaplastic astrocytoma (each 11%); of pts with known IDH/MGMT status, 3/29 pts had IDH1 mutations and 8/17 had MGMT promoter methylation. Prior therapies included radiotherapy (98%), surgery, and chemotherapy (93% each). Median (range) follow-up was 12.7 (1.1-56.1) months (mo); ORR was 33% (3 CR, 12 PR); median DOR was 36.9 mo (95% CI, 7.4-44.2). Median PFS and OS were 3.8 mo (95% CI, 1.8-9.2) and 17.6 mo (95% CI, 9.5-45.2), respectively. The LGG cohort enrolled 13 pts (4 male); 7 discontinued, 5 remained on treatment, 1 was in follow up. Most common histologies were ganglioglioma (31%), diffuse astrocytoma, and pleomorphic xanthoastrocytoma (each 15%); of pts with known IDH/MGMT status, 1/8 pts had IDH1 mutation and 0/2 had MGMT promoter methylation. Prior therapies included surgery (92%), radiotherapy (62%), and chemotherapy (38%). Median (range) follow-up was 32.2 (0.8-71.8) mo; ORR was 69% (1 CR, 6 PR, 2 MR); median DOR, PFS, and OS were not reached. Overall, 54/58 pts (93%) experienced adverse events (AEs) across cohorts, most commonly (≥30%) fatigue (50%), headache (43%), nausea (34%), and pyrexia (33%); 31 pts (53%) had grade ≥3 AEs, most commonly (≥5%) fatigue, decreased neutrophil count (9% each), headache, and neutropenia (5% each). Next-generation sequencing showed a heterogenous landscape and low tumor mutation burden. Conclusions: Dabrafenib + trametinib demonstrated promising efficacy in pts with BRAF V600E mutation-positive recurrent/refractory HGG and LGG. The safety profile was consistent with the known safety profile for other indications. Citation Format: Vivek Subbiah, Alexander Stein, Martin van den Bent, Antje Wick, Filip Y. de Vos, Nikolas von Bubnoff, Myra E. van Linde, Albert Lai, Gerald W. Prager, Mario Campone, Angelica Fasolo, Jose A. Lopez-Martin, Tae Min Kim, Ralf-Dieter Hofheinz, Jean-Yves Blay, Daniel C. Cho, Anas Gazzah, Damien Pouessel, Jeffrey Yachnin, Aislyn Boran, Paul Burgess, Palanichamy Ilankumaran, Eduard Gasal, Patrick Y. Wen. Dabrafenib plus trametinib in BRAF V600E-mutant high-grade (HGG) and low-grade glioma (LGG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr CT025.

Introduction Tumor Treating Fields (TTFields) therapy is a clinically applied anti-neoplastic treatment modality, approved for the treatment of glioblastoma and malignant pleural mesothelioma. TTFields are low intensity (1-3 V/cm) alternating electric fields within the intermediate frequency range (100-300 kHz), that are applied loco-regionally and non-invasively to the tumor site. Although TTFields therapy has demonstrated improved progression free survival and overall survival, tumor recurrence still occurs in some patients. To evaluate the cellular mechanisms of treatment resistance to TTFields, we analyzed changes in signaling pathway mediators in treated human glioma (U-87 MG) and ovarian (A2780) cancer cell lines. Also, we examined the in vitro efficacy of TTFields in combination with therapies that target molecular candidates that may confer resistance to TTFields. Methods To establish TTFields resistant glioma and ovarian cancer cell lines in vitro, 2 approaches were tested: 1) cyclic repetitions, where TTFields treatment was repeatedly applied for 72 hours with 24 hours breaks between treatment cycles; and 2) continuous, long-duration application of TTFields (1 or 2 weeks, depending on the cancer cell line type) without treatment breaks. Luminex assay followed by Western blot analysis were used to study changes in signaling pathways following TTFields treatment. Finally, we examined the in vitro efficacy of continuous, long-duration application of TTFields in combination with BGT226 (a phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dual inhibitor). Results Application of TTFields using cyclic repetition significantly decreased proliferation of human glioma and ovarian cancer cells, whereas cells treated using continuous, long-duration application of TTFields showed reduced sensitivity to TTFields. Luminex analysis clearly showed activation of the PI3K/mTOR/AKT signaling pathway in response to TTFields treatment. To clarify the importance of this signaling pathway on regulating treatment resistance to TTFields, we examined the effect of TTFields, alone and in combination, with a PI3K/mTOR inhibitor (BGT226) on cancer cell proliferation, apoptosis, and activation of AKT. While long-duration application of TTFields led to Akt activation, TTFields in combination with BGT226 resulted in reduction of p-AKT S473, with a corresponding blockade of proliferation, induction of apoptosis, and decrease in the clonogenic potential. Conclusions We propose here a new mechanism of resistance to prolonged TTFields treatment mediated by the PI3K/mTOR/AKT signaling pathway in glioma and ovarian cancer cells. We demonstrate that combination therapy of TTFields and a targeted PI3K/mTOR dual inhibitor (BGT226) inhibited AKT S473 phosphorylation and sensitized cancer cells to “long-duration” TTFields application. Citation Format: Anat Klein-Goldberg, Tali Voloshin, Efrat Zemer-Tov, Rom Paz, Lilach Koren, Alexandra Volodin, Boris Brant, Moshe Giladi, Uri Weinberg, Yoram Palti. Targeting Akt signaling pathway potentiates the antitumor effect of Tumor Treating Fields (TTFields) in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1382.

Enhanced expression of TERT in gliomas is a result of two hotspot mutations, C228T and C250T, at the promoter region. GA-binding proteins selectively bind at these positions, respectively causing an activation of the promoter and overexpression of TERT. GABP is a multimeric protein consisting of GABPA and GABPB with its isoforms GABPB1, GABPB1-L, GABPB1-S, GABPB2. In this study, we investigated the mRNA expression and association between TERT and GABPA/B isoforms in tumor samples of different glioma grades. The expression was determined by quantitative real-time PCR and the results were statistically analyzed. We present that TERT is mainly expressed in primary glioblastomas. All GA-binding proteins progress through the glioma grades and have the highest expression levels in secondary glioblastomas. In secondary glioblastomas after chemotherapy, GABPB1 and GABPB1-L are lower expressed than without treatment. In high grades, TERT and GABPA, GAPB1, GABPB1-L, GABPB1-S are upregulated compared to low grades. Between primary and secondary glioblastomas with and without chemotherapy, TERT is elevated in the former while GABPB1 is increased in the secondary glioblastomas. GABPA and GABPB1, GABPB1-L and GABPB1-S positive correlate in primary glioblastomas. The present study confirms the upregulation of TERT in primary glioblastomas while all GABP proteins rise with the malignancy of the gliomas. TERT and all GABP proteins seem to play a key role in the gliomagenesis. Citation Format: Marco Timmer, Efthymios Papazacharias, Roland Goldbrunner. Overexpression of the TERT binding proteins GABPA/B in Glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2164.