Pediatric Glioma Cell Research Articles

Histone H3.3K27M Mobilizes Multiple Cancer/Testis (CT) Antigens in Pediatric Glioma.

Lysine to methionine mutations at position 27 (K27M) in the histone H3 (H3.3 and H3.1) are highly prevalent in pediatric high-grade gliomas (HGG) that arise in the midline of the central nervous system. H3K27M perturbs the activity of polycomb repressor complex 2 and correlates with DNA hypomethylation; however, the pathways whereby H3K27M drives the development of pediatric HGG remain poorly understood. To understand the mechanism of pediatric HGG development driven by H3.3K27M and discover potential therapeutic targets or biomarkers, we established pediatric glioma cell model systems harboring H3.3K27M and performed microarray analysis. H3.3K27M caused the upregulation of multiple cancer/testis (CT) antigens, such as ADAMTS1, ADAM23, SPANXA1, SPANXB1/2, IL13RA2, VCY, and VCX3A, in pediatric glioma cells. Chromatin immunoprecipitation analysis from H3.3K27M cells revealed decreased H3K27me3 levels and increased H3K4me3 levels on the VCX3A promoter. Knockdown of VCX3A by siRNA significantly inhibited the growth of pediatric glioma cells harboring H3.3K27M. Overexpression of VCX3A/B genes stimulated the expression of several HLA genes, including HLA-A, HLA-B, HLA-E, HLA-F, and HLA-G The expression of VCX3A in pediatric HGG was confirmed using a tissue microarray. Gene set enrichment analysis revealed that CT antigens are enriched in pediatric HGG clinical specimens with H3.3K27M, with the upregulation of IL13RA2 contributing to the enrichment significantly. These results indicate that the upregulation of CT antigens, such as VCX3A and IL13RA2, correlates with pediatric gliomagenesis. Mol Cancer Res; 16(4); 623-33. ©2018 AACR.

Abstract 3466: ALT-positive pediatric high grade glioma cells display chemosensitivity to ATR pathway inhibition

Abstract Overcoming the end-replication problem is a major hurdle for cancer cells due to progressive telomere shortening that results from excessive cell division during tumorigenesis. While most cancers activate telomerase to maintain their telomere length, about five percent of cancers use a telomerase-independent telomere maintenance mechanism, termed alternative lengthening of telomeres (ALT). Despite its low overall frequency in cancer, ALT is enriched in several cancer subtypes, including pediatric high grade glioma (HGG). Previous work in our laboratory suggests that nearly half of pediatric HGG utilize the ALT mechanism. To date, therapeutic options are largely ineffective for children with HGG, reflected in the dismal five-year survival rate, which is less than 33 percent. Our goal is to better harness ALT as a clinical marker in pediatric HGG, specifically by identifying drugs that target ALT-positive cancers. In order to study ALT in this context, we obtained and characterized a panel of six pediatric HGG cell lines. Two of the six cell lines in this panel display hallmarks of the ALT pathway. To assess the potential of ALT as a therapeutic biomarker in pediatric HGG, we measured cell viability in the presence of inhibitors, stratifying our analysis by ALT status. ALT is predicted to occur via a homologous-recombination-based mechanism. As such, we focused our attention on inhibitors of the DNA damage response, as well as agents that induce DNA damage. Treatment with inhibitors of DNA-PK, RAD51, and MRE11 did not result in significant differences in cell viability when stratified by ALT status, nor did treatment with temozolomide or hydroxyurea. However, inhibitors of ATR (VE-821, VE-822/VX-970, and AZD-6738) and CHK1 (MK-8776) led to significantly greater reductions in cell viability in ALT-positive cells. In order to further our understanding of the mechanism through which ATR inhibition preferentially targets ALT-positive cell lines, we examined the effect of these inhibitors on ALT-specific properties, such as the presence of ultrabright telomeric foci and extrachromosomal telomeric DNA (c-circles). Preliminary results indicate that ATR inhibition does not diminish these ALT-associated properties and, therefore, the differential sensitivity to these agents is unlikely to be due to directly blocking the ALT telomere maintenance mechanism. Continued study of the effect of ATR/CHK1 inhibition on ALT-positive cells will yield further insight into the mechanism of ALT-specific toxicity. Overall, we have identified ATR/CHK1 as a promising pathway to target for ALT-positive pediatric HGG. Our goal is to better understand the relationship between ATR/CHK1 signaling and ALT in order to effectively translate this observation to the clinic, both for pediatric HGG and for other ALT-positive cancers. Citation Format: Jacqueline A. Brosnan-Cashman, Anthony J. Rizzo, Ezgi Goger, Kaylar M. Myers, Reza Zarinshenas, Christine Davis, Ming Yuan, Dinesh Rakheja, Eric H. Raabe, Charles G. Eberhart, Christopher M. Heaphy, Alan K. Meeker. ALT-positive pediatric high grade glioma cells display chemosensitivity to ATR pathway inhibition [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 3466. doi:10.1158/1538-7445.AM2017-3466

MicroRNA profiling of low-grade glial and glioneuronal tumors shows an independent role for cluster 14q32.31 member miR-487b

Low-grade (WHO I-II) gliomas and glioneuronal tumors represent the most frequent primary tumors of the central nervous system in children. They often have a good prognosis following total resection, however they can create many neurological complications due to mass effect, and may be difficult to resect depending on anatomic location. MicroRNAs have been identified as molecular regulators of protein expression/translation that can repress multiple mRNAs concurrently through base pairing, and have an important role in cancer, including brain tumors. Using the NanoString digital counting system, we analyzed the expression levels of 800 microRNAs in nine low-grade glial and glioneuronal tumor types (n=45). A set of 61 of these microRNAs were differentially expressed in tumors compared with the brain, and several showed levels varying by tumor type. The expression differences were more accentuated in subependymal giant cell astrocytoma, compared with other groups, and demonstrated the highest degree of microRNA repression validated by RT-PCR, including miR-129-2-3p, miR-219-5p, miR-338-3p, miR-487b, miR-885-5p, and miR-323a-3p. Conversely, miR-4488 and miR-1246 were overexpressed in dysembryoplastic neuroepithelial tumors compared with the brain and other tumors. The cluster 14q32.31 member miR-487b was variably under-expressed in pediatric glioma lines compared with human neural stem cells. Overexpression of miR-487b in a pediatric glioma cell line (KNS42) using lentiviral vectors led to a decrease in colony formation in soft agar (30%) (P<0.05), and decreased expression of known predicted targets PROM1 and Nestin (but not WNT5A). miR-487b overexpression had no significant effect on cell growth, proliferation, sensitivity to temozolomide, migration, or invasion. In summary, microRNA regulation appears to have a role in the biology of glial and glioneuronal tumor subtypes, a finding that deserves further investigation.

Parvovirus H-1 prolongs response to antineoplastc treatment with TMZ in pediatric high-grade glioma

Immunotherapeutic approaches have recently entered clinical routine in oncology. Oncolytic viruses such as Parvovirus H-1 (H-1PV) combine direct cytotoxic effects with enhanced anti-tumor immune responses. Here, we characterized the cytotoxic effects induced by the oncolytic parvovirus H-1 in conventional pediatric high-grade glioma (HGG) cell culture models and the combinatorial effects in simultaneous TMZ treatment in vitro and in vivo.

Abstract 2466: Development of a novel gene targeting and clone screening platform to engineer common pediatric glioma mutations into model cell lines

Abstract The utility of CRISPR/Cas9 gene targeting tools to alter endogenous genomic loci has revolutionized biomedical research across numerous disease focus areas, with especially large gains made in the development of disease models. However, the introduction of specific mutations using CRISPR/Cas9 can be associated with low yields of single cell clones harboring the desired homozygous or heterozygous mutations. Conventional clone screening methods can be a labor intensive and expensive process and thus, better approaches are needed to identify cells with the desired genotype(s). In parallel, there is a dearth of human tumor cell model systems for pediatric cancers, especially for Diffuse Intrinsic Pontine Glioma (DIPG). To address these needs, we recently designed and validated a platform which utilizes a novel, inducible Cas9 expression system coupled with a high-resolution DNA melt (HRM) analysis protocol to rapidly identify mutant clones. We applied our platform to model several common DIPG mutations, including truncating mutations within exon 6 of the gene, protein phosphatase 1D (PPM1D). Using our HRM analysis protocol, clones containing activating mutations in PPM1D were detectable and grouped distinctly from wild-type and non-functional clones; as validated through conventional Sanger sequencing and Ion Torrent Next Generation Sequencing. Importantly, multiple PPM1D mutant clones were generated in an expedited manner (&amp;lt; 4 weeks), using our unique HRM process-flow. We subsequently validated these clones in a collection of secondary assays, and we confirmed that the mutant proteins were functionally active. Several unique findings were identified in these studies, including substantial cell cycle-associated defects and basal activation of key proteins in the DNA damage response network. To our knowledge, we have created the first set of astrocyte cell lines harboring engineered PPM1D mutations at the endogenous gene locus. We are now testing these cell lines in high-throughput synthetic lethal screens to identify novel inhibitors of PPM1D-mutant DIPG tumors. These findings highlight the utility and power of our unique approach to rapidly create pertinent mutant cell lines for use in mutation-targeted biological assays. Our platform likely will become an invaluable tool for the development of better pediatric glioma cell line models in the future. Citation Format: Nathan R. Fons, Yulia Surovtseva, Gregory A. Breuer, Ranjini K. Sundaram, Ranjit S. Bindra. Development of a novel gene targeting and clone screening platform to engineer common pediatric glioma mutations into model cell lines. [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 2466.

Cell culture models of pediatric high-grade glioma are susceptible to parvovirus-1 induced cytotoxicity

Oncolytic viruses have entered the field of pediatric clinical research with the first early phase clinical trial recently published. Based on promising pre-clinical studies, the oncolytic parvovirus H-1 (H-1PV) has been applied to adult glioblastoma patients in a phase I/IIa clinical trial, indicating clinical safety of intracranial and intravenous virus application. Here, we characterized the cytotoxic effects induced by the oncolytic parvovirus H-1 in conventional pediatric high-grade glioma (HGG) cell culture models and neurosphere cultures derived thereof.

HG-12 * LIN28 AND MYC EXPRESSING PEDIATRIC GLIOBLASTOMAS ARE SENSITIVE TO INHIBITORS OF GLUTAMINE METABOLISM

We have previously shown that LIN28A protein is highly expressed in a subset of pediatric glioblastoma (GBM). Knockdown of LIN28A using lentiviral shRNA in GBM cell lines inhibits their invasion, growth and clonogenicity. LIN28A downregulates the let-7 family of tumor suppressing microRNAs, and these in turn inhibit multiple oncogenes including MYC. LIN28 is also known to regulate cellular metabolism in normal and transformed cells. We hypothesized that LIN28 would contribute to GBM tumorigenicity in part by modifying cellular metabolism. Pediatric GBM express MYC and NMYC, and these oncogenes also promote tumorigenicity through increasing proliferation, invasion and altering cellular metabolism. Addition of LIN28A to the JHH- GBM14 cell line leads to upregulation of MYC and glutaminase (GLS) as measured by western blot. The inhibitors of glutamine metabolism DON (6-Diazo-5-oxo-L-norleucine) and acivicin suppress the growth of LIN28A-transduced GBM14 cells to a greater extent than control empty-vector transduced cells in MTT assays (p = 0.008 for 5 uM DON, Student's t-test; p = 0.03, 5 uM acivicin, Student's t-test). We found that the pediatric high grade glioma cell lines SF188, UW479, SJ-GBM2, and KNS42 express varying levels of LIN28, MYC, and NMYC and have robust GLS expression by western blot. Acivicin treatment decreased the growth of SF188, KNS42 and UW479 in MTT Assays (P < .005 10 uM acivicin, Student's t-test). DON and acivicin have been tested in phase I clinical trials in children, but never extensively investigated in aggressive pediatric brain tumors. Our data suggest that LIN28 can alter GBM cellular metabolism in a way that confers sensitivity to inhibitors of glutamine metabolism and that these agents may be of therapeutic use in pediatric high grade gliomas.

PM-11 * DEVELOPMENT OF A NOVEL GENE TARGETING AND CLONE SCREENING PLATFORM TO ENGINEER COMMON PEDIATRIC GLIOMA MUTATIONS INTO MODEL CELL LINES

The utility of CRISPR/Cas9 gene targeting tools to alter endogenous genomic loci has revolutionized biomedical research across numerous disease focus areas, with especially large gains made in the development of disease models. However, the introduction of specific mutations using donor DNA templates can be associated with low yields of single cell clones harboring homozygous versus heterozygous mutations. Conventional clone screening methods can be a labor intensive and expensive process, and thus better approaches are needed to identify cells with the desired genotype(s). In parallel, there is a dearth of human tumor cell model systems for pediatric cancers. To address these needs, we recently designed and validated a platform which utilizes a novel, inducible Cas9 expression system coupled with a high-resolution DNA melt (HRM) analysis protocol to rapidly identify mutant clones. We applied our platform to model several common DIPG mutations, including the Lys27Met missense mutation within histone variant H3.3 (H3F3A), and truncating mutations within protein phosphatase 1D (PPM1D) exon 6. Using our HRM protocol, both heterozygous point mutations in H3F3A, and indels in PPM1D were detectable and grouped distinctly from wild-type samples. These findings were validated using conventional Sanger sequencing and topo cloning, which confirmed that this approach could differentiate between mutant and wild-type clones. Multiple clones were generated in under four weeks using this process-flow. These findings highlight the utility and power of our unique approach to rapidly create engineered mutant cell lines. Our platform likely will become an invaluable tool for the development of better pediatric glioma cell line models in the future.

A preclinical study demonstrating the efficacy of nilotinib in inhibiting the growth of pediatric high-grade glioma

Solid tumors arising from malignant transformation of glial cells are one of the leading causes of central nervous system tumor-related death in children. Recurrence in spite of rigorous surgical and chemoradiation therapies remains a major hurdle in management of these tumors. Here, we investigate the efficacy of the second-generation receptor tyrosine kinase inhibitor nilotinib as a therapeutic option for the management of pediatric gliomas. We have utilized two independent pediatric high-grade glioma cell lines with either high platelet-derived growth factor receptor alpha (PDGFRα) or high PDGFRβ expression in in vitro assays to investigate the specific downstream effects of nilotinib treatment. Using in vitro cell-based assays we show that nilotinib inhibits PDGF-BB-dependent activation of PDGFRα. We further show that nilotinib is able to decrease cell proliferation and anchorage-independent growth via suppression of AKT and ERK1/2 signaling pathways. Our results suggest that nilotinib may be effective for management of a PDGFRα-dependent group of pediatric gliomas.Electronic supplementary materialThe online version of this article (doi:10.1007/s11060-015-1744-y) contains supplementary material, which is available to authorized users.

Nilotinib inhibits pediatric high-grade glioma cell growth by blocking PDGFR

Nilotinib inhibits pediatric high-grade glioma cell growth by blocking PDGFR

Activity of the polyamine-vectorized anti-cancer drug F14512 against pediatric glioma and neuroblastoma cell lines.

The poor prognosis of children with high-grade glioma (HGG) and high-risk neuroblastoma, despite multidisciplinary therapeutic approaches, demands new treatments for these indications. F14512 is a topoisomerase II inhibitor containing a spermine moiety that facilitates selective uptake by tumor cells via the Polyamine Transport System (PTS) and increases topoisomerase II poisoning. Here, F14512 was evaluated in pediatric HGG and neuroblastoma cell lines. PTS activity and specificity were evaluated using a fluorescent spermine-coupled probe. The cytotoxicity of F14512, alone or in combination with ionizing radiation and chemotherapeutic agents, was investigated in vitro. The antitumor activity of F14512 was assessed in vivo using a liver-metastatic model of neuroblastoma. An active PTS was evidenced in all tested cell lines, providing a specific and rapid transfer of spermine-coupled compounds into cell nuclei. Competition experiments confirmed the essential role of PTS in the cell uptake and cytotoxicity of F14512. This cytotoxicity appeared greater in neuroblastoma cells compared with HGG cells but appeared independent of PTS activity levels. In vivo evaluation confirmed a marked and prolonged antitumoral effect in neuroblastoma cells. The combinations of F14512 with cisplatin and carboplatin were often found to be synergistic, and we demonstrated the significant radiosensitizing potential of F14512 in the MYCN-amplified Kelly cell line. Thus, F14512 appears more effective than etoposide in pediatric tumor cell lines, with greater efficacy in neuroblastoma cells compared with HGG cells. The synergistic effects observed with platinum compounds and the radiosensitizing effect could lead to a clinical development of the drug in pediatric oncology.

Cilengitide targets pediatric glioma and neuroblastoma cells through cell detachment and anoikis induction

The prognosis of children with high-grade glioma or high-risk neuroblastoma remains poor. Cilengitide is a selective antagonist of αvβ3 and αvβ5 integrins, which are involved in tumor growth and development of metastasis. We have evaluated the effects of cilengitide on pediatric glioma and neuroblastoma cell lines for the first time. Expression levels of αvβ3 and αvβ5 were determined by flow cytometry in three neuroblastoma and five pediatric glioma cell lines compared with adult U87-MG before and after irradiation. Cell detachment, cytotoxicity, and cell growth under nonadhesive conditions were measured using the MTS assay. Cell death and apoptosis were assessed by annexin-V/propidium iodide staining. The varying αvβ3 and αvβ5 expression levels were unrelated to tumor grade. Irrespective of the αvβ5 expression level, the pediatric cells expressing αvβ3 were dose dependently sensitive to cilengitide. UW479 cells expressed only αvβ5 integrin and were not sensitive to cilengitide, suggesting that cilengitide's action largely depends on αvβ3 inhibition. Cell detachment resulted in a higher cytotoxicity in pediatric glioma compared with U87-MG cells, which seem able to grow despite the significant cilengitide-induced cell detachment. Growth kinetics on polyHEMA showed that only pediatric glioma cells were sensitive to anoikis and so died after cilengitide-induced detachment. Furthermore, irradiation of glioma cells increased αvβ3 expression slightly but not cilengitide sensitivity. Cilengitide's action on glioma and neuroblastoma cells appears to be dependent on αvβ3 expression and sensitivity to anoikis. Cilengitide is able to target pediatric glioma and neuroblastoma cells in vitro directly and efficiently. Tumor context could validate these promising observations.

Preclinical Evaluation of Dipotassium Bisperoxo (Picolinato) Oxovanadate V for the Treatment of Pediatric Low-Grade Gliomas

The treatment of pediatric low-grade gliomas with current treatment modalities still remains ineffective among a subset of patients; hence, justifying the need to further investigate more effective therapies. Dipotassium bisperoxo (picolinato) oxovanadate V (Bpv[pic]), is a derivative of the trace metal vanadium and a potent inhibitor of protein tyrosine phosphatases, which are important mediators of oncogenic and tumor suppressive activities in cancers. In this study, we undertook a preclinical evaluation of the antineoplastic functions of Bpv(pic) in the treatment of pediatric low-grade gliomas. We utilized pediatric low-grade glioma cell lines (Res186, Res259 and R286) in a wide variety of cancer assays to determine whether Bpv(pic) can abrogate the neoplastic properties of these cells. Our preclinical evaluation of the antineoplastic properties of Bpv(pic) in pediatric low-grade gliomas reveals a significant dose-dependent decrease in cell viability as a consequence of decreased proliferation and sustained induction of growth arrest and apoptosis. Bpv(pic) significantly decreases cell migration/invasion and anchorage-independent growth in soft agarose. Within cells, Bpv(pic) functions by attenuating CDC25A activity, and by decreasing the expression of multiple protein tyrosine phosphatases, DNA repair genes, microtubule-associated genes, such as PLK1, AURKA and HDAC6, and conversely augmenting the expression of proapoptotic mediators such as BAK, AIFM and CTSL1. Collectively, our data strongly suggest novel evidence of Bpv(pic) being a potent antineoplastic drug and a suitable alternative for the treatment of pediatric low-grade gliomas.

ABT-263 Enhances Sensitivity to Metformin and 2-Deoxyglucose in Pediatric Glioma by Promoting Apoptotic Cell Death

Pediatric high grade glioma is refractory to conventional multimodal treatment, highlighting a need to develop novel efficacious therapies. We investigated tumor metabolism as a potential therapeutic target in a panel of diverse pediatric glioma cell lines (SF188, KNS42, UW479 and RES186) using metformin and 2-deoxyglucose. As a single agent, metformin had little effect on cell viability overall. SF188 cells were highly sensitive to 2-deoxyglucose however, combination of metformin with 2-deoxyglucose significantly reduced cell proliferation compared to either drug alone in all cell lines tested. In addition, the combination of the two agents was associated with a rapid decrease in cellular ATP and subsequent AMPK activation. However, increased cell death was only observed in select cell lines after prolonged exposure to the drug combination and was caspase independent. Anti-apoptotic BCL-2 family proteins have been indicated as mediators of resistance against metabolic stress. Therefore we sought to determine whether pharmacological inhibition of BCL-2/BCL-xL with ABT-263 could potentiate apoptosis in response to these agents. We found that ABT-263 increased sensitivity to 2-deoxyglucose and promoted rapid and extensive cell death in response to the combination of 2-deoxyglucose and metformin. Furthermore, cell death was inhibited by the pan-caspase inhibitor, z-VAD-FMK suggesting that ABT-263 potentiated caspase-dependent cell death in response to 2-deoxyglucose or its combination with metformin. Overall, these data provide support for the concept that targeting metabolic and anti-apoptotic pathways may be an effective therapeutic strategy in pediatric glioma.

Abstract 5048: mTORC1/mTORC2 signaling in pediatric low grade glioma and pilocytic astrocytoma.

Abstract Pilocytic astrocytoma is the most frequent low grade glioma in children and young adults. Previous studies support a role for MAPK pathway signaling, and more recently AKT/mTOR, in its biology. mTOR operates as part of two different multiprotein signaling complexes, mTORC1 and mTORC2. In the current study we evaluated mTORC1/mTORC2 pathway activity in pediatric low grade astrocytoma. We used formalin-fixed paraffin embedded human pilocytic astrocytomas for immunohistochemical studies in tissue microarrays. Moderate (2+) to strong (3+) immunostaining was observed for pS6 in 66/114(58%), p4EBP1 in 35/116(30%), pElF4G in 66/113 (58%), and mTOR (total) in 52/114 (46%), supporting mTOR pathway activation in a subset of pediatric astrocytomas. Specifically, there was increased immunoreactivity in pilocytic astrocytomas of the optic nerve proper (pS6 39/54(72%), p4EBP1 22/54 (41%), 4IF4G 41/53 (77%), and mTOR 28/54 (52%)), a difference that was statistically significant for pS6, p4E-BP1 and pEIF4G (p&amp;lt;0.05, Chi Square). Next, we studied total levels of Raptor (mTORC1 component) and Rictor (mTORC2 component). We observed moderate to strong Raptor reactivity in 81/142 (57%) and Rictor in 69/141(49%) pilocytic astrocytomas. By contrast, we observed increased Rictor immunostaining in 90/144 (63%) diffuse gliomas grade II-IV and Raptor in 28/147 (19%), a difference that was statistically significant between the two groups (p&amp;lt;0.001, Chi Square). Next we studied in vitro the effect of mTORC1 blockade in pediatric low grade glioma cell lines Res 186 and Res 259 with a TORC1 inhibitor. Using the MTT assay, Res 186 cell growth was inhibited with 1nM and 10nM drug concentrations, compared with DMSO (p&amp;lt;0.005, t-test), but the effect was less pronounced in Res 259. Additionally, pS6 levels were decreased by Western Blot in Res 186 cells, but not in Res 259, consistent with mTORC1 inhibition of Res 186 cells. The findings suggest that the mTOR pathway is active in a subset of pediatric low grade gliomas, that it varies by clinicopathologic subtype, and that it is primarily mediated by mTORC1 compared with other glioma subtypes. Furthermore, mTOR may represent a therapeutic target in pediatric low grade glioma that merits further investigation. Citation Format: Smit Shah, Marianne Hutt, Matthias A. Karajannis, Eric Raabe, Charles G. Eberhart, Fausto J. Rodriguez. mTORC1/mTORC2 signaling in pediatric low grade glioma and pilocytic astrocytoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5048. doi:10.1158/1538-7445.AM2013-5048

Investigation of Targetin, a Microtubule Binding Agent which Regresses the Growth of Pediatric High and Low Grade Gliomas.

BackgroundPediatric gliomas, the most common solid childhood neoplasm, manifest unique molecular signatures that distinguish them from adult gliomas. Unfortunately, most studies have focused on adult gliomas and extrapolate the findings to treat pediatric gliomas. In this study, we assessed the efficacy of Targetin, a folate conjugated analogue of Noscapine, on the treatment of pediatric low and high grade gliomas.MethodAn assortment of standard cancer assays were used with different drug doses and experimental durations.ResultsWe found that pediatric glioma cells are more susceptible to lower doses of Targetin than parental Noscapine. Targetin functions by disrupting the microtubule network, and can likewise perturb DNA synthesis, delay the cellular transition within the S and G2M cell cycle phases, diminish anchorage independent growth and the migratory/invasiveness of pediatric glioma cells. Moreover, Targetin impairs the expression of several regulators of cancer progression belonging to prominent signalling pathways in pediatric gliomas; including Platelet Derived Growth Factor alpha and some members of the Mitogen Activated Protein Kinase cascade.ConclusionTargetin has an excellent anti-neoplastic profile and functions to modulate the expression of several genes belonging to key cancer progression pathways in pediatric gliomas. Collectively, findings from this study highlight the usefulness of Targetin for the treatment of pediatric high and low grade gliomas.

Viability screen on pediatric low grade glioma cell lines unveils a novel anti-cancer drug of the steroid biosynthesis inhibitor family

Pediatric low grade gliomas are the most common central nervous system tumors and are still incurable among a subset of patients despite current treatment modalities. Steroid biosynthesis occurs in a wide variety of organs including the brain, to mediate an assortment of functions, including a proposed role in the growth of gliomas. Hence, targeting steroid biosynthesis and/or their signaling pathways, is anticipated as an effective approach for treating gliomas. In this study, we investigated whether our chemical library of steroid inhibitors can modulate the growth of pediatric low grade glioma cell lines (Res186, Res259, R286), and subsequently identified a potent inhibitor of 17β-hydroxysteroid dehydrogenase type 3, referred to as DK16, which functions by attenuating cell viability, proliferation, migration/invasion and anchorage independent growth and conversely induces apoptosis and cell cycle arrest in a dose and duration dependent manner. Further investigations into the mechanisms of how DK16 functions showed that this drug increased the BAX/BCL2 expression ratio, induced phosphatidylserine externalization, and mitochondrial membrane depolarizations culminating to the release and nuclear translocation of AIF. In addition, treatments of low grade glioma cell lines with DK16 increased the expression of pro-apoptotic mediators including CDK2 and CTSL1, and with the converse diminished expression of pro-survival and migratory/invasion genes like PRKCA, TERT, MAPK8, MMP1 and MMP2. Our findings collectively demonstrate the potent anti-neoplastic properties of DK16, a steroid biosynthesis inhibitor, on the growth of pediatric low grade gliomas.

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

Abstract 463: MAP4K4 (HGK) plays a key role in tumor progression

Abstract MAP4K4/HGK is an STE kinase that was identified in an siRNA screen for modulators of tumor cell motility and in a five-gene signature predicting metastasis and survival in colon cancer. Interestingly, its catalytic function is required for anchorage-independent growth in 3D soft agar (but not in 2D monolayers) of 3T3 fibroblasts and rat intestinal epithelial cells. The aim of the present study was to evaluate MAP4K4 expression in a range of human tumor cell lines and further explore its role in cell motility and invasion. Using western blotting analysis, MAP4K4 protein expression was measured in a panel of approximately 50 human tumor cell lines and 3 non-transformed cell lines. Selected high-expressing MAP4K4 cell lines were used for genetic manipulation. Lentiviral shRNA inducible (TRIPZ-RFP) and stable (GIPZ-GFP) knockdowns (KD) permitted the selection of isogenic cell lines for functional studies. Novel high-throughput 96-well plate-based 3D assays were used to analyze: (i) 3D spheroid growth, (ii) tumor cell dissemination over gelatin or collagen type I and (iii) invasion into Matrigel™. A fully automated image analysis system was validated against standard microscopy-based methods and applied to these studies. Cell viability was assessed in 2D and 3D by the Cell Titer Glo assay. Pharmacodynamic biomarkers were analyzed in KD and control cells to correlate MAP4K4 expression to the activation/inhibition status of putative downstream signalling pathways. MAP4K4 protein expression levels were high to moderate in 60% of the cell lines tested (including glioma, neuroblastoma, hepatocellular, prostate, colorectal, ovarian, squamous and breast carcinomas) and undetectable in PNT2 and MCF10A non-transformed prostate and breast epithelial cells. It was also expressed in activated human endothelial cells. The SF188 pediatric glioma cell line was prioritized for further study based on its strong expression of MAP4K4 (and activated downstream signalling pathways), the ability to generate reproducible tumor spheroids and the highly invasive nature of this cancer type. Additional confirmatory studies utilized the metastatic triple-negative human breast cancer cell line MDA MB 231. Both stable and inducible KD were achieved as described above. Functional studies showed that MAP4K4 KD induced between 30-50% inhibition of cell viability, motility and/or invasion. Inhibition of MAP4K4 expression in SF188 cells also down-regulated key downstream signalling molecules such as pJNK, pSTAT3, pTAK1 and MMP2. In MDA MB 231, MAP4K4 was upregulated when cells were cultured in 3D spheroids and again, stable knockdown inhibited 3D migration by ∼ 50%. Conclusions: In the present study we provide evidence that MAP4K4 plays an important role in functions associated with tumor progression (ability to grow in 3D, migration and invasion) in two cancer types where invasion and/or metastasis contribute to their notoriously poor prognosis. 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 463. doi:1538-7445.AM2012-463

Abstract 196: Functional role of death-associated protein 3 in high grade gliomas of childhood

Abstract Background: High grade gliomas (HGG) are highly invasive tumors and respond poorly to conventional treatments. Although significant progress has been made in understanding the molecular pathways that lead to the development of HGG in adults, comparatively few data is available for gliomas of childhood. The death-associated protein 3 (DAP3) is localized on the chromosomal region 1q21-22 that has been previously described as a region of frequent chromosomal gain associated with poor outcome in HGG. Moreover, DAP3 has been reported to be overexpressed and to play anti-apoptotic role in invasive glioblastoma cells. Methods: In the current study to investigate the functional role of DAP3 in pediatric versus adult HGG, we suppressed the endogenous DAP3 protein using DAP3-specific short hairpin RNA (shRNA) or chemical inhibitors of protein kinase C, and analyzed the phenotypic effect on glioma cell proliferation, migration, mitochondrial morphology and apoptosis. Results: We found that knockdown of DAP3 by shRNA induced mitochondrial fragmentation and apoptosis in pediatric glioma cell lines while in adult glioma cell lines it suppressed cell proliferation and reduced mitochondrial potential without inducing mitochondrial fragmentation. Specific chemical inhibition of the two classical PKC isoforms (PKCα and PKCβ) significantly reduced the expression of DAP3 protein in both pediatric and adult glioma cell lines. The inhibition of PKCα changed cell morphology, mitochondrial status and significantly suppressed proliferation and migration of both pediatric and adult glioma cell lines without associated caspase activation indicating that DAP3 may mediate nonapoptotic form of cell death in HGG. The suppression of PKCβ rendered pediatric glioma cells more sensitive to the apoptotic effect of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) through a caspase-dependent process as compared with adult glioma cells suggesting a possible implication of DAP3 in intrinsic and extrinsic pathways for apoptosis in pediatric HGG. Conclusion: Our findings confirmed the anti-apoptotic role of DAP3 in glioma cell biology suggesting that specific PKC isoforms mediated the DAP3 suppression of apoptosis in HGG and pointed out the specific functional role of this protein in mitochondrial maintenance. DAP3 may represent a novel target for the treatment of gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 196. doi:10.1158/1538-7445.AM2011-196