Patient-derived Glioma Cells Research Articles

The glutamate release inhibitor riluzole increases DNA damage and enhances cytotoxicity in human glioma cells, in vitro and in vivo.

PurposeHigh-grade gliomas are lethal malignancies that cause morbidity and mortality due to local progression rather than metastatic spread. Our group has previously demonstrated that human GRM1 (hGRM1) is ectopically expressed in melanocytes leading to a transformed phenotype. Riluzole, a glutamate release inhibitor, leads to apoptotic cell death via DNA damage. Recent work has demonstrated the pathological significance of the related mGluR3/GRM3 (protein or gene: hGRM3) in gliomas. We evaluated the effect of riluzole on glioma cells.Experimental DesignWestern blot analysis and immunofluorescence was performed to assess for GRM3 expression in commercially available and patient-derived glioma cells and for functional analysis of GRM3 using receptor agonist/antagonists and downstream effectors, ERK and AKT phosphorylation, as the read-out. Glutamate secretion by glioma cells was measured using ELISA. Flank and intracranial mouse xenograft models were used to assess growth delay with the glutamate release inhibitor, riluzole (RIL). Immunofluorescence was used to evaluate 53BP1 or γ-H2AX foci after RIL.ResultsGRM3 was expressed in most tested glioma samples, and strongly expressed in some. Glioma cells were found to secrete glutamate in the extracellular space and to respond to receptor stimulation by activating downstream ERK. This signaling was abrogated by pretreatment with RIL. Treatment with RIL caused an increase in DNA damage markers, and an increase in cellular cytotoxicity in vitro and in vivo.ConclusionsWe have demonstrated that pretreatment with the glutamate-release inhibitor riluzole sensitizes glioma cells to radiation and leads to greater cytotoxicity; these results have clinical implications for patients with glioblastoma.

Pathogenic mutations in neurofibromin identifies a leucine-rich domain regulating glioma cell invasiveness

Glioblastoma (GBM) is the most aggressive tumor of the brain. NF1, a tumor suppressor gene and RAS-GTPase, is one of the highly mutated genes in GBM. Dysregulated NF1 expression promotes cell invasion, proliferation, and tumorigenesis. Loss of NF1 expression in glioblastoma is associated with increased aggressiveness of the tumor. Here, we show that NF1-loss in patient-derived glioma cells using shRNA increases self-renewal, heightens cell invasion, and promotes mesenchymal subtype and epithelial mesenchymal transition-specific gene expression that enhances tumorigenesis. The neurofibromin protein contains at least four major domains, with the GAP-related domain being the most well-studied. In this study, we report that the leucine-rich domain (LRD) of neurofibromin inhibits invasion of human glioblastoma cells without affecting their proliferation. Moreover, under conditions tested, the NF1-LRD fails to hydrolyze Ras-GTP to Ras-GDP, suggesting that its suppressive function is independent of Ras signaling. We further demonstrate that rare variants within the NF1-LRD domain found in a subset of the patients are pathogenic and reduce NF1-LRD’s invasion suppressive function. Taken together, our results show, for the first time, that NF1-LRD inhibits glioma invasion, and provides evidence of a previously unrecognized function of NF1-LRD in glioma biology.

Methylation-dependent Tissue Factor Suppression Contributes to the Reduced Malignancy of IDH1-mutant Gliomas.

Gliomas with isocitrate dehydrogenase 1 mutations (IDH1mut) are less aggressive than IDH1 wild-type (IDH1wt) gliomas and have global genomic hypermethylation. Yet it is unclear how specific hypermethylation events contribute to the IDH1mut phenotype. Previously, we showed that the gene encoding the procoagulant tissue factor (TF), F3, is among the most hypermethylated and downregulated genes in IDH1mut gliomas, correlating with greatly reduced thrombosis in patients with IDH1mut glioma. Because TF also increases the aggressiveness of many cancers, the current study explored the contribution of TF suppression to the reduced malignancy of IDH1mut gliomas.Experimental Design: TF expression was manipulated in patient-derived IDH1mut and IDH1wt glioma cells, followed by evaluation of in vitro and in vivo behavior and analyses of cell signaling pathways. A demethylating agent, decitabine, increased F3 transcription and TF-dependent coagulative activity in IDH1mut cells, but not in IDH1wt cells. TF induction enhanced the proliferation, invasion, and colony formation of IDH1mut cells, and increased the intracranial engraftment of IDH1mut GBM164 from 0% to 100% (P = 0.0001). Conversely, TF knockdown doubled the median survival of mice engrafted with IDH1wt/EGFRvIIIamp GBM6, and caused complete regression of IDH1wt/EGFRamp GBM12 (P = 0.001). In vitro and in vivo effects were linked to activation of receptor tyrosine kinases (RTK) by TF through a Src-dependent intracellular pathway, even when extracellular RTK stimulation was blocked. TF stimulated invasion predominately through upregulation of β-catenin. These data show that TF suppression is a component of IDH1mut glioma behavior, and that it may therefore be an attractive target against IDH1wt gliomas.

Abstract 5871: Efficacy and selectivity of novel dually targeted kinase inhibitors for therapy of adult and pediatric high-grade glioma

Abstract High grade gliomas (HGGs) in adults and children confer very poor prognosis, with median survival rates under two years post diagnosis. Genetic alterations in receptor tyrosine kinases (RTKs) are seen in both adult and pediatric glioblastoma. Amplification of epidermal growth factor receptor (EGFR), as well as mutations in the PI3K/AKT/mTOR pathway, are frequent and can be targeted by kinase inhibitor based strategies. However, clinical trials singly targeting either EGFR or PI3K have had marginal impact on HGG patient outcomes. Given data showing that compensatory kinase activation is responsible for refractory disease, we developed therapeutics that target more than one kinase. Employing a computational modeling approach, we exploited the known binding modes of structurally related ATP binding site inhibitors of EGFR and PI3K to design small molecules that simultaneously and selectively inhibit both kinases. A panel of novel inhibitors has been synthesized, screened for metabolic stability and characterized with respect to kinase inhibition. Six compounds with varying degrees of inhibition for EGFR and PI3K were evaluated in the NCI-60 COMPARE cell line panel, whereupon two molecules, MTX-216 and MTX-241, exhibited strong in vitro activity against a series of human HGG cell lines. We further evaluated cytotoxicity in five human glioblastoma (GBM) lines, some of which bear EGFRvIII, the truncation mutation frequently seen, and in three patient-derived pediatric diffuse intrinsic pontine glioma (DIPG) cells. Treatment with either MTX-216 or MTX-241 resulted in potent growth inhibition relative to targeting of EGFR alone or PI3K alone using gefitinib, lapatinib or alpelisib. Cytotoxic activity against DIPG patient-derived cell lines was observed at lower concentrations compared to adult GBM cell lines, suggesting that dosing could be de-escalated in children, thereby reducing risks of toxicities. Importantly, selectivity of these compounds was demonstrated against malignant cells versus normal human astrocytes. Furthermore, we have identified unique metabolism targeting features of these compounds in GBM and DIPG lines, indicating suppression of both glycolytic pathways and oxidative phosphorylation, which is not seen with clinically relevant EGFR or PI3K inhibitors. Given the documented metabolic dependencies of brain tumor cells on glycolytic pathways, future studies will evaluate whether biomarkers of sensitivity to the dual inhibitors can be identified. Taken together, our findings point toward proof that a single small molecule inhibitor with dual specificity for EGFR and PI3K represents a rational and promising treatment strategy for recurrent adult and pediatric HGG. Despite the knowledge that HGG is associated with deregulated EGFR and PI3K activity, no current treatment exists targeting both of these critical oncogenes with a single molecule approach. Citation Format: Trever R. Carter, Cavan P. Bailey, Yusha Y. Sun, Alexsandra B. Espejo, Christopher E. Whitehead, Judith Leopold, Joya Chandra. Efficacy and selectivity of novel dually targeted kinase inhibitors for therapy of adult and pediatric high-grade glioma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5871.

Abstract 5443: Methylation-dependent suppression of Tissue Factor is a key contributor to the less aggressive phenotype in IDH1 mutant versus IDH1 wild-type gliomas

Abstract Gliomas with mutations in isocitrate dehydrogenase 1 (IDH1mut) have global genomic hypermethylation, are less aggressive than IDH1 wild-type (IDH1wt) gliomas, and generally grow poorly in vitro and in vivo. Yet little data exist that connect specific hypermethylation events to this unique phenotype. We previously reported that the gene encoding Tissue Factor (TF), F3, is among the most hypermethylated and downregulated genes in IDH1mut gliomas relative to IDH1wt gliomas. TF was originally described in blood coagulation, but also can enhance tumor malignancy via protease-activated receptor 2 (PAR2) signaling, though its role in gliomas is not as well understood. In this study, we further explored the significance of TF on malignancy in IDH1wt and IDH1mut gliomas, and showed that receptor tyrosine kinases (RTKs) and β-catenin are downstream effectors of TF-PAR2 signaling. Studies were performed using 6 patient derived glioma cell lines, 3 IDH1wt (GBM6, GBM12, GBM43) and 3 IDH1mut (TB09, BT142, GBM164). DNA methylation was assessed by Illumina Human 850K. TF procoagulant activity (PCA) was determined by FXa generation, using conditioned medium. Markers of malignancy included BrdU incorporation for cell proliferation, transwell invasion, and soft agar colony formation. Nude mice (N≥6/group) were transplanted with glioma cells and monitored for growth using bioluminescence imaging. The TF gene, F3, was significantly hypermethylated in IDH1mut cells compared to IDH1wt cells, with corresponding lower TF protein in IDH1mut cells. Treatment of IDH1mut glioma cells with a demethylating agent, decitabine, increased F3 transcription 5-fold and PCA 19-fold in IDH1mut cells. TF increased the in vivo "take rate" of IDH1mut GBM164 from 0% to 100% (P=0.0001, Fisher's exact test). Conversely, TF knockdown in IDH1wt cells greatly reduced cell proliferation, invasion, colony formation, and extended median survival of engrafted mice by 86% (P=0.001). In both IDH1wt and IDH1mut cells, TF-PAR2 activated β-catenin, ERK1/2, and Akt, and was associated with multiple pathways that increase high-grade behavior. Disrupting TF-PAR2 signaling had the greatest impact on tumor growth in IDH1wt gliomas driven by epidermal growth factor receptor (EGFR), a major receptor tyrosine kinase (RTK) involved in glioma malignancy. TF-PAR2 transactivated EGFR through a Src-dependent intracellular pathway, even when extracellular stimulation of EGFR was blocked, and further enhanced phosphorylation of the constitutively activated EGFRvIII. In IDH1mut cells, TF-PAR2 signaled through another RTK, PDGFR. In contrast, TF-PAR2 stimulated invasion in all glioma cell lines through an RTK-independent, β-catenin-dependent mechanism. These results demonstrate the importance of TF-PAR2 in gliomas, and show that its suppression is a critical component of the IDH1mut phenotype. Citation Format: Dusten J. Unruh, Snezana Mirkov, Seamus Caragher, Jann Sarkaria, Atique Ahmed, C. David James, Craig Horbinski. Methylation-dependent suppression of Tissue Factor is a key contributor to the less aggressive phenotype in IDH1 mutant versus IDH1 wild-type gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5443.

Investigation of the aryl hydrocarbon receptor and the intrinsic tumoral component of the kynurenine pathway of tryptophan metabolism in primary brain tumors.

There is mounting evidence supporting the role of tryptophan metabolism via the kynurenine pathway (KP) in the pathogenesis of primary brain tumors. Under normal physiological conditions, the KP is the major catabolic pathway for the essential amino acid tryptophan. However, in cancer cells, the KP becomes dysregulated, depletes local tryptophan, and contributes to an immunosuppressive tumor microenvironment. We examined the protein expression levels (in 73 gliomas and 48 meningiomas) of the KP rate-limiting enzymes indoleamine 2,3-dioxygenase (IDO) 1, IDO2, and tryptophan 2,3-dioxygenase (TDO2), as well as, the aryl hydrocarbon receptor (AhR), a carcinogenic transcription factor activated by KP metabolites. In addition, we utilized commercially available small-molecules to pharmacologically modulate IDO1, IDO2, TDO2, and AhR in patient-derived glioma and meningioma cell lines (n = 9 each). We observed a positive trend between the grade of the tumor and the average immunohistochemical staining score for IDO1, IDO2, and TDO2, with TDO2 displaying the strongest immunostaining. AhR immunostaining was present in all grades of gliomas and meningiomas, with the greatest staining intensity noted in glioblastomas. Immunocytochemical staining showed a positive trend between nuclear localization of AhR and histologic grade in both gliomas and meningiomas, suggesting increased AhR activation with higher tumor grade. Unlike enzyme inhibition, AhR antagonism markedly diminished patient-derived tumor cell viability, regardless of tumor type or grade, following in vitro drug treatments. Collectively, these results suggest that AhR may offer a novel and robust therapeutic target for a patient population with highly limited treatment options.

Modeling the diffusion of D-2-hydroxyglutarate from IDH1 mutant gliomas in the central nervous system.

Among diffusely infiltrative gliomas in adults, 20%-30% contain a point mutation in isocitrate dehydrogenase 1 (IDH1mut), which increases production of D-2-hydroxyglutarate (D2HG). This is so efficient that D2HG often reaches 30 mM within IDH1mut gliomas. Yet, while up to 100 µM D2HG can be detected in the circulating cerebrospinal fluid of IDH1mut glioma patients, the exposure of nonneoplastic cells within and surrounding an IDH1mut glioma to D2HG is unknown and difficult to measure directly. Conditioned medium from patient-derived wild type IDH1 (IDH1wt) and IDH1mut glioma cells was analyzed for D2HG by liquid chromatography-mass spectrometry (LC-MS). Mathematical models of D2HG release and diffusion around an IDH1mut glioma were independently generated based on fluid dynamics within the brain and on previously reported intratumoral and cerebrospinal D2HG concentrations. LC-MS analysis indicates that patient-derived IDH1mut glioma cells release 3.7-97.0 pg D2HG per cell per week. Extrapolating this to an average-sized tumor (30 mL glioma volume and 1 × 108 cells/mL tumor), the rate of D2HG release by an IDH1mut glioma (SA) is estimated at 3.2-83.0 × 10-12 mol/mL/sec. Mathematical models estimate an SA of 2.9-12.9 × 10-12 mol/mL/sec, within the range of the in vitro LC-MS data. In even the most conservative of these models, the extracellular concentration of D2HG exceeds 3 mM within a 2 cm radius from the center of an IDH1mut glioma. The microenvironment of an IDH1mut glioma is likely being exposed to high concentrations of D2HG, in the low millimolar range. This has implications for understanding how D2HG affects nonneoplastic cells in an IDH1mut glioma.

The role of CXCR3/LRP1 cross-talk in the invasion of primary brain tumors

CXCR3 plays important roles in angiogenesis, inflammation, and cancer. However, the precise mechanism of regulation and activity in tumors is not well known. We focused on CXCR3-A conformation and on the mechanisms controlling its activity and trafficking and investigated the role of CXCR3/LRP1 cross talk in tumor cell invasion. Here we report that agonist stimulation induces an anisotropic response with conformational changes of CXCR3-A along its longitudinal axis. CXCR3-A is internalized via clathrin-coated vesicles and recycled by retrograde trafficking. We demonstrate that CXCR3-A interacts with LRP1. Silencing of LRP1 leads to an increase in the magnitude of ligand-induced conformational change with CXCR3-A focalized at the cell membrane, leading to a sustained receptor activity and an increase in tumor cell migration. This was validated in patient-derived glioma cells and patient samples. Our study defines LRP1 as a regulator of CXCR3, which may have important consequences for tumor biology.

IMMU-32. NON-INFLAMMATORY EFFECT OF DIPG ON TUMOR-ASSOCIATED MICROGLIA

Diffuse intrinsic pontine glioma (DIPG) is a diffusely infiltrating high-grade glioma that has a peak incidence from ages 6-8 and only a 9-12 month median survival. Like adult gliomas, DIPG exhibits a significant expansion of myeloid cells, with microglia/macrophages accounting for ~30% of the tumor cell mass (Caretti & Monje, 2014). In adult glioma, these glioma-associated macrophages have been implicated as supporting angiogenesis and tumor cell invasion, proliferation, and survival (Glass & Synowitz, 2014). However, genomic and epigenomic characterizations of adult and pediatric high-grade gliomas revealed striking differences in the molecular nature of these tumors (Sturm et al., 2014), and traditional chemotherapeutic approaches to adult glioblastoma are not effective in DIPG, suggesting these are unique diseases with distinct pathophysiology. Our data demonstrates that tumor-associated macrophages from primary DIPG samples exhibit gene expression signatures that are associated with pro-tumorigenic functions, but are also significantly less inflammatory than tumor-associated macrophages isolated from adult glioblastoma samples. Moreover, the cytokine secretion profiles of patient-derived DIPG cell cultures are dramatically less inflammatory when compared to patient-derived adult high-grade glioma cell cultures, and are more similar to the profile of human neural precursor cell cultures. This suggests that DIPG is less immunogenic than adult GBM, which may allow the tumor cells to evade detection and infiltrate healthy tissue. We additionally show that DIPG cells express CD200, a ligand that inhibits myeloid cell activation. Knockdown of DIPG expression of CD200 increases primary microglial cell phagocytosis in direct co-culture experiments, and also inhibits DIPG progression in in vivo xenograft models. Thus, CD200 may represent a mechanism by which DIPG evades normal immune responses to tumor growth. By understanding the DIPG-microglial interaction in molecular detail, we will gain insight into the pathophysiology of DIPG that could direct future targeted therapies.

Abstract 1345: Mutant IDH1 and tissue factor in gliomas

Abstract Background: IDH1 mutations (IDH1mut) occur in 20-30% of gliomas, induce DNA hypermethylation, and are associated with a better prognosis than IDH1 wild-type (IDH1wt) gliomas, yet the basis for this remains unclear. Furthermore, venous thromboemboli (VTE) are a serious complication in glioma patients, and the mechanism for this is also unclear. We recently discovered that IDH1mut gliomas are much less likely to develop VTE compared to IDH1wt gliomas. Our data suggests that suppression of Tissue Factor (TF), the primary initiator of coagulation, is a key reason for this. TF also directly enhances tumor malignancy via protease-activated receptor 2 (PAR2). In this study, we further explored the significance of TF on malignancy and thrombosis in IDH1wt and IDH1mut gliomas. Methods: Assays were performed using 6 patient derived glioma cells, 3 IDH1wt (GBM6, GBM12, GBM43) and 3 IDH1mut (TB09, BT142, GBM164). DNA methylation was analyzed by Illumina Human 850K. TF procoagulant activity (PCA) was determined by FXa generation, using preoperative glioma patient arterial plasma, conditioned medium, or cells. In vivo models of cancer induced thrombosis were created via surgical stenosis of the inferior vena cava of mice engrafted with IDH1wt or IDH1mut gliomas. In vitro markers of malignancy were measured by BrdU incorporation for cell proliferation, serum gradient Matrigel-coated transwell inserts for invasion, soft agar colony formation for clonogenicity. Nude mice (N≥6/group) were orthotopically transplanted with IDH1wt glioma cells and monitored for growth using bioluminescence imaging. Results: The TF gene, F3, was significantly hypermethylated in IDH1mut cells compared to IDH1wt cells, and IDH1mut glioma cells had lower levels of TF protein and TF PCA compared to IDH1wt cells. Exogenous treatment with the product of IDH1mut enzyme, D-2-hydroxyglutarate, directly suppressed TF PCA by 38% in IDH1wt cells (P=0.03), as did transgene IDH1mut expression. Mice with IDH1mut glioma xenografts produced fewer and smaller IVC thrombi than mice with IDH1wt xenografts (0.9±0.9 vs 10.3±3.6 mg, P=0.02). Patients whose gliomas contained IDH1mut had lower TF PCA compared to IDH1wt (0.5±0.2 vs 1.2±0.2 pg/mL, P=0.04), and there was a strong link between levels of TF PCA and subsequent development of VTE (P=0.03). TF knockdown greatly reduced cell proliferation, invasion, and colony formation of IDH1wt cells. Likewise, the PAR2 antagonist, GB83, inhibited IDH1wt cell proliferation by 54% (P<0.001) and reduced cell invasion by 25% (P<0.05). TF knockdown also reduced in vivo growth of IDH1wt gliomas by 96% at 28 days post engraftment (P=0.03), and extended median survival of engrafted mice by 86% (P=0.001). Conclusions: IDH1mut mediated suppression of TF expression and PCA may be a critical component of the less thrombogenic, and less malignant, IDH1mut phenotype. Targeting TF-PAR2 signaling may therefore represent a novel therapeutic strategy to reduce IDH1wt glioma malignancy. Citation Format: Dusten Unruh, Snezana Mirkov, Charles D. James, Craig Horbinski. Mutant IDH1 and tissue factor in gliomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1345. doi:10.1158/1538-7445.AM2017-1345

Abstract LB-290: Oncometabolites induce a BRCAness state that can be exploited by PARP inhibitors

Abstract 2-Hydroxyglutarate (2HG) exists as two enantiomers, R-2HG and S-2HG, and both are implicated in tumor progression via their inhibitory effects on α-ketoglutarate (αKG)-dependent dioxygenases. The former is an oncometabolite that is induced by the neomorphic activity conferred by isocitrate dehydrogenase-1 and -2 (IDH1/2) mutations, while the latter is produced under pathologic process such as hypoxia. Recurring IDH1/2 mutations were first identified gliomas and acute myeloid leukemia (AML), and subsequently they were found in multiple other tumor types. Many IDH1/2-mutant tumors are known to be chemo- and radiosensitive, although the mechanisms underlying this enhanced sensitivity have been elusive. Here, we report that IDH1/2 mutations induce a homologous recombination (HR) defect which renders tumor cells exquisitely sensitive to Poly (ADP-Ribose) polymerase (PARP) inhibitors. Remarkably, this “BRCAness” phenotype can be completely reversed by small molecule mutant IDH1/2 inhibitors, and it can be entirely recapitulated by treatment with either 2HG enantiomer in cells with intact IDH1/2. We performed a comprehensive series of studies directly implicate two αKG-dependent dioxygenases, KDM4A and KDM4B, as key mediators of the observed phenotype. In addition, we demonstrate that 2HG-induced HR suppression cannot be explained by mutant IDH1/2-associated alterations in NAD+ levels. We have demonstrated IDH1/2-dependent PARP inhibitor sensitivity in a range of clinically relevant models, including primary patient-derived glioma cells and AML bone marrow cultures in vitro, as well as genetically-matched tumor xenografts in vivo. Finally, we have extended these findings to several structurally related and clinically relevant oncometabolites. We demonstrate profound synthetic lethality with PARP inhibitors in tumors which produce these other oncometabolites, and our data suggest a similar mechanism of action via which HR is suppressed. Small molecule inhibition of oncogenic kinases is a pillar of precision medicine in modern oncology, and this approach has been extrapolated to treat IDH1/2-mutant and other oncometabolite-producing cancers with small molecule inhibitors which block the neomorphic activity of the mutant proteins. The findings present here directly challenge this therapeutic strategy, and they instead provide a novel approach to treat these tumors oncometabolite-producing tumors with DNA repair inhibitors. Furthermore, our results uncover an unexpected link between oncometabolites, altered DNA repair and genetic instability. We previously reported that hypoxia suppresses HR, driving genetic instability and conferring a BRCAness phenotype in hypoxic tumor cells. It is tempting to speculate that the findings reported here provide a novel commonality between hypoxia and IDH1/2 mutations as mediating a “hit-and-run” mechanism for genetic instability and tumor progression through 2HG, but at the same time bestowing a vulnerability to PARP inhibition that can be therapeutically exploited. Based on these findings, we are planning a multi-center Phase II trial testing the efficacy of olaparib for the treatment of recurrent IDH1/2-mutant tumors, and we anticipate this trial will be open for enrollment later this year. Citation Format: Parker Sulkowski, Christopher Corso, Nathaniel Robinson, Susan Scanlon, Karin Purshouse, Hanwen Bai, Yanfeng Liu, Ranjini Sundaram, Denise Hegan, Nathan Fons, Gregory Breuer, Yuanbin Song, Ketu Mishra, Henk De Feyter, Robin de Graaf, Yulia Surovtseva, Maureen Kachman, Stephanie Halene, Murat Gunel, Peter Glazer, Ranjit S. Bindra. Oncometabolites induce a BRCAness state that can be exploited by PARP inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-290. doi:10.1158/1538-7445.AM2017-LB-290

Self-inflicted DNA double-strand breaks sustain tumorigenicity and stemness of cancer cells

DNA double-strand breaks (DSBs) are traditionally associated with cancer through their abilities to cause chromosomal instabilities or gene mutations. Here we report a new class of self-inflicted DNA DSBs that can drive tumor growth irrespective of their effects on genomic stability. We discover a mechanism through which cancer cells cause DSBs in their own genome spontaneously independent of reactive oxygen species or replication stress. In this mechanism, low-level cytochrome c leakage from the mitochondria leads to sublethal activation of apoptotic caspases and nucleases, which causes DNA DSBs. In response to these spontaneous DNA DSBs, ATM, a key factor involved in DNA damage response, is constitutively activated. Activated ATM leads to activation of transcription factors NF-κB and STAT3, known drivers of tumor growth. Moreover, self-inflicted DNA DSB formation and ATM activation are important in sustaining the stemness of patient-derived glioma cells. In human tumor tissues, elevated levels of activated ATM correlate with poor patient survival. Self-inflicted DNA DSBs therefore are functionally important for maintaining the malignancy of cancer cells.

Spatiotemporal genomic architecture informs precision oncology in glioblastoma.

Precision medicine in cancer proposes that genomic characterization of tumors can inform personalized targeted therapies. However, this proposition is complicated by spatial and temporal heterogeneity. Here we study genomic and expression profiles across 127 multisector or longitudinal specimens from 52 individuals with glioblastoma (GBM). Using bulk and single-cell data, we find that samples from the same tumor mass share genomic and expression signatures, whereas geographically separated, multifocal tumors and/or long-term recurrent tumors are seeded from different clones. Chemical screening of patient-derived glioma cells (PDCs) shows that therapeutic response is associated with genetic similarity, and multifocal tumors that are enriched with PIK3CA mutations have a heterogeneous drug-response pattern. We show that targeting truncal events is more efficacious than targeting private events in reducing the tumor burden. In summary, this work demonstrates that evolutionary inference from integrated genomic analysis in multisector biopsies can inform targeted therapeutic interventions for patients with GBM.

Serglycin as a potential biomarker for glioma: association of serglycin expression, extent of mast cell recruitment and glioblastoma progression

Serglycin is an intracellular proteoglycan with a unique ability to adopt highly divergent structures by glycosylation with variable types of glycosaminoglycans (GAGs) when expressed by different cell types. Serglycin is overexpressed in aggressive cancers suggesting its protumorigenic role. In this study, we explored the expression of serglycin in human glioma and its correlation with survival and immune cell infiltration. We demonstrate that serglycin is expressed in glioma and that increased expression predicts poor survival of patients. Analysis of serglycin expression in a large cohort of low- and high-grade human glioma samples reveals that its expression is grade dependent and is positively correlated with mast cell (MC) infiltration. Moreover, serglycin expression in patient-derived glioma cells is significantly increased upon MC co-culture. This is also accompanied by increased expression of CXCL12, CXCL10, as well as markers of cancer progression, including CD44, ZEB1 and vimentin.In conclusion, these findings indicate the importance of infiltrating MCs in glioma by modulating signaling cascades involving serglycin, CD44 and ZEB1. The present investigation reveals serglycin as a potential prognostic marker for glioma and demonstrates an association with the extent of MC recruitment and glioma progression, uncovering potential future therapeutic opportunities for patients.

TMIC-28. HETEROGENEOUS GLIOBLASTOMA CELL RESPONSES TO TNF DEPEND ON MOLECULAR SUBTYPE

Glioblastoma multiforme (GBM), which displays wide variation between patients, has been stratified into subtype classifications that are anchored at two ends by proneural (PN) and mesenchymal (Mes) subtypes. PN tumors are associated with preferential expression of neural stem and progenitor cell markers, and Mes tumors with mesenchymal markers and markers of epithelial-to-mesenchymal transition (EMT). Regions within a tumor may vary in subtype, and for GBM up to one-third of total cells may be microglia and tumor associated macrophages. We examined how GBM molecular subtype and differentiation status influence invasive behaviors. The microglia/macrophages associated with GBM secrete a number of proinflammatory cytokines including tumor necrosis factor (TNF). In vitro, TNF up-regulates expression of the adhesion molecule VCAM-1 (Mahadev et al., PLoS One 9: e95123; 2014). We utilized a cohort of patient-derived primary glioma cell lines characterized by TCGA signature gene expression as PN or Mes (Brown et al., PLoS One 8: e7769; 2013), grown in culture with stem-like or differentiated cell properties (Brown et al., Cancer Res 69: 8886-8893; 2009), in a “dot migration assay” that recapitulates in vivobrain tumor dissemination along blood vessels and associated ECM (Baghdadchi et al., in preparation). We measured by immunofluorescence spatial dependence of tumor cell morphology and physical interactions with other tumor cells, migration, and expression of tumor adhesion molecules and other markers. VCAM-1 expression responses to TNF by proneural (PBT003) and mesenchymal (PBT017) GBM cell lines differed markedly. Specifically, VCAM-1 immunoreactivity was initially low on (PN) PBT003 cells and markedly enhanced by exposure (6 days) to TNF (10 ng/ml). In contrast, VCAM-1 immunoreactivity was initially higher on (Mes) PBT017 cells and decreased on chronic exposure to TNF. While preliminary, these observations suggest that within patient tumors, responses to activated microglia and macrophages may be regionally disparate depending on local molecular subtype

TMOD-14. ESTABLISHMENT AND MOLECULAR CHARACTERIZATION OF PATIENT DERIVED CELL LINES FROM LOW GRADE GLIOMA

Low Grade Gliomas (LGGs), although they are rare in incidence, usually affect younger adults (20-40 years of age) with patient survival varying between 5-15 years. The in vitro culturing of LGG and subsequent mutational profiling would be an invaluable tool for: (1) understanding mechanisms underpinning progression; (2) identifying novel diagnostic, prognostic and predictive biomarkers; and (3) identifying new treatment strategies using drug screening. Our aim is to develop and characterize patient derived LGG in vitro models through phenotypic, genetic and epigenetic analyses. Fresh LGG specimens were collected from the Prince of Wales Private Hospital, Sydney. All tumor samples were dissociated and cultured in serum free media supplemented with EGF and FGF. The minimum time to expand the cells from one passage to the next was at least 3 weeks. Successful cell lines were harvested at different passages. Molecular characterization of the tissue and cell lines were conducted using Sanger sequencing, Western blot, immunocytochemistry, short tandem repeat analyses and methylation arrays. 15 IDH1 mutant LGG cell lines were developed from 34 freshly collected specimens. IDH1 mutation exclusively reverted back to wild type in all cell lines within several passages. Further molecular characterization of LGG associated genes (for example p53, ATRX, TERT) and epigenetic studies through methylation arrays of paired tumor tissue samples and cell lines enabled us to better determine the selective pressures associated with in vitro culturing of patient derived glioma cell lines. We have established and characterized LGG cell lines derived from patient tumor material and provided a unique resource to better understand LGG biology and improve targeted treatment strategies whilst also detailing the extent to which in vitro models of patient derived glioma cell lines mimic their tumor of origin.

Therapeutic Strategy for Targeting Aggressive Malignant Gliomas by Disrupting Their Energy Balance

Although abnormal metabolic regulation is a critical determinant of cancer cell behavior, it is still unclear how an altered balance between ATP production and consumption contributes to malignancy. Here we show that disruption of this energy balance efficiently suppresses aggressive malignant gliomas driven by mammalian target of rapamycin complex 1 (mTORC1) hyperactivation. In a mouse glioma model, mTORC1 hyperactivation induced by conditional Tsc1 deletion increased numbers of glioma-initiating cells (GICs) in vitro and in vivo Metabolic analysis revealed that mTORC1 hyperactivation enhanced mitochondrial biogenesis, as evidenced by elevations in oxygen consumption rate and ATP production. Inhibition of mitochondrial ATP synthetase was more effective in repressing sphere formation by Tsc1-deficient glioma cells than that by Tsc1-competent glioma cells, indicating a crucial function for mitochondrial bioenergetic capacity in GIC expansion. To translate this observation into the development of novel therapeutics targeting malignant gliomas, we screened drug libraries for small molecule compounds showing greater efficacy in inhibiting the proliferation/survival of Tsc1-deficient cells compared with controls. We identified several compounds able to preferentially inhibit mitochondrial activity, dramatically reducing ATP levels and blocking glioma sphere formation. In human patient-derived glioma cells, nigericin, which reportedly suppresses cancer stem cell properties, induced AMPK phosphorylation that was associated with mTORC1 inactivation and induction of autophagy and led to a marked decrease in sphere formation with loss of GIC marker expression. Furthermore, malignant characteristics of human glioma cells were markedly suppressed by nigericin treatment in vivo Thus, targeting mTORC1-driven processes, particularly those involved in maintaining a cancer cell's energy balance, may be an effective therapeutic strategy for glioma patients.

Abstract LB-308: Effects of the IDH1 R132H mutation on redox status and metabolism are cell type dependent but independent from D-2-hydroxyglutarate accumulation

Abstract IDH1 R132H mutations are considered to play a key role in the development of low grade gliomas and therefrom derived secondary glioblastomas (GBM). Wild type IDH1 converts isocitrate to α-ketoglutarate (a-KG) while reducing NADP+. IDH1R132H has a neomorphic enzymatic function using a-KG to generate high amounts of the oncometabolite D-2-hydroxyglutarate (D-2-HG). While the effects of D-2-HG have been subject to intense research, D-2-HG independent effects of IDH1R132H on energy homeostasis and redox status are not well studied. Here we demonstrate that IDH1R132H transduction but not D-2-HG alone leads to significantly decreased Krebs cycle metabolite concentrations and proliferation in U87 and the primary GBM cell line HT7606 as well as in immortalized astrocytes SVGp12. Furthermore, IDH1R132H mutation, but not D-2-HG treatment, resulted in a significant drop in NADPH levels in tumor cells (U87 and HT7606), whereas immortalized astrocytes retained normal NADPH levels. Since NAPDH levels can be restored via the reaction of the NAD-kinase, we analyzed NAD levels and enzymes involved in NAD-synthesis in our cell lines. Indeed we found a significant drop of NAD levels and in the activity of the NAD-dependent enzyme sirtuin in IDH1R132H mutant U87 and HT7606 but not in NADPH stable SVGp12-IDH1R132H. Interestingly, there were marked differences in expression of NAD-synthesis enzymes between the different cell-lines. In particular, NAMPT-levels were much higher in U87 and HT7606 then in astrocytes (SVGp12) and significantly decreased in U87-IDH1R132H and HT7606-IDH1R132H. Importantly, we also found decreased levels of NAMPT in primary tumor tissues and patient derived glioma cell lines with IDH1 R132H compared to wild type gliomas. Altogether our results for the first time show that the IDH1 mutation directly affects energy homeostasis and redox status in a cell-type dependent manner. We hypothesize that this leads to a drop in NADPH and NAD-levels during malignant progression, resulting in a disadvantage for proliferating tumor cells. This is in line with the favorable prognosis and good response to chemo- and radiation therapy clinically observed in IDH-mutated gliomas. Our findings suggest that the impaired metabolism in IDH1-mutant tumors might be a promising target for future therapies. Citation Format: Evelin Schrock, Julia Biedermann, Khalil Abou-El-Ardat, Matthias Lehmann, Marina Conde, Mirko Peitzsch, Susan Richter, Ralf Wiedemuth, Matthias Meinhardt, William P. J. Leenders, Christel Herold-Mende, Graeme Eisenhofer, Simone P. Niclou, Leoni Kunz-Schughart, Achim Temme, Barbara Klink. Effects of the IDH1 R132H mutation on redox status and metabolism are cell type dependent but independent from D-2-hydroxyglutarate accumulation. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-308.

Mechanical confinement triggers glioma linear migration dependent on formin FHOD3.

Glioblastomas are extremely aggressive brain tumors with highly invasive properties. Brain linear tracks such as blood vessel walls constitute their main invasive routes. Here we analyze rat C6 and patient-derived glioma cell motility in vitro using micropatterned linear tracks to mimic blood vessels. On laminin-coated tracks (3-10 μm), these cells used an efficient saltatory mode of migration similar to their in vivo migration. This saltatory migration was also observed on larger tracks (50-400 μm in width) at high cell densities. In these cases, the mechanical constraints imposed by neighboring cells triggered this efficient mode of migration, resulting in the formation of remarkable antiparallel streams of cells along the tracks. This motility involved microtubule-dependent polarization, contractile actin bundles and dynamic paxillin-containing adhesions in the leading process and in the tail. Glioma linear migration was dramatically reduced by inhibiting formins but, surprisingly, accelerated by inhibiting Arp2/3. Protein expression and phenotypic analysis indicated that the formin FHOD3 played a role in this motility but not mDia1 or mDia2. We propose that glioma migration under confinement on laminin relies on formins, including FHOD3, but not Arp2/3 and that the low level of adhesion allows rapid antiparallel migration.

BKM-120 (Buparlisib): A Phosphatidyl-Inositol-3 Kinase Inhibitor with Anti-Invasive Properties in Glioblastoma.

Glioblastoma is an aggressive, invasive tumor of the central nervous system (CNS). There is a widely acknowledged need for anti-invasive therapeutics to limit glioblastoma invasion. BKM-120 is a CNS-penetrant pan-class I phosphatidyl-inositol-3 kinase (PI3K) inhibitor in clinical trials for solid tumors, including glioblastoma. We observed that BKM-120 has potent anti-invasive effects in glioblastoma cell lines and patient-derived glioma cells in vitro. These anti-migratory effects were clearly distinguishable from cytostatic and cytotoxic effects at higher drug concentrations and longer durations of drug exposure. The effects were reversible and accompanied by changes in cell morphology and pronounced reduction in both cell/cell and cell/substrate adhesion. In vivo studies showed that a short period of treatment with BKM-120 slowed tumor spread in an intracranial xenografts. GDC-0941, a similar potent and selective PI3K inhibitor, only caused a moderate reduction in glioblastoma cell migration. The effects of BKM-120 and GDC-0941 were indistinguishable by in vitro kinase selectivity screening and phospho-protein arrays. BKM-120 reduced the numbers of focal adhesions and the velocity of microtubule treadmilling compared with GDC-0941, suggesting that mechanisms in addition to PI3K inhibition contribute to the anti-invasive effects of BKM-120. Our data suggest the CNS-penetrant PI3K inhibitor BKM-120 may have anti-invasive properties in glioblastoma.