High-grade Glioma Cell Research Articles

Development and Optimization of Irinotecan-Loaded PCL Nanoparticles and Their Cytotoxicity against Primary High-Grade Glioma Cells.

Background: High-grade gliomas (HGGs) are highly malignant tumors with a poor survival rate. The inability of free drugs to cross the blood–brain barrier and their off-target accumulation result in dose-limiting side effects. This study aimed at enhancing the encapsulation efficiency (EE) of irinotecan hydrochloride trihydrate (IRH) within polycaprolactone (PCL) nanoparticles with optimized size and charge. Materials and Methods: IRH-loaded PCL nanoparticles were formulated using either the single emulsion (O/W, W/O and O/O) or double emulsion (W/O/O and W/O/W) solvent evaporation techniques. The nanoparticles were characterized for their size, zeta potential and EE, with the optimized nanoparticles being characterized for their drug release and cytotoxicity. Results: The amorphization of PCL and the addition of electrolytes to the aqueous phases of the W/O/W emulsion produced spherical nanoparticles with a mean diameter of 202.1 ± 2.0 nm and an EE of 65.0%. The IRH-loaded nanoparticles exhibited zero-order release and were cytotoxic against primary HGG cells. Conclusion: The amorphization of PCL improves its EE of hydrophilic drugs, while the addition of electrolytes to the aqueous phases of the W/O/W emulsion enhances their EE further. IRH-loaded PCL nanoparticles have the potential to deliver cytotoxic levels of IRH over a sustained period of time, enhancing the cell death of HGGs.

The Tumor Suppressor MTUS1/ATIP1 Modulates Tumor Promotion in Glioma: Association with Epigenetics and DNA Repair.

Simple SummaryDespite multidisciplinary treatments, survival remains poor in glioma patients. Although novel therapeutic approaches are being explored, no outstanding effects on the survival have been achieved so far, which substantiates the need to develop new therapeutic strategies. To understand the mechanisms responsible for its high malignancy and obligatory recurrence, we examined the impact of MTUS1, a tumor-suppressor gene (TSG), coding for ATIP1, in glioma malignancy as well as how its expression might influence glioma therapy. We confirmed that in glioma cells, elevated ATIP1 expression damps tumor progression by mitigating proliferation and motility. Additionally, MTUS1/ATIP1 can be used as a biological marker to predict therapy outcomes. In glioma cell lines, glioma sphere cultures (GSC), high-grade glioma (HGG) and especially in glioma recurrence, MTUS1/ATIP1 expression is downregulated, probably by promoter hypermethylation. However, in GBM, high ATIP1 expression might interfere with radiation-therapy since elevated expression of MTUS1/ATIP1 drives double-strand break (DSB) DNA repair. Glioblastoma (GBM) is a highly aggressive brain tumor. Resistance mechanisms in GBM present an array of challenges to understand its biology and to develop novel therapeutic strategies. We investigated the role of a TSG, MTUS1/ATIP1 in glioma. Glioma specimen, cells and low passage GBM sphere cultures (GSC) were analyzed for MTUS1/ATIP1 expression at the RNA and protein level. Methylation analyses were done by bisulfite sequencing (BSS). The consequence of chemotherapy and irradiation on ATIP1 expression and the influence of different cellular ATIP1 levels on survival was examined in vitro and in vivo. MTUS1/ATIP1 was downregulated in high-grade glioma (HGG), GSC and GBM cells and hypermethylation at the ATIP1 promoter region seems to be at least partially responsible for this downregulation. ATIP1 overexpression significantly reduced glioma progression by mitigating cell motility, proliferation and facilitate cell death. In glioma-bearing mice, elevated MTUS1/ATIP1 expression prolonged their survival. Chemotherapy, as well as irradiation, recovered ATIP1 expression both in vitro and in vivo. Surprisingly, ATIP1 overexpression increased irradiation-induced DNA-damage repair, resulting in radio-resistance. Our findings indicate that MTUS1/ATIP1 serves as TSG-regulating gliomagenesis, progression and therapy resistance. In HGG, higher MTUS1/ATIP1 expression might interfere with tumor irradiation therapy.

Inhibition of Invasion by Polyphenols from Citrus Fruit and Berries in Human Malignant Glioma Cells In Vitro

The outlook for patients with high grade glioma (HGG) remains dismal. Hence, attention has focused on numerous innovative treatments. Our group has proposed a strategy on the use of a combination of polyphenols, as anti-invasive agents for the management of these neoplasms. The aim of this study was to evaluate the in vitro effects of citrus flavonoids (tangeretin, nobiletin, naringin and limonin) and berry extracts (chokeberry, elderberry and bilberry) on selected mediators of invasion in 2 HGG cell cultures. The IC50 values could only be determined for tangeretin and chokeberry extract. The rest were non-functional in this context. Immunocytochemistry and flow cytometry results showed that chokeberry extract was most effective in down-regulating the expression of CD44. Similarly, RT-PCR data supported its ability to reduce gene expression of MMP-14 and EGFR. 2D invasion assays confirmed that inhibition is greater with chokeberry extract. Both polyphenols have anti-invasive potential but chokeberry extract is a stronger agent for glioma management.

Targeting glioblastoma using a novel peptide specific to a deglycosylated isoform of brevican.

Glioblastoma multiforme (GBM) is the most common and deadliest form of brain tumor and remains amongst the most difficult cancers to treat. Brevican (Bcan), a central nervous system (CNS)-specific extracellular matrix protein, is upregulated in high-grade glioma cells, including GBM. A Bcan isoform lacking most glycosylation, dg-Bcan, is found only in GBM tissues. Here, dg-Bcan is explored as a molecular target for GBM. In this study, we screened a d-peptide library to identify a small 8-amino acid dg-Bcan-Targeting Peptide (BTP) candidate, called BTP-7 that binds dg-Bcan with high affinity and specificity. BTP-7 is preferentially internalized by dg-Bcan-expressing patient-derived GBM cells. To demonstrate GBM targeting, we radiolabeled BTP-7 with 18F, a radioisotope of fluorine, and found increased radiotracer accumulation in intracranial GBM established in mice using positron emission tomography (PET) imaging. dg-Bcan is an attractive molecular target for GBM, and BTP-7 represents a promising lead candidate for further development into novel imaging agents and targeted therapeutics.

Inhibition of microglial EZH2 leads to anti-tumoral effects in pediatric diffuse midline gliomas.

BackgroundDiffuse intrinsic pontine gliomas (DIPG), within diffuse midline gliomas are aggressive pediatric brain tumors characterized by histone H3-K27M mutation. Small-molecule inhibitors for the EZH2-H3K27 histone methyltransferase have shown promise in preclinical animal models of DIPG, despite having little effect on DIPG cells in vitro. Therefore, we hypothesized that the effect of EZH2 inhibition could be mediated through targeting of this histone modifying enzyme in tumor-associated microglia.MethodsPrimary DIPG tissues, and cocultures between microglia and patient-derived DIPG or -pediatric high-grade glioma (pHGG) cell lines, were used to establish the H3-K27M status of each cell type. Antisense RNA strategies were used to target EZH2 gene expression in both microglia and glioma cells. Microglia anti-tumoral properties were assessed by gene expression profile, tumor cell invasion capacity, microglial phagocytic activity, and associated tumor cell death.ResultsIn primary DIPG tissues, microglia do not carry the H3-K27M mutation, otherwise characteristic of the cancer cells. Activation of a microglial tumor-supportive phenotype by pHGG, independently of their H3-K27M status, is associated with a transient H3K27me3 downregulation. Repression of EZH2 in DIPG cells has no impact on tumor cell survival or their ability to activate microglia. However, repression of EZH2 in microglia induces an anti-tumor phenotype resulting in decreased cancer cell invasion capability, increased microglial phagocytosis, and tumor-related cell death.ConclusionsThese results indicate that microglia, beyond the tumor cells, contribute to the observed response of DIPG to EZH2 inhibition. Results highlight the potential importance of microglia as a new therapeutic avenue in DIPG.

Multicellular "hotspots" harbor high-grade potential in lower-grade gliomas.

BackgroundLower-grade gliomas may be indolent for many years before developing malignant behavior. The mechanisms underlying malignant progression remain unclear.MethodsWe collected blocks of live human brain tissue donated by people undergoing glioma resection. The tissue blocks extended through the peritumoral cortex and into the glioma. The living human brain tissue was cut into ex vivo brain slices and bathed in 5-aminolevulinic acid (5-ALA). High-grade glioma cells avidly take up 5-ALA and accumulate high levels of the fluorescent metabolite, Protoporphyrin IX (PpIX). We exploited the PpIX fluorescence emitted by higher-grade glioma cells to investigate the earliest stages of malignant progression in lower-grade gliomas.ResultsWe found sparsely distributed “hot-spots” of PpIX-positive cells in living lower-grade glioma tissue. Glioma cells and endothelial cells formed part of the PpIX hotspots. Glioma cells in PpIX hotspots were IDH1 mutant and expressed nestin suggesting they had acquired stem-like properties. Spatial analysis with 5-ALA-conjugated quantum dots indicated that these glioma cells replicated adjacent to blood vessels. PpIX hotspots were formed in the absence of angiogenesis.ConclusionOur data show that PpIX hotspots represent microdomains of cells with high-grade potential within lower-grade gliomas and identify locations where malignant progression could start.

Terminally sialylated and fucosylated complex N-glycans are involved in the malignant behavior of high-grade glioma.

Gliomas are the most common intracranial primary tumors, for which very few therapeutic options are available. The most malignant subtype is the glioblastoma, a disease associated with a 5-year survival rate lower than 5%. Given that research in glycobiology continues highlighting the role of glycans in tumor cell biology, it offers an interesting niche for the search of new therapeutic targets. In this study, we characterized aberrant glycosylation and its impact on cell biology over a broad panel of high- and low-grade glioma cell lines. Results show high expression of terminal Lewis glycans, mainly SLex, and overexpression of sialyl- and fucosyltransferases involved in their biosynthesis in high-grade glioma cell lines. Moreover, we report an association of complex multi-antennary N-glycans presenting β1,6-GlcNAc branches with the high-grade glioma cells, which also overexpressed the gene responsible for these assemblies, MGAT5. In addition, downmodulation of N-glycosylation by treatment with the inhibitors Tunicamycin/Swainsonine or MGAT5 silencing decreased SLex expression, adhesion and migration in high-grade glioma cells. In contrast, no significant changes in these cell capacities were observed in low-grade glioma after treatment with the N-glycosylation inhibitors. Furthermore, inhibition of histone deacetylases by Trichostatin A provoked an increase in the expression of SLex and its biosynthetic related glycosyltransferases in low-grade glioma cells. Our results describe that aggressive glioma cells show high expression of Lewis glycans anchored to complex multi-antennary N-glycans. This glycophenotype plays a key role in malignant cell behavior and is regulated by histone acetylation dependent mechanisms.

DIPG-51. ACVR1 MUTATIONS PROMOTE TUMOR GROWTH IN MODELS OF DIFFUSE INTRINSIC PONTINE GLIOMA

Mutations in the gene encoding activin A receptor type 1 (ACVR1) are found in approximately 25% of diffuse intrinsic pontine gliomas (DIPGs), a pediatric glioma with 2-year survival rate of less than 10%. ACVR1mutations frequently coincide with activating PIK3CA or PIK3R1 mutations, indicating a potential cooperative effect of BMP and PI3K signaling in gliomagenesis. We used genetically engineered mice with inducible knock-in of Acvr1R206H or Pik3caE545K alleles, such that cre-mediated recombination resulted in expression of the gain of function mutated genes from their endogenous promoters at physiological levels. Cre-mediated deletion in GFAP-CreER;Pik3caE545K/+;p53cKO mice (Pik3ca;p53) mediated Trp53 deletion and expression of Pik3caE545K in glial progenitors, and spontaneously induced high-grade glioma (HGG) in mice with complete penetrance. Heterozygous knock-in of the Acvr1R206H allele accelerated tumorigenesis and impaired survival in Pik3ca;p53 mice (Acvr1;Pik3ca;p53). Transcriptomic analysis of Acvr1;Pik3ca;p53 tumors compared to Pik3ca;p53 littermate controls, as in patient-derived tumors, revealed broad molecular signatures associated with cell fate commitment and chromosome maintenance. Pharmacologic inhibition of ACVR1 was sufficient to impair growth in human patient-derived DIPG cell lines. Together, our studies show that ACVR1 activation promotes tumor growth in spontaneous mouse HGG and patient-derived DIPG cells, suggesting that ACVR1 inhibition may produce a clinically significant therapeutic effect in DIPG.

HGG-27. ANTI-CANCER POTENTIAL OF ARGINASE FOR HIGH-GRADE GLIOMA IN VITRO & IN-VIVO

BACKGROUNDHigh-grade glioma is currently incurable. It was reported that glioma may be auxotrophic to arginine due to the lack of urea cycle genes expressions, suggesting arginase may be a potential agent for high grade glioma. AIM: We investigated the efficacy of pegylated arginase I (pegArg-I) or in combination with other anti-cancer drugs for high-grade glioma in vitro and in vivo.METHODS4 high-grade glioma cell lines (U87, U373, U138, D54) were treated with pegArg-I in vitro. The molecular mechanism of pegArg-I-induced cytotoxicity was tested in U87. The ultra-morphological changes of pegArg-I-treated U87 was investigated by both scanning and transmission electron microscopy. Orthotopic glioma xenograft model with luciferase-transfected U87 cell line was tested for anti-cancer efficacy of peg-Arg I in vivo.RESULTSWe showed that pegArg-I induced significant cell death in all 4 cell lines in vitro. Temozolomide, difluoromethyornithine and chloroquine (CQ) were then tested together with pegArg-I in U87 in vitro. We found that only CQ showed additive effect with pegArg-I against glioma in vitro. Such additive cytotoxic effect may be associated with enhanced autophagy and necrosis as shown in transmission electron microscopy and autophagy markers’ expression by Western blotting. PegArg-I prolonged the survival of glioma mice, suggesting its possible anti-glioma efficacy. However, CQ+pegArg-I didn’t show further significant anti-cancer efficacy in vivo.CONCLUSIONPegArg-I may be useful in slowing the progression of glioma, but additional drug candidate which works synergistically with pegArg-I remains to be explored.

MBRS-31. COMBINING IRRADIATION AND ANTI-CD47 TO ENHANCE THE TREATMENT OF GROUP 3 MEDULLOBLASTOMA

Medulloblastoma (MB) is the most common malignant primary pediatric brain tumor. The Group 3 molecular subgroup of Medulloblastoma (Group 3 MB) is the deadliest with only 30% long term survival. Irradiation for Group 3 Medulloblastoma is required for long term survival of children. Methods to enhance the effect of irradiation against Group 3 MB are an active area of investigation. Immunotherapy using the anti-CD47 treatment has shown promise in treating Group 3 MB. We recently demonstrated that irradiation significantly enhanced anti-CD47-mediated phagocytosis of high-grade glioma cells in vitro. Furthermore, mice engrafted with human high-grade glioma that received anti-CD47 combined with irradiation showed a significant increase in the survival rate and a significant decrease in tumor growth than those that received a single treatment. We have now extended these studies to demonstrate the enhancement of anti-CD47-dependent phagocytosis of human Group 3 MB with irradiation. We also analyzed normal human neural stem cells exposed to the same treatments to assess for the potential toxicity that uniquely exists with this treatment combination.

Identification of the immune gene expression signature associated with recurrence of high-grade gliomas.

High-grade gliomas (HGGs), the most common and aggressive primary brain tumors in adults, inevitably recur due to incomplete surgery or resistance to therapy. Intratumoral genomic and cellular heterogeneity of HGGs contributes to therapeutic resistance, recurrence, and poor clinical outcomes. Transcriptomic profiles of HGGs at recurrence have not been investigated in detail. Using targeted sequencing of cancer-related genes and transcriptomics, we identified single nucleotide variations, small insertions and deletions, copy number aberrations (CNAs), as well as gene expression changes and pathway deregulation in 16 pairs of primary and recurrent HGGs. Most of the somatic mutations identified in primary HGGs were not detected after relapse, suggesting a subclone substitution during the tumor progression. We found a novel frameshift insertion in the ZNF384 gene which may contribute to extracellular matrix remodeling. An inverse correlation of focal CNAs in EGFR and PTEN genes was detected. Transcriptomic analysis revealed downregulation of genes involved in messenger RNA splicing, cell cycle, and DNA repair, while genes related to interferon signaling and phosphatidylinositol (PI) metabolism are upregulated in secondary HGGs when compared to primary HGGs. In silico analysis of the tumor microenvironment identified M2 macrophages and immature dendritic cells as enriched in recurrent HGGs, suggesting a prominent immunosuppressive signature. Accumulation of those cells in recurrent HGGs was validated by immunostaining. Our findings point to a substantial transcriptomic deregulation and a pronounced infiltration of immature dendritic cells in recurrent HGG, which may impact the effectiveness of frontline immunotherapies in the GBM management. KEY MESSAGES: Most of the somatic mutations identified in primary HGGs were not detected after relapse. Focal CNAs in EGFR and PTEN genes are inversely correlated in primary and recurrent HGGs. Transcriptomic changes and distinct immune-related signatures characterize HGG recurrence. Recurrent HGGs are characterized by a prominent infiltration of immature dendritic and M2 macrophages.

IMMU-03. EFFECT OF COMBINING IRRADIATION AND ANTI-CD47 TREATMENT ON THE PHAGOCYTOSIS OF NORMAL BRAIN CELLS

Abstract Immunotherapy using CD47 blockade has shown promise in treating adult and pediatric malignant primary central nervous system tumors. We recently demonstrated that irradiation significantly enhanced anti-CD47-dependent phagocytosis of high-grade glioma cells in vitro. Furthermore, mice engrafted with high-grade human glioma that received anti-CD47 combined with irradiation showed a significant increase in the survival rate and a significant decrease in tumor growth than those that received a single treatment. However, the use of irradiation may result in potential toxicity to normal CNS cells that are not susceptible to macrophage phagocytosis in anti-CD47 monotherapy. We have now extended these studies to test the effect of combining anti-CD47 and irradiation on macrophage-mediated phagocytosis of central nervous system cells. We analyzed phagocytosis of normal human neural stem cells exposed to different irradiation doses in combination with anti-CD47 treatment to assess for the potential toxicity that uniquely exists with this treatment combination.

Abstract PO-102: A novel disseminated tumor cell identified in myriad cancer harbors tumor initiating properties

Abstract The identification of a disseminated tumor cell generated by heterotypic cell fusion, a circulating hybrid cell (CHCs) is a biologically relevant analyte of disease across myriad of cancers. Detected at elevated levels in peripheral blood, this novel population can be leveraged to understand mechanisms driving metastatic progression and predict metastatic spread. Our long term goal is to leverage evolving knowledge of mechanisms of metastatic progression to identify biologically relevant tumor markers for clinical disease assessment strategies. We therefore detected and delineated the role of CHCs as a measure of disease across a multitude of solid tumors. Human blood specimens were collected and analyzed with IRB approved protocols. Peripheral blood was obtained from patients with cancer in 14 different organ sites: ampullary adenocarcinoma, breast adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, esophageal cancer, high grade glioma (pediatric & adult), head and neck squamous cell carcinoma, pancreatic ductal adenocarcinoma, pancreatic neuroendocrine tumor, prostate adenocarcinoma, rectal adenocarcinoma and uveal melanoma and healthy subjects. Peripheral blood mononuclear cells were isolated and prepared for flow cytometry or immunofluorescence microscopy analyses with antibodies to CD45 and panCytokeratin, EpCAM, NKI Beteb, chromogranin A (CHGA)/synaptophysin (SYP) or glial fibrillary acid protein (GFAP). Discrete cellular populations including CHCs and circulating tumor cells (CTCs) were identified by protein expression. Cyclic immunofluorescence (cycIF) techniques were performed on a subset of specimens, for indepth interrogation of phenotypic heterogeneity in both the tumor and among CHCs. CHCs, defined by co-expression of CD45+ and a tumor associated protein were detected in all subjects across all disease sites at statistically significantly higher numbers than in controls samples using both detection paradigms. Furthermore, higher levels of CHCs compared with CTCs (that were CD45-) across disease sites. CycIF analysis of tumor tissue and dissemnated tumor cells from a triple negative breast cancer subject revealed a heterogeneous population of CHCs as well as complex tumor ecosystem. The phenotypic diversity of CHCs included CD44 and androgen receptor (AR) expression, which aligned with those detected in the proliferative tumor compartment. AR expression in triple negative breast cancer is known to correlate with treatment resistance and indeed, our subject rapidly succumb to disease. Taken together, these data suggest that tumors disseminate a heterogeneous population of CHCs that reflect the overall phenotype of viable tumor, supporting the value of phenotypically profiling CHCs for liquid biopsy to anticipate disease evolution and treatment resistance. This study established an isolation and detection platform for measuring CHCs in myriad of cancers and methods of phenotypic profiling CHC heterogeneity to identify discrete subpopulations. Citation Format: Matthew S. Dietz, Thomas Sutton, Brett Walker, Young Hwan Chang, Koei Chin, Melissa H. Wong. A novel disseminated tumor cell identified in myriad cancer harbors tumor initiating properties [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-102.

Everolimus improves the efficacy of dasatinib in PDGFRα-driven glioma.

Pediatric and adult high-grade gliomas (HGGs) frequently harbor PDGFRA alterations. We hypothesized that cotreatment with everolimus may improve the efficacy of dasatinib in PDGFRα-driven glioma through combinatorial synergism and increased tumor accumulation of dasatinib. We performed dose-response, synergism, P-glycoprotein inhibition, and pharmacokinetic studies in in vitro and in vivo human and mouse models of HGG. Six patients with recurrent PDGFRα-driven glioma were treated with dasatinib and everolimus. We found that dasatinib effectively inhibited the proliferation of mouse and human primary HGG cells with a variety of PDGFRA alterations. Dasatinib exhibited synergy with everolimus in the treatment of HGG cells at low nanomolar concentrations of both agents, with a reduction in mTOR signaling that persisted after dasatinib treatment alone. Prolonged exposure to everolimus significantly improved the CNS retention of dasatinib and extended the survival of PPK tumor-bearing mice (mutant TP53, mutant PDGFRA, H3K27M). Six pediatric patients with glioma tolerated this combination without significant adverse events, and 4 patients with recurrent disease (n = 4) had a median overall survival of 8.5 months. Our results show that the efficacy of dasatinib treatment of PDGFRα-driven HGG was enhanced with everolimus and suggest a promising route for improving targeted therapy for this patient population.

Quantitative analysis for the differences in vasculogenic activity and sensitivity to angiogenic stimulants between human glioma cells and normal endothelial cells

Neovascularization is a histological feature of glioma, especially of glioblastoma (GBM), being associated with tumor invasiveness and poor prognosis. However, current anti-angiogenic therapies targeting vascular endothelial cells (ECs), has exhibited poor efficacy in some GBM cases. This may be at least partially attributed to the potential of glioblastoma cells to construct blood supply chain via vasculogenic mimicry or endothelial differentiation. This study aims to explore differences in vasculogenic activity and sensitivity to angiogenic stimulants between normal human ECs and glioma cells of different grades. We found that grade IV U87 GBM cells showed highly inducible vasculogenic activity either in the orthotopic xenograft model or under in vitro angiogenic stimulants as compared with grade II CHG5 glioma cells. The hypoxia mimetic more strongly induced in vitro vasculogenic capacity and endothelial marker expression of U87 GBM cells than the stimulation with multiple proangiogenic growth factors (vascular endothelial growth factor, basic fibroblast growth factor and epidermal growth factor). In contrast, proangiogenic effect of hypoxia on human umbilical vein endothelial cells (HUVECs) was weaker than on U87 GBM cells. In addition, it was also observed that the in vitro vasculogenic process of U87 cells started later but lasted longer than that of HUVECs. These results demonstrate that when compared with normal ECs, high-grade glioma cells basically possess weaker vasculogenic activity, but exhibit higher sensitivity and longer-lasting response to angiogenic stimulants, especially to hypoxia. This may be helpful to develop novel anti-angiogenic strategies targeting both vascular ECs and vasculogenic glioma cells.

β-arrestin 1 transfection induced cell death in high grade glioma in vitro

ABSTRACT The best known functions of β-arrestins (β-arr) are to regulate G protein-coupled receptors (GPCR) signaling through receptor desensitization and internalization. Many reports also suggest that β-arrs play important role in immune regulation and inflammatory responses, under physiological and pathological conditions. Recent studies have shown that β-arr 1 silencing halts proliferation and increases temozolomide (TMZ) response in glioblastoma (GBM) cells. The focus of this paper is to analyze the role of β-arr 1 overexpression in the 18 high grade glioma (HGG) cell line in terms of viability and their response to TMZ treatment. For this reason, the cell line was transfected with β-arr 1 and the effect was analyzed after 24 h, 48 h and 72 h in terms of proliferation and treatment response. We observed that β-arr 1 overexpression induced a time and dose dependant inhibition in the HGG cells. Unexpectedly, β-arr transfection resulted in a very mild increase in TMZ toxicity after 24 h, becoming non-statistically significant at 72 h. In conclusion, we showed that β-arr 1 overexpression inhibits cell proliferation in the 18 cell line but only has a very modest effect on treatment response with the alkylating agent TMZ.

Abstract A53: Synergistic interaction of HDACi and PLK1i in Group 3 MYC-amplified medulloblastoma

Abstract Medulloblastoma (MB) is one of the common malignant brain tumors in children. Patients with MYC-amplified Group 3 MBs exhibit particularly poor survival rates even after intensive therapy. Surviving patients often suffer from long-term side effects. This calls for new therapeutic strategies, such as targeted therapy options. The sensitivity of MYC-amplified MBs to class I histone deacetylase (HDAC) inhibition was previously shown. In order to elucidate potential combination partners, we have identified PLK1 as one of the top regulated genes following treatment of MYC-amplified Group 3 MB cells with class I HDACi entinostat. Our aim here is to investigate possible combination treatments with entinostat and several PLK1i (volasertib, GSK461364, onvansertib). The cell metabolic activity was evaluated using MYC-amplified and nonamplified MB, high-grade glioma (HGG) and neuroblastoma (NB) cell lines after single and combination treatments. The interaction effect was determined by combination index (CI) (Chou-Talalay CI calculation method). Results were validated assessing cell viability, cell cycle profile, and caspase activity upon treatment with single agents or combination. On-target activity was examined using immunoblotting for pTCTP and H3K27ac. We have confirmed our findings in an inducible MYC cell line. The gene expression profile was analyzed in HD-MB03 cell line after entinostat, volasertib, or combination treatment. We demonstrate that the MYC target gene PLK1 is significantly downregulated upon HDACi treatment. Based on this, we hypothesized that inhibition of both class I HDACs and PLK1 could have synergistic effects. MYC-amplified cell lines were more sensitive than nonamplified cell lines to treatment with all PLK1i investigated, showing 3 to 10 times lower IC50. PLK1i and entinostat interacted synergistically (CI below 0.9) at lower concentrations in MYC-amplified compared to non-amplified MB cell lines. Similar results were obtained for MYC or N-MYC-amplified HGG and NB cell lines. We also observed the loss of viability and loss of fraction of cells in G1 phase in MYC-amplified MB cells after treatment with entinostat and PLK1i. MYC target genes were significantly downregulated in the MYC-amplified MB cell line HD-MB03 after treatment with PLK1i and entinostat. Moreover, we demonstrated reduction of MYC levels and faster MYC degradation upon volasertib treatment. Our data suggest that MYC-amplification might serve as a predictive biomarker for PLK1i treatment in MB and other entities. The combination of entinostat and PLKi could be a candidate therapy for future clinical trials for MYC-amplified Group 3 MB, and possibly other tumors harboring MYC amplification. Citation Format: Gintvile Valinciute, Jonas Ecker, Thomas Hielscher, Christin Schmidt, Marc Remke, Gianluca Sigismondo, Jeroen Krijgsveld, Stefan M. Pfister, Olaf Witt, Till Milde. Synergistic interaction of HDACi and PLK1i in Group 3 MYC-amplified medulloblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A53.

Abstract IA20: Neuronal activity promotes proliferation of normal and neoplastic glial cells

Abstract Neuronal activity regulates the proliferation and differentiation of oligodendrocyte precursor cells during development and in adulthood. In the healthy brain, this results in activity-regulated plasticity of myelination and subsequent modulation of neural circuit function evident in oligodendrogenesis-dependent behavioral changes. The robust mitogenic effect of neuronal activity on normal neural precursor and oligodendroglial precursor cells, a putative cellular origin of pediatric high-grade gliomas (HGG), suggests that dysregulated or “hijacked” mechanisms of myelin plasticity might similarly promote proliferation in this devastating group of childhood brain cancers. Using in vivo and in situ optogenetic techniques together with patient-derived high-grade glioma cell cultures and xenograft models, we have demonstrated that neuronal activity similarly promotes proliferation and growth of both pediatric and adult high-grade glioma subtypes. Crucial mechanisms mediating activity-regulated high-grade glioma growth include secretion of Brain Derived Neurotrophic Factor (BDNF) and the synaptic protein neuroligin-3 (NLGN3), which induces multiple oncogenic signaling pathways together with robust changes in synaptic gene expression in glioma cells. Nlgn3 is necessary for the growth of high-grade glioma xenografts in the mouse brain, and NLGN3 expression levels in human HGG negatively correlate with patient overall survival. Thus, neuronal activity not only modulates the structure and function of the brain’s myelinated infrastructure, but neurons also play an important role in the brain tumor microenvironment, with activity-regulated secretion of NLGN3 emerging as an unexpected and therapeutically targetable mechanism underlying axon-glioma interactions and promoting neuronal activity-regulated cancer growth.

Granulocyte-macrophage colony-stimulating factor enhances effect of temozolomide on high-grade glioma cells.

In the present study, to delve into the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) combined with temozolomide (TMZ) on high-grade glioma cells and related mechanism, six cases of high-grade glioma cells from patient's tumor tissues were cultured. 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay was performed to detect cell proliferation and toxicity. Flow cytometry was performed to ascertain cell cycle and apoptosis rate. To detect the expressions of O6-methylguanine-DNA methyltransferase (MGMT) methylation status and MGMT protein, respectively, specific PCR and immunofluorescence were performed. According to the results of MTT assay, compared with the results of control group, GM-CSF group exhibited enhanced cell viability in varying degrees. In three cases of cells (MGMT gene methylation), the combination group [(67.67 ± 1.16), (68.13 ± 1.06), (68.42 ± 1.73)] had noticeably lower cell viability than the corresponding TMZ group [(90.00 ± 1.73), (82.33 ± 1.53), (82.67 ± 2.11)] (P < 0.01). Nevertheless, the two groups showed no significant difference in another three cases (MGMT gene unmethylated) (P > 0.05). In combination group, the apoptosis rate of the MGMT methylation cells was higher than that in the corresponding TMZ group (P < 0.01), which is consistent with MTT assay results. In all six cases of primary glioma cells, the fraction of cells in G1 phase of GM-CSF-treated group was noticeably down-regulated and was up-regulated in S phase (P < 0.01). GM-CSF could induce high-grade glioma cells to rapidly enter the cell cycle, thereby enhancing the lethal effect of TMZ on glioma cells with MGMT gene promoter methylation. However, this effect is not ideal on glioma cells with MGMT unmethylation.

Anti-PD-1 for patients with leptomeningeal metastasis from advanced solid tumors: Efficacy, safety, and biomarkers of response.

e14506 Background: Leptomeningeal metastasis (LMM) from solid tumors are rare and often refractory to standard therapies. Pembrolizumab (anti-PD-1) has efficacy in patients (pts) with brain metastases from ICI-responsive tumors, however pts with LMM are often excluded from ICI trials. We hypothesized that: 1. Pembrolizumab will lead to CNS response in LMM, and 2. Genomic/immunologic analyses on CSF could identify CNS biomarkers of response. Methods: We conducted an investigator-initiated open-label phase 2 trial of Pembrolizumab in pts with LMM from any solid tumor (NCT03091478). Eligible pts had: LMM on MRI ( &gt; 3mm lesion) or CSF cytology, ECOG PS ≤1, and were PD-naïve. Prior RT to LMM was allowed &gt; 3 months before study start or to non-target areas. Pembrolizumab was administered IV 200mg q3W until disease progression/unacceptable toxicity. The primary endpoint was CNS response after 12 weeks, defined as radiologic (RECIST 1.1)/cytologic/clinical response to therapy. Serial CSF samples were assessed by chromosomal copy number changes using a PCR-based approach (RealSeqS) to detect tumor-derived DNA (CSF-tDNA), and 16-color flow cytometry. Results: Thirteen of a planned 18 pts were treated 04/2017-12/2019, the study was closed early for low accrual. Median age was 58 years (22-79), 53% were female. Pts had breast carcinoma (38%, n = 5), NSCLC (23%, n = 3), high-grade glioma (23%, n = 3), HNSCC (8%, n = 1) and cutaneous squamous cell carcinoma (8%, n = 1). Median no. of prior therapies was 4 (0-8), 76% of pts had prior RT to LMM. CNS response was seen in 38% of pts (5/13: 2 = complete response (NSCLC; Squamous skin); 3 = stable disease (NSCLC, Glioma, HER2+BC) by RECIST 1.1. Treatment-related adverse events were seen in 31% (4/13) of pts, 15% (2/13) G3+ (fatigue = 1, infection = 1). CSF cytology was negative at ICI start in 6 cases, but positive by CSF-tDNA in 5/6 cases. In addition, CSF-tDNA levels can correlate with disease status. CSF flow cytometry demonstrated increase in CD45R0+ activated memory T-cells, and a shift from immature to antibody-secreting B-cells in both pts with CRs in LMM. Conclusions: Pembrolizumab was well-tolerated and demonstrated anti-tumor activity in pts with LMM in ICI-responsive tumors. Correlative analyses identified CSF-tDNA can potentially be used from diagnosis to longitudinally monitor LMM; and that T and B cell populations may be enriched in the CSF of pts whose LMM regressed. These findings support inclusion of pts with LMM in ICI studies and interrogation of CSF biomarkers. Clinical trial information: NCT03091478.