Pediatric Glioblastoma Cell Lines Research Articles

IMMU-21. B7-H3 NANOBODY CAR-T CELL THERAPY IN PEDIATRIC GLIOBLASTOMA

Abstract BACKGROUND Immunotherapy is a developing but challenging field of research for the treatment of central nervous system malignancies. Chimeric antigen receptor (CAR)-T cell therapy has been effective in treating hematologic malignancies, but that success has not been clinically translated to solid tumors in children. B7-H3 has been identified as a promising immunotherapy target as it is highly expressed in pediatric glioblastoma and correlates with tumor progression and poor prognosis. We aim to evaluate a nanobody-based CAR-T cell therapy that binds B7-H3 with high affinity and hypothesize that anti-B7-H3 nanobody CAR-T cells can effectively kill B7-H3-expressing tumor cells. METHODS B7-H3 nanobodies were isolated from camel phage libraries and cloned into lentiviral CAR-T cell constructs. Human T cells were transduced by lentivirus to express CAR on the surface. Nanobody CAR-T cell functionality was evaluated by an impedance-based cytotoxicity assay and cytokine release was measured by ELISA. CAR-T avidity was quantified by the Lumicks z-Movi assay. In vivo studies are evaluating the efficacy of B7-H3 CAR-T cells in an orthotopic glioblastoma model in immunodeficient mice. Mice were treated with B7-H3 CAR-T cells by intracerebroventricular or intravenous delivery. RESULTS We confirmed high expression of B7-H3 on the surface of three pediatric glioblastoma cell lines by flow cytometry and Quantibrite staining. We confirmed effective killing of B7-H3-expressing tumor cells and the release of high levels of cytokines. B7-H3 nanobody CAR-T cells were observed to have high avidity compared to control CAR-T cells. In vivo studies are currently ongoing and treatment response will be evaluated by luminescence imaging and endpoint histologic analysis. CONCLUSION We have shown that B7-H3 nanobody CAR-T cells demonstrate significant preclinical efficacy. It is critical to study CAR-T cell therapy in immunocompetent in vivo models for further development and clinical application.

IMMU-20. EVALUATION OF B7-H3 NANOBODY CAR-T CELLS IN PEDIATRIC GLIOBLASTOMA

Abstract Chimeric antigen receptor (CAR)-T cell therapies are an undoubted success in childhood acute lymphoblastic leukemia; however, this success has not translated clinically to any childhood solid tumor. B7-H3 has been found to be highly expressed in pediatric brain tumors and correlates with tumor progression and poor prognosis. We utilized a novel nanobody based CAR-T cell which can bind tumor antigens with high affinity. We hypothesize that anti-B7-H3 nanobody CAR-T cells mediate more potent antitumor effects both in vitro and in vivo compared to existing antibody-based B7-H3 CAR-T cells. High expression of B7-H3 was confirmed on the surface of two pediatric glioblastoma cell lines by flow cytometry and Quantibrite staining. B7-H3 nanobodies isolated from camel phage libraries were cloned into lentiviral CAR-T cell constructs. We manufactured CAR-T cells by lentiviral transduction of human T cells. Glioblastoma tumor cells were co-cultured with B7-H3 CAR-T cells vs. control T cells. We confirmed B7-H3 specific cytokine release by CAR-T cells via ELISA and effective tumor cell killing via two different cytotoxicity assays (impedance or luciferase based). Nanobody based B7-H3 CAR-T cells exhibited statistically significant higher cytokine release and cytotoxicity compared to antibody based B7-H3 CAR-T cells. In vivo studies are currently evaluating the efficacy of intracerebroventricular delivery of B7-H3 CAR-T cells against two orthotopic glioblastoma models utilizing multiple human donor T cells in immunocompromised mice. We are simultaneously developing a syngeneic orthotopic glioblastoma model to evaluate the nanobody based B7-H3 CAR-T cell cross-reactive in mice utilizing an immunocompetent model. Careful investigation of CAR-T cell therapy in appropriate immunocompetent in vivo models is needed to develop effective immunotherapy for clinical application.

HGG-04. ZINC ENHANCES TEMOZOLOMIDE CYTOTOXICITY IN PEDIATRIC GLIOBLASTOMA MULTIFORME MODEL SYSTEM

BACKGROUNDTemozolomide (TMZ) is an alkylating agent that has become the mainstay treatment of the most malignant brain cancer, glioblastoma multiforme (GBM). Unfortunately only a limited number of patients respond to it positively. We have shown that zinc metal reestablishes chemosensitivity in adult GBM in vitro and also in vivo but this effect has not been tested with pediatric GBM.METHODSUsing Human pediatric glioblastoma cell lines- KNS42 (mutant p53/ MGMT [+]) and SF188 (mutant p53/ MGMT [-]), we investigated whether addition of zinc to TMZ enhances its cytotoxicity against GBM.RESULTS In vitro cell viability analysis showed that the cytotoxic activity of TMZ was substantially increased with addition of zinc and this response was accompanied by an elevation of p21, PUMA, BAX and a decrease in growth fraction as manifested by low ki67. Beta gal analysis showed that most of the remaining cells after the combination therapy are in senescence state. In order to eliminate the senescent population created as a result of the combined treatment of TMZ and Zinc, we decided to use a senolytic agent Navitoclax (ABT-263) that was demonstrated to be effective in reducing senescent cells by specific inhibition of Bcl-2, Bcl-XL and Bcl-w. Following the addition of Navitoclax to the combined treatment, SF188 cells, but not KNS42, show a significance reduction in viability compare to the combination treatment.CONCLUSIONSOur results suggest that zinc may serve as a potentiator of TMZ therapy in pediatric GBM patients and using a second hit with senolytic drug in some cases may be even more beneficial.

Abstract B09: Epigenetic changes mediated by H3.3 G34R mutation in a CRISPR-edited pediatric glioblastoma cell line

Abstract Background: Pediatric glioblastomas (pGBM) are aggressive and lethal brain tumors with few effective treatment options. Since the discovery of recurrent histone H3 mutations in pGBMs, multiple groups have sought to uncover the mechanism by which these mutations drive tumorigenesis. One striking aspect of histone H3 mutations is their tumor location and age-specificity: the K27M mutation occurs in midline brain structures in infants and young children, whereas the G34R/V mutations occur in the cerebral hemisphere of adolescents. Efforts to elucidate the mechanism underlying K27M tumorigenesis have centered on K27M-mediated inhibition of the PRC2 complex and the resulting drastic loss of the repressive H3K27me3 mark in these tumors. Studies have shown that G34 mutations affect methylation of the adjacent K36 residue, resulting in a local decrease of the active H3K36me3 mark on the mutant histone. It remains uncertain whether the G34R/V mutations promote global changes in the epigenome that drive tumorigenesis. Methods: To investigate the epigenetic consequences of the G34R mutation, we generated an isogenic in vitro model using CRISPR/Cas9 to correct the G34R mutation in a pGBM-derived cell line. We then comprehensively characterized and compared the epigenome and transcriptome of CRISPR-unedited clones (carrying the G34R mutation) and CRISPR-edited (wild-type) clones. Using ChIP-seq, we profiled H3K36me3 and H3K27me3 marks to study G34R-mediated regulation of these antagonistic marks. Results: ChIP-seq profiling revealed that CRISPR editing of the G34R mutation had minimal effects on total levels or genomic distribution and profile of H3K36me3 and H3K27me3 marks. In contrast, we previously showed that CRISPR editing of the K27M mutation in pGBM cell lines rescued total H3K27me3 levels and genomic distribution to that of histone wild-type pGBMs. In agreement with their similar epigenetic profiles, there were very few transcriptomic differences between G34R and wildtype cells. Conclusions: Our results suggest that the epigenome in G34R-mutant pGBM cells may be less dynamic or amenable to epigenetic perturbation from removal of the initial driver oncomutation than K27M-mutant tumors. These findings underscore that different strategies may be required to target and treat G34R and K27M-mutant pGBM tumors. Citation Format: Shriya Deshmukh, Carol C.L. Chen, Amira Ouanouki, Nada Jabado. Epigenetic changes mediated by H3.3 G34R mutation in a CRISPR-edited pediatric glioblastoma cell line [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 B09.

Abstract 3499: Dianhydrogalactitol (VAL-083) in combination with AZD1775 increases survival in diffuse intrinsic pontine glioma (DIPG), in vivo

Abstract OBJECTION: VAL-083 is a structurally unique bi-functional DNA targeting agent that readily crosses the blood-brain barrier and accumulates in brain tumor tissue. VAL-083 induces interstrand crosslinks leading to DNA double-strand breaks and S/G2 cell cycle arrest. By inhibiting the G2 checkpoint regulator kinase Wee1, AZD1775 allows a cancer cell with DNA damage to progress past the G2 checkpoint and into premature mitosis leading to cancer cell death. Herein we assess the activity of VAL-083 as single agent as well as in combination with Wee1 Kinase inhibitor AZD1775 in patient derived model systems of DIPG. METHODS: DIPG derived cell lines SF8628 and NEM157 (H3.3K27) as well as SF10693 (H3.1K27M) and pediatric glioblastoma cell lines SF188 (H3.3K27 wildtype) were treated with increasing concentrations of single agent VAL-083 as well as in combination with AZD1775. To determine potential synergistic activity, we applied the Chou-Talalay method, which allows the quantitative determination of drug interactions by calculating a combination index (CI). In vivo activity of VAL-083 (3 mg/kg) as single agent as well as in combination with AZD1775 (60 mg/kg) was assessed in an orthotopic engraftment model of pediatric DIPG (SF8628). RESULTS: The IC50 of VAL-083 as single agent ranged from 1µM to 10µM. VAL-083 exhibited synergistic activity in combination with AZD1775 in cell lines SF8628 and SF188 and additive effect in NEM157 with CI values ranging from 0.04 to 0.95, with CI < 1 indicating synergy. In vivo, treatment with VAL-083 as single agent and in combination with AZD1775 conferred significant survival benefit to mice with engrafted DIPG tumors compared to control as well as single agent treatment with AZD1775. The median survival for mice treated with VAL-083 alone was 54.5 days (p=0.0004, VAL-083 vs. control) and for the VAL-083/AZD1775 combination it was 62 days (p<0.0001, VAL-083/AZD1775 vs. control), compared to 44 days for control and 47 days for mice treated with AZD1775 alone (p=0.0839, AZD1775 vs. control). CONCLUSION: Our present study highlights that the combination of VAL-083 and AZD1775 might be a promising new therapeutic strategy for children with DIPG. Ongoing studies continue to assess the in vivo activity in other DIPG models as well explore the underlying mechanism of action of the combination strategy. Citation Format: Anne Steino, Xiaodong Yang, Cassie Kline, Jeffrey Bacha, Dennis M. Brown, Sabine Mueller. Dianhydrogalactitol (VAL-083) in combination with AZD1775 increases survival in diffuse intrinsic pontine glioma (DIPG), in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3499.

Using PIK3CB and connexin-43 inhibition to sensitize pediatric glioblastoma cells to temozolomide.

e13572 Background: Glioblastoma (GBM) is one of the most common high grade gliomas in pediatric patients, accounting for 2.8% of central nervous system tumors. Standard of care for glioblastoma includes surgical resection, radiation, and temozolomide chemotherapy. However, many pediatric patients are not suitable candidates for surgery, and their tumors inevitably develop temozolomide resistance. Despite aggressive treatment, temozolomide (TMZ) resistance leads to tumor recurrence in 90% of patients. Thus, outcome for children with pediatric GBM remains poor with a 2-year survival rate ranging from 10-30%, highlighting the need for new therapeutic approaches. Connexin-43 (Cx43) and PIK3CB are proteins involved in conferring temozolomide resistance in glioblastoma. Combined inhibition of these proteins could improve temozolomide sensitivity and help develop a selective therapy that significantly improves quality of life and prognosis in pediatric glioblastoma patients. Methods: Primary GBM cells were plated in a 96-well plate and treated with vehicle (DMSO), TMZ (50 μmol/L), TGX-221 (20 μmol/L), αCT1 (30 μmol/L), or a combination of the latter three drugs. Cell viability was measured using the MTS assay. Primary pediatric GBM cell lines SJ-GBM2 and CHLA-200 were tested. These drug combinations were also tested in normal human astrocytes to make sure there were no off-target effects. Results: We found that targeting PIK3CB and Cx43 in primary pediatric GBM increases their sensitivity to TMZ. The triple combination of TMZ, TGX-221, and αCT1 significantly decreased cell viability than each drug alone while sparing normal human astrocytes. Conclusions: Our findings suggest that inhibiting PIK3CB and Cx43 enhances therapeutic response to TMZ and limits off-target effects in pediatric GBM. These findings closely mirror our results using the triple combination in adult glioblastoma as well, thus laying the groundwork for a novel combination therapeutic that can be used across age groups.

Irradiation of pediatric glioblastoma cells promotes radioresistance and enhances glioma malignancy via genome-wide transcriptome changes.

Pediatric glioblastoma (GBM) is a relatively rare brain tumor in children that has a dismal prognosis. Surgery followed by radiotherapy is the main treatment protocol used for older patients. The benefit of adjuvant chemotherapy is still limited due to a poor understanding of the underlying molecular and genetic changes that occur with irradiation of the tumor. In this study, we performed total RNA sequencing on an established stable radioresistant pediatric GBM cell line to identify mRNA expression changes following radiation. The expression of many genes was altered in the radioresistant pediatric GBM model. These genes have never before been reported to be associated with the development of radioresistant GBM. In addition to exhibiting an accelerated growth rate, radioresistant GBM cells also have overexpression of the DNA synthesis-rate-limiting enzyme ribonucleotide reductase, and pro-cathepsin B. These newly identified genes should be concertedly studied to better understand their role in pediatric GBM recurrence and progression after radiation. It was observed that the changes in multiple biological pathways protected GBM cells against radiation and transformed them to a more malignant form. These changes emphasize the importance of developing a treatment regimen that consists of a multiple-agent cocktail that acts on multiple implicated pathways to effectively target irradiated pediatric GBM. An alternative to radiation or a novel therapy that targets differentially expressed genes, such as metalloproteases, growth factors, and oncogenes and aim to minimize oncogenic changes following radiation is necessary to improve recurrent GBM survival.

MutT homolog 1 counteracts the effect of anti-neoplastic treatments in adult and pediatric glioblastoma cells

Glioblastoma, a fatal disease in both adult and pediatric patients, currently has limited treatment options that offer no more than temporary relief. Our experiments with adult and pediatric glioblastoma cell lines showed that radiation induces a dose-dependent increase in the level of MutT homolog 1 (MTH1) - an enzyme that hydrolyzes oxidized purine nucleoside triphosphates. Similarly, the combination of vorinostat, which is a histone deacetylase inhibitor, and ABT-888, which is a PARP-1 inhibitor, enhanced clonogenic death and increased the MTH1 level, relative to each treatment alone. This result suggests that the MTH1 level is directly related to the damage that is inflicted upon the cells, and its activity protects them against anti-neoplastic therapy. Indeed, the MTH1 inhibitor TH588 and MTH1 siRNA increased glioblastoma's response to both radiation and the combination of vorinostat and ABT-888. TH588 also inhibited glioblastoma's capacity for migration and invasion. In normal fibroblasts, low radiation doses and the combination of vorinostat and ABT-888 decreased the level of the enzyme. TH588 did not alter the fibroblasts’ response to radiation and only mildly affected their response to the combination of vorinostat and ABT-888.In summary, the inhibition of MTH1 is required to better realize the therapeutic potential of anti-neoplastic treatments in glioblastoma.

Pediatric and adult glioblastoma radiosensitization induced by PI3K/mTOR inhibition causes early metabolic alterations detected by nuclear magnetic resonance spectroscopy.

Poor outcome for patients with glioblastomas is often associated with radioresistance. PI3K/mTOR pathway deregulation has been correlated with radioresistance; therefore, PI3K/mTOR inhibition could render tumors radiosensitive. In this study, we show that NVP-BEZ235, a dual PI3K/mTOR inhibitor, potentiates the effects of irradiation in both adult and pediatric glioblastoma cell lines, resulting in early metabolic changes detected by nuclear magnetic resonance (NMR) spectroscopy. NVP-BEZ235 radiosensitises cells to X ray exposure, inducing cell death through the inhibition of CDC25A and the activation of p21cip1(CDKN1A). Lactate and phosphocholine levels, increased with radiation, are decreased after NVP-BEZ235 and combination treatment, suggesting that inhibiting the PI3K/mTOR pathway reverses radiation induced metabolic changes. Importantly, NVP-BEZ235 potentiates the effects of irradiation in a xenograft model of adult glioblastoma, where we observed a decrease in lactate and phosphocholine levels after seven days of combination treatment. Although tumor size was not affected due to the short length of the treatment, a significant increase in CASP3 mRNA was observed in the combination group. Taken together, our data suggest that NMR metabolites could be used as biomarkers to detect an early response to combination therapy with PI3K/mTOR inhibitors and radiotherapy in adult and pediatric glioblastoma patients.

Penfluridol suppresses glioblastoma tumor growth by Akt-mediated inhibition of GLI1.

Glioblastoma (GBM) is the most common brain tumor with poor survival rate. Our results show that penfluridol, an antipsychotic drug significantly reduced the survival of ten adult and pediatric glioblastoma cell lines with IC50 ranging 2–5 μM after 72 hours of treatment and induced apoptosis. Penfluridol treatment suppressed the phosphorylation of Akt at Ser473 and reduced the expression of GLI1, OCT4, Nanog and Sox2 in several glioblastoma cell lines in a concentration-dependent manner. Inhibiting Akt with LY294002 and siRNA, or inhibiting GLI1 using GANT61, cyclopamine, siRNA and CRISPR/Cas9 resulted in enhanced cell growth suppressive effects of penfluridol. On the other hand, overexpression of GLI1 significantly attenuated the effects of penfluridol. Our results further demonstrated that penfluridol treatment inhibited the growth of U87MG tumors by 65% and 72% in subcutaneous and intracranial in vivo glioblastoma tumor models respectively. Immunohistochemical and western blot analysis of tumors revealed reduced pAkt (Ser 473), GLI1, OCT4 and increase in caspase-3 cleavage and TUNEL staining, confirming in vitro findings. Taken together, our results indicate that overall glioblastoma tumor growth suppression by penfluridol was associated with Akt-mediated inhibition of GLI1.

The histone deacetylase inhibitor PCI-24781 as a putative radiosensitizer in pediatric glioblastoma cell lines.

BackgroundGlioblastoma (GBM) is considered to be one of the most aggressive tumors of the central nervous system (CNS). Even with the use of modern treatment protocols, the prognosis remains reserved, with children with GBM having a mean survival of 12–15 months.MethodsIn the present study we investigated the potential radiosensitizing effect of PCI-24781, a potent pan-histone deacetylase inhibitor (HDACi), on the SF188 and KNS42 cell lines of pediatric GBM. Cell proliferation rates, clonogenicity and apoptosis were compared in the presence and absence of treatment with PCI-24781. We also compared the clonogenicity rates of the irradiated SF188 and KNS42 cell lines with or without previous treatment with PCI-24781 at the doses of 0.25–16 μM. In addition, we investigated the effects of PCI-24781 on the expression of some of the main proteins responsible for the repair of double-strand DNA breaks caused by irradiation.ResultsThe inhibitor blocked cell proliferation, induced death by apoptosis and reduced the colony forming capacity of the cell lines, both of them showing a significant decrease of colony formation at all irradiation doses. The expression of the Rad51 protein, important for the homologous recombination (HR) repair pathway, and of the DNA-PKcs, Ku70 and Ku86 proteins, important for the non-homologous end joining (NHEJ) repair pathway, was more reduced when the irradiated cell line was previously treated with PCI-24781 than when it was treated exclusively with radiotherapy.ConclusionsThese findings demonstrate that HDACi PCI-24781 has a radiosensitizing profile that compromises the repair of double-strand DNA breaks in cells of pediatric GBM treated with radiotherapy.

HG-17 * THE HISTONE DEACETYLASE INHIBITOR PCI-24781 SHOWS ANTIPROLIFERATIVE, PRO-APOPTOTIC AND RADIOSENSITIZING EFFECTS ON PEDIATRIC GLIOBLASTOMA CELL-LINES

The acetylation of DNA can modulate the expression of genes involved in the development and progression of malignancies. Histone deacetylase (HDACs) inhibitors are clinically active and well tolerated in the treatment of a wide variety of tumors. HDACs inhibitors can sensitize cell response to ionizing radiation, potentially reducing treatment doses and side effects. OBJECTIVES: To assess the potential radiosensitizer effect of abexinostat (PCI-24781), a new and potent pan-HDAC inhibitor in two pediatric glioblastoma cell-lines (SF188 and KNS42). METHODS: The effect of PCI-24781 on proliferation rates, clonogenic capacity and apoptosis were compared prior and following cell irradiation. The clonogenic assay was performed without irradiation (48h of incubation) at the doses of 0.25, 0.5, 1 and 2uM of PCI-24781. The clonogenic assay combined with irradiation was performed at 48h using the inhibitor IC30 for each cell-line in combination with irradiation doses of 0.5, 1, 2 and 4Gy. For cell proliferation assays, the intervals of 24h, 48h, 72h and 96h at the doses of 0.5, 1, 2, 4, 8 and 16uM of PCI-24781 were selected for study. Apoptosis were evaluated at 48h-cultures at the doses of 2, 4, 8 and 16uM. RESULTS: The HDAC inhibitor repressed cell proliferation, displaying a more significant effect at 48h in the dose of 2uM (p < 0.05). PCI-24781 also induced apoptosis (p < 0,05); the percentage of cell death for SF188 and KNS42 (at 16uM) was 40% and 55% respectively. Cells demonstrated massive decrease in colony formation with PCI-24781; this effect was more pronounced when drug was combined with irradiation (p < 0.001). CONCLUSION: These data demonstrate potential radiosensitizer and antiproliferative effects of PCI- 24781 on pediatric glioblastoma cell-lines. This study will now focus on key proteins responsible for the double-strand breaks repair caused by irradiation, trying to elucidate epigenetic pathways potentially involved in the therapy with PCI-24781. Financial Support: Fapesp (process no. 2013/15891-8).

Abstract B15: Pediatric glioblastoma cell lines maintain response to developmental growth factors

Abstract Glioblastoma (GBM) is a highly invasive brain tumor arising from an astrocytic cell lineage. In the United States, nearly 500 children each year develop GBMs. Pediatric GBM has a different mutational, methylation, and gene expression spectra than adult GBM, implicating a strong role for developmental processes in disease pathogenesis. The developmental growth factors that define the astrocytic lineage include epidermal growth factor (EGF) and platelet-derived growth factor (PDGF). Expression of these growth factors also distinguishes pediatric versus adult GBM. Here, we use EGF or PDGF as a stimulus for malignant behavior in GBM cell lines isolated from either adult or pediatric patients to understand whether the cell line models parallel growth factor receptor signaling preferences observed clinically. In response to these stimuli, pediatric cell lines demonstrate distinct protein signaling and invasion phenotypes from adult cell lines in vitro, and generate invasive, multifocal lesions in an orthotopic xenograft model. Thus, distinguishing features of adult and pediatric GBM are maintained in the cell line models that will be used to test therapeutic regiments preclinically. Both of the receptors activated by EGF and PDGF are known to interact with a promising therapeutic target in adult glioblastoma: signal transducer and activator 3 (STAT3). STAT3 acts as either an oncogene or tumor suppressor depending on cellular context such as the activating receptor complex and phosphatase and tensin homolog (PTEN) mutational status. Our studies will therefore address the differential implications of STAT3 therapy for pediatric versus adult GBM. Citation Format: Marie Renee Mooney. Pediatric glioblastoma cell lines maintain response to developmental growth factors. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B15.

Lactate and choline metabolites detected in vitro by nuclear magnetic resonance spectroscopy are potential metabolic biomarkers for PI3K inhibition in pediatric glioblastoma.

The phosphoinositide 3-kinase (PI3K) pathway is believed to be of key importance in pediatric glioblastoma. Novel inhibitors of the PI3K pathway are being developed and are entering clinical trials. Our aim is to identify potential non-invasive biomarkers of PI3K signaling pathway inhibition in pediatric glioblastoma using in vitro nuclear magnetic resonance (NMR) spectroscopy, to aid identification of target inhibition and therapeutic response in early phase clinical trials of PI3K inhibitors in childhood cancer. Treatment of SF188 and KNS42 human pediatric glioblastoma cell lines with the dual pan-Class I PI3K/mTOR inhibitor PI-103, inhibited the PI3K signaling pathway and resulted in a decrease in phosphocholine (PC), total choline (tCho) and lactate levels (p<0.02) as detected by phosphorus (31P)- and proton (1H)-NMR. Similar changes were also detected using the pan–Class I PI3K inhibitor GDC-0941 which lacks significant mTOR activity and is entering Phase II clinical trials. In contrast, the DNA damaging agent temozolomide (TMZ), which is used as current frontline therapy in the treatment of glioblastoma postoperatively (in combination with radiotherapy), increased PC, glycerophosphocholine (GPC) and tCho levels (p<0.04). PI-103-induced NMR changes were associated with alterations in protein expression levels of regulatory enzymes involved in glucose and choline metabolism including GLUT1, HK2, LDHA and CHKA. Our results show that by using NMR we can detect distinct biomarkers following PI3K pathway inhibition compared to treatment with the DNA-damaging anti-cancer agent TMZ. This is the first study reporting that lactate and choline metabolites are potential non-invasive biomarkers for monitoring response to PI3K pathway inhibitors in pediatric glioblastoma.

MerTK inhibition is a novel therapeutic approach for glioblastoma multiforme

Glioblastoma is an aggressive tumor that occurs in both adult and pediatric patients and is known for its invasive quality and high rate of recurrence. Current therapies for glioblastoma result in high morbidity and dismal outcomes. The TAM subfamily of receptor tyrosine kinases includes Tyro3, Axl, and MerTK. Axl and MerTK exhibit little to no expression in normal brain but are highly expressed in glioblastoma and contribute to the critical malignant phenotypes of survival, chemosensitivity and migration. We have found that Foretinib, a RTK inhibitor currently in clinical trial, inhibited phosphorylation of TAM receptors, with highest efficacy against MerTK, and blocked downstream activation of Akt and Erk in adult and pediatric glioblastoma cell lines, findings that are previously unreported. Survival, proliferation, migration, and collagen invasion were hindered in vitro. Foretinib treatment in vivo abolished MerTK phosphorylation and reduced tumor growth 3-4 fold in a subcutaneous mouse model. MerTK targeted shRNA completely prevented intracranial and subcutaneous glioma growth further delineating the impact of MerTK inhibition on glioblastoma. Our findings provide additional target validation for MerTK inhibition in glioblastoma and demonstrate that robust MerTK inhibition can be achieved with the multi-kinase inhibitor Foretinib as an innovative and translational therapeutic approach to glioblastoma.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Abstract B61: Picropodophyllin (PPP) increases glucose metabolism and lactate production in paediatric glioblastoma cells

Abstract Introduction: Picropodophyllin (PPP) is of interest as an anti-cancer agent, as it has been shown to induce growth inhibition and apoptosis in a variety of human cancer cells and xenografts. Recently, PPP was found to have dramatic antitumor effects in human glioblastoma cells and in subcutaneous and intracerebral xenografts and reported to be an insulin-like growth factor-1 receptor inhibitor; however, its full mechanisms of action remain unclear. Aims: To examine the effect of PPP on glucose metabolism in cancer cells and to use hyperpolarized [1-13C]pyruvate 13C-magnetic resonance spectroscopy (MRS) and conventional 1H-MRS to measure lactate formation/production in real-time and in steady state, respectively, in order to develop a non-invasive biomarker of response for PPP treatment in a pediatric glioblastoma cell line. Methods: Pediatric glioblastoma KNS42 cells were treated for 24 hours with 2.4μM (5×IC50) of PPP. Real-time 13C exchange from hyperpolarized [1-13C] pyruvate to lactate was performed by DNP-13C MRS. Culture media and extracts from the PPP-treated and control KNS42 cells were analyzed by 1H-MRS to examine the effect of PPP on glucose metabolism and lactate production. Lactate dehydrogenase (LDH) expression was examined by western blot and LDH activity was also measured enzymatically. Cellular protein content and size were also evaluated. Results and Discussion: The rate of real-time 13C exchange from pyruvate to lactate measured by DNP-13C MRS showed a significant increase in the PPP-treated group (P = 0.027 versus control group). These DNP changes in PPP-treated KNS42 cells are consistent with the significant increase in intracellular lactate (P = 0.003) and lactate excretion to the culture media (P &amp;lt; 0.001). This increase in lactate production is also consistent with significantly elevated LDH activity (P = 0.003) in the PPP-treated group. However, no change in LDH expression was observed in the PPP-treated group. Significant increases in intracellular glucose (P = 0.006) and glucose uptake (P = 0.008) were also found in PPP-treated KNS42 cells. These changes indicate an up-regulation of glucose metabolism following PPP treatment and a subsequent increase in lactate production. Twenty-four hours PPP treatment caused a significant decrease in cell number when compared to controls (∼40%, P = 0.001). No significant difference in cellular protein concentration and size were found in PPP-treated cells when compared to controls, indicating that the observed increases in glucose metabolism, lactate production and LDH activity are due to changes in cellular metabolism following PPP treatment and not from changes in cellular size or protein concentration. The increase in lactate production following PPP treatment found in this study is an unusual observation, as most cancer targeting-treatments cause a decrease in lactate production. Hence, the use of DNP and 13C MRS to measure lactate production could provide a more specific non-invasive biomarker of response for PPP treatment. Conclusions: PPP treatment resulted in increased glucose metabolism and lactate production in KNS42 cells, as shown by both real time and steady-state measurements. These changes have potential as specific biomarkers of response for PPP treatment. We acknowledge the support received from the CRUK and EPSRC Cancer Imaging Centre with the MRC and Department of Health (England) grant C1060/A10334, NHS funding to the NIHR Biomedical Research Centre.

Abstract 2501: Inhibition of the PI3K pathway potentiates temozolomide effects in pediatric glioblastoma and results in alterations in glucose and choline metabolism detected by MRS

Abstract Introduction: Children with high grade-glioma have a poor outcome with a 5-year survival rate of less than 20%. The use of adjuvant temozolomide (TMZ) has now been well accepted as a standard of care in glioblastoma. However, it does not seem to improve survival in children. We have recently shown that resistance to TMZ in pediatric glioma cell lines can be reversed by PI3K pathway inhibition. The aim of this study was to identify whether combination of PI3K pathway inhibition with TMZ would result in metabolic changes detectable by magnetic resonance spectroscopy (MRS), and potentially can be used as a noninvasive method of monitoring response in early stage clinical trials in children with glioma. Methods: The MGMT-independent TMZ-resistant pediatric glioblastoma cell line KNS42 was treated for 48h with dual PI3K/mTOR inhibitor PI-103 (2xGI50), TMZ (2xGI50) or with the combination of both (each at 2xGI50). Control and treated cells were extracted and the aqueous fractions were analyzed by 1H MRS. Results and Discussion: The number of treated KNS42 cells was significantly reduced relative to their controls, to 79±8% and 65±10% (P&amp;lt;0.02) following treatment with PI-103 and TMZ respectively. Combination of PI-103 and TMZ further reduced the number of treated vs. control cells (down to 54±6%, P&amp;lt;0.01). 1H MRS analysis showed that PI-103 treatment caused a decrease in lactate, phosphocholine (PC) and total choline (tCho) levels down to 48±17%, 52±11%, 64±14% (P&amp;lt;0.02) respectively. In contrast, treament with TMZ resulted in an increase in glycerolphosphocholine (GPC) up to 176±43% (P=0.05). Combination of PI-103 with TMZ retained the combined metabolic effects of both agents including a decrease in the levels of lactate (49±12%, P&amp;lt;0.01) and PC (71±11%, P&amp;lt;0.02) while an increase in GPC (190±40%, P&amp;lt;0.03) levels. tCho was normalized by the opposing changes in PC and GPC and hence was similar to controls. To explore potential mechanisms underlying the detected metabolic changes, immunoblotting was used to assess treatment effects on protein levels of enzymes involved in glycolysis and choline metabolism. The decrease in PC levels was associated with a decrease in the protein levels of Choline kinase (ChoK), the enzyme responsible for choline phosphorylation into PC. ChoK was not affected by TMZ single treatment leading to no change in PC levels. Furthermore, a decrease in hexokinase II protein was detected following PI-103 treatment and the combination, but not with TMZ alone, suggesting a mechanism for depletion of lactate. Conclusions: Our results show that using MRS we can detect distinct biomarkers following treatment with PI-103 compared to TMZ. Choline metabolites and lactate are potential noninvasive biomarkers for monitoring response to PI3K/mTOR inhibitors both as single agents and in combination with cytotoxic therapy in children with glioma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2501. doi:1538-7445.AM2012-2501

Pediatric glioblastoma cell line shows different patterns of expression of transmembrane ABC transporters after in vitro exposure to vinblastine

Resistance to drug is a major cause of treatment failure in pediatric brain cancer. The multidrug resistance (MDR) phenotype can be mediated by the superfamily of adenosine triphosphate-binding cassette (ABC) transporters. The dynamics of expression of the MDR genes after exposure to chemotherapy, especially the comparison between pediatric brain tumors of different histology, is poorly described. To compare the expression profiles of the multidrug resistance genes ABCB1, ABCC1, and ABCG2 in different neuroepithelial pediatric brain tumor cell lines prior and following short-term culture with vinblastine. Immortalized lineages from pilocytic astrocytoma (R286), anaplasic astrocytoma (UW467), glioblastoma (SF188), and medulloblastoma (UW3) were exposed to vinblastine sulphate at different schedules (10 and 60 nM for 24 and 72 h). Relative amounts of mRNA expression were analyzed by real-time quantitative polymerase chain reaction. Protein expression was assessed by immunohistochemistry for ABCB1, ABCC1, and ABCG2. mRNA expression of ABCB1 increased together with augmenting concentration and time of exposure to vinblastine for R286, UW467, and UW3 cell lines. Interestingly, ABCB1 levels of expression diminished in SF188. Following chemotherapy, mRNA expression of ABCC1 decreased in all cell lines other than glioblastoma. ABCG2 expression was influenced by vinblastine only for UW3. The mRNA levels showed consistent association to protein expression in the selected sets of cell lines analyzed. The pediatric glioblastoma cell line SF188 shows different pattern of expression of multidrug resistance genes when exposed to vinblastine. These preliminary findings may be useful in determining novel strategies of treatment for neuroepithelial pediatric brain tumors.