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Supplemental Figure 6. A. Drug naive and panobinostat and bortezomib-resistant (P + B) U87 cells were treated with gefitinib (2 µmol/L), enzastaurin (2 µmol/L), dasatinib (0.1 µmol/L), dinaciclib (0.18 µmol/L), PI 103 (1 µmol/L), PD 0325901 (2 µmol/L), HSP 990 (0.025 µmol/L), ABT 737 (2 µmol/L), JQ 1 (2 µmol/L), TP 0903 (2 µmol/L), Bay 11-7082 (2 µmol/L), YM 155 (0.025 µmol/L), cucurbitacin-I (0.2 µmol/L), vincristine (0.01 µmol/L), vinblastine (0.01 µmol/L), taxol (0.05 µmol/L), topotecan (0.025 µmol/L), temozolomide (25 µmol/L), gemcitabine (0.25 µmol/L) and 5-fluorouracil (20 µmol/L).

A, MTT cell viability relative absorbance values across multiple cell lines after a 7-day incubation with Veliparib, Olaparib, or Niraparib. Boxes with an asterisk denote that an indicated concentration of PARP inhibitor significantly reduced cell viability relative vehicle treated cells. B, MTT cell viability heat map with numerical values. C, Linear regression analysis between PARP1 protein levels, relative to GAPDH, across all cell lines vs. IC50 values calculated from MTT viability assay on day 7. D, Linear regression analysis between PARP1 activity levels, relative to GAPDH, across all cell lines vs. IC50 values calculated from MTT viability assay on day 7. E, Different exposure of PARP1 Western blot in SJG2, SF188, 462, and 626 cells demonstrating variable PARP1 protein expression. *, **, ***, **** Denotes significance of p

Supplemental File 2: Genes common to all resistant U87 cell lines compared to drug naive control

Supplemental File 5

Supplemental Figure 4. Volcano plot representing differentially expressed genes for U87 naive versus resistant cells with a minimum of a 1.5-fold change, P-value < 0.05 and FDR < 0.10 between control and A.

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Supplemental Figure 2. Drug naive U87 or panobinostat-resistant, bortezomib-resistant or panobinostat and bortezomib-resistant cells were treated with the combination of panobinostat (25 nmol/L) plus bortezomib (2.5 nmol/L) for 24 h.

A and C, Representative immunofluorescence staining for γH2AX and ki67 in SJG2, SF188, KNS42, and DIPG58 cells after a 24 hour incubation with either DMSO vehicle (V) or 5 μM Niraparib (N). DAPI was used as a nuclear counterstain. B and D, Quantification of γH2AX and ki67 in SJG2, SF188, KNS42, and DIPG58 cells. γH2AX images were taken at 63X magnification, scale bar 12 μm, ki67 images were taken at 25X magnification, scale bar 30 μm. **, ***, **** Denotes significance of p

A, Several representative time points of SJG2 cells after immunofluorescent staining for γH2AX with DAPI as a counterstain after 1μM Niraparib, 2 Gy gamma radiation, or 2 Gy radiation pre-treated with 1μM Niraparib. Images were taken at indicated time points at 64X magnification.

PDF file - 432KB, Supplementary Figure 1: Validation of Temozolomide (TMZ) siRNA Screen. Supplementary Figure 2: Base Excision Repair correlates with IC50 of alkylating agents. Supplementary Figure 3: Loss of MPG promotes sensitivity to alkylating agents. Supplementary Figure 4: Over-expression of MPG promotes resistance to TMZ. Supplementary Figure 5: Targeting multiple DNA repair proteins has an additive effect on TMZ sensitivity. Supplementary Figure 6: ATM interacts with MPG and regulates MPG glycosylase activity. Supplementary Figure 7: Methoxyamine enhances cytotoxicity of TMZ in pGBM cell lines. Supplementary Figure 8: Methoxyamine enhances cytotoxicity of TMZ in primary pGBM cultures. Supplementary Figure 9: Normal astrocytes have redundant pathways limiting cytotoxicity when the ATM-MPG pathways are inhibited.

Supplemental Figure 1. Schematic of the approach taken to achieve drug resistance.

Supplemental Figure 5. Drug naive or panobinostat and bortezomib resistant (Pano + Bort) U87 or SJG 2 cells were either non-target siRNA (NT siRNA) or QPRT siRNA-2 as described in the Materials and Methods.

Supplemental Figure 3. Drug naive U87 or panobinostat-resistant, bortezomib-resistant, or panobinostat and bortezomib-resistant (P + B) cells were cotreated with panobinostat (25 nmol/L) plus bortezomib (2.5 nmol/L) for 24 h.

Supplementary Figure Legends

Supplemental File 1: Differential Gene Expression between drug naive versus resistant/"recovery" U87 cell line

Supplemental Figure Legends 1-7

Supplemental File 4: DIPG007 Genes, SJG2 Genes, U87 Genes

A, B, MTT viability relative absorbance values of SJG2 and SF188 after being treated with an indicated concentration of Niraparib for 72h. C, Western blot for cleaved PARP1 in SJG2 and SF188 after being treated with an indicated concentration of Niraparib for 72h. D, E, Propidium Iodide DNA staining analysis in SJG2, SF188 cells, respectively, after a 72h incubation with an indicated concentration of Niraparib. *, ***, **** Denotes significance of p

A, Representative immunohistochemical staining for PARP1 in pHGA and DIPG patient samples. Pictures were taken at 10X magnification. Scale bar 100 μm.

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Supplemental Figure 7. Bar graph showing QPRT mRNA expression levels (absolute value) between drug naive and panobinostat + bortezomib resistant DIPG 007, SJG 2 and U87 cells.

Supplemental File 3: Gene-Ontology Analysis

Glioblastoma (GBM) is the most prevalent and aggressive type of primary human brain tumours originating in the central nervous system. Despite the fact that current treatments involve surgery, chemotherapy (Temozolomide), and radiation therapy, the prognosis for patients diagnosed with GBM remains extremely poor. The standard treatment is not only unable to completely eradicate the tumour cells, but also tumour recurrence after surgical resection presents a major challenge. Furthermore, adjuvant therapies including radiation and chemotherapy have high cytotoxicity which causes extensive damage to surrounding healthy tissues and treatment is usually halted before GBM is fully eradicated. Finally, most GBM cases demonstrate temozolomide resistance, a common reason for GBM treatment failure. Therefore, there is an urgent need to develop a suitable alternative therapy that targets GBM specifically and has low cytotoxicity for healthy cells. We previously reported that transient receptor potential melastatin 7 (TRPM7) channels are aberrantly upregulated in GBM, and inhibition of TRPM7 reduced GBM cellular functions including proliferation, migration, and invasion. This suggests TRPM7 is a potential therapeutic target for GBM treatment. In this study, we investigated the effects of the TRPM7 inhibitor, carvacrol, on human GBM cell lines U87 and U251 in vivo. With the use of a flank xenograft GBM mouse model, we demonstrated that carvacrol significantly reduced the tumour size in both mice injected with U87 and U251 cells, decreased p-Akt protein level and increased p-GSK3β protein levels. Therefore, these results suggest that carvacrol may have therapeutic potential for GBM treatment.

Suplemental Figure Legends for supplemental data

Supplemental Figure 1: Related to Figure 1: Characterization of Gene-Trapped Clones Supplemental Figure 2 Related to Figure 2: PINK1 alters normal astrocyte metabolism Supplemental Figure 3 Related to Figure 2: PINK1 alters normal astrocyte metabolism Supplemental Figure 4: Related to Figure 3: PINK1 alters normal astrocyte metabolism and stabilizes HIF1a Supplemental Figure 5: Related to Figure 3: PINK1 overexpression stops GBM cell growth and inhibits glycolysis. Supplemental Figure 6: Related to Figure 3: PINK1 overexpression inhibits glycolysis. Supplemental Figure 7: Related to Figure 3: PINK1 overexpression inhibits glycolysis. Supplemental Figure 8: Related to Figure 4: Selective targeting in PINK1 expressing GBM cells leads to reduced viability Supplemental Figure 9: Related to Figure 4: Targeting of PINK1 in GBM cells leads to reduced colony formation and caspase activity Supplemental Figure 10: Related to Figure 5 and 6. PINK1 expression in vivo. Supplemental Table 1: Genes identified from gene-trap screen.

Proliferating cancer cells are characterized by high rates of glycolysis, lactate production, and altered mitochondrial metabolism. This metabolic reprogramming provides important metabolites for proliferation of tumor cells, including glioblastoma. These biological processes, however, generate oxidative stress that must be balanced through detoxification of reactive oxygen species (ROS). Using an unbiased retroviral loss-of-function screen in nontransformed human astrocytes, we demonstrate that mitochondrial PTEN-induced kinase 1 (PINK1) is a regulator of the Warburg effect and negative regulator of glioblastoma growth. We report that loss of PINK1 contributes to the Warburg effect through ROS-dependent stabilization of hypoxia-inducible factor-1A and reduced pyruvate kinase muscle isozyme 2 activity, both key regulators of aerobic glycolysis. Mechanistically, PINK1 suppresses ROS and tumor growth through FOXO3a, a master regulator of oxidative stress and superoxide dismutase 2. These findings highlight the importance of PINK1 and ROS balance in normal and tumor cells. PINK1 loss was observed in a significant number of human brain tumors including glioblastoma (n > 900) and correlated with poor patient survival. PINK1 overexpression attenuates in vivo glioblastoma growth in orthotopic mouse xenograft models and a transgenic glioblastoma model in Drosophila. Cancer Res; 76(16); 4708–19. ©2016 AACR.

Suplemental methods for experiments appearing in supplemental data and expanded details from methods in main manuscript.

To improve therapeutic responses in patients with glioma, new combination therapies that exploit a mechanistic understanding of the inevitable emergence of drug resistance are needed. Intratumoral heterogeneity enables a low barrier to resistance in individual patients with glioma. We reasoned that targeting two or more fundamental processes that gliomas are particularly dependent upon could result in pleiotropic effects that would reduce the diversity of resistant subpopulations allowing convergence to a more robust therapeutic strategy. In contrast to the cytostatic responses observed with each drug alone, the combination of the histone deacetylase inhibitor panobinostat and the proteasome inhibitor bortezomib synergistically induced apoptosis of adult and pediatric glioma cell lines at clinically achievable doses. Resistance that developed was examined using RNA-sequencing and pharmacologic screening of resistant versus drug-naïve cells. Quinolinic acid phosphoribosyltransferase (QPRT), the rate-determining enzyme for de novo synthesis of NAD+ from tryptophan, exhibited particularly high differential gene expression in resistant U87 cells and protein expression in all resistant lines tested. Reducing QPRT expression reversed resistance, suggesting that QPRT is a selective and targetable dependency for the panobinostat–bortezomib resistance phenotype. Pharmacologic inhibition of either NAD+ biosynthesis or processes such as DNA repair that consume NAD+ or their simultaneous inhibition with drug combinations, specifically enhanced apoptosis in treatment-resistant cells. Concomitantly, de novo vulnerabilities to known drugs were observed. Implications: These data provide new insights into mechanisms of treatment resistance in gliomas, hold promise for targeting recurrent disease, and provide a potential strategy for further exploration of next-generation inhibitors.

Primary central nervous system (CNS) tumors are now the most common cause of childhood cancer–related deaths. Pediatric high-grade gliomas (pHGGs) are among the most lethal brain tumors with a 5-year survival rate of only 20%. MYCN pHGGs represent one subgroup with an unmet need for therapeutics. MYCN belongs to the family of MYC transcription factors that regulate numerous cancer hallmarks such as proliferation, apoptosis, and metabolism. While no direct inhibitors of MYCN are in clinical trial, current strategies focus on targeting the MYCN mediated transcriptional machinery or cell cycle regulators. Lack of relevant pHGG models for pre-clinical testing contribute to limited therapeutic efficacy. To address these knowledge gaps, we developed a novel mouse model of MYCN pHGG using the FLEx-Cre switch system, whereby neural stem cells are selectively delivered with MYCN cDNA and shRNA targeting the tumor suppressor genes p53 and Pten and form tumors in vivo. We identified that this model harbors hyper-activation of the PI3K/AKT/mTOR signaling pathway. We demonstrate that dual PI3K-mTOR blood brain barrier penetrant inhibitors are effective in reducing pHGG growth and MYCN protein levels. Because treatment-resistance is a fundamental feature of pHGGs, we developed a novel drug-resistance model of MYCN pHGG as a mechanistic tool to identify relevant resistance mechanisms. Using transcriptome analysis, we identified the insulin growth factor signaling pathway as our top mechanism of resistance. We hypothesized that MYCN is a critical driver of pHGG and can be effectively targeted via dual inhibition of PI3K-mTOR and IGF/Insulin signaling pathways. We tested next generation inhibitors of IGF and PI3K/mTOR pathways and performed genetic and pharmacological assays in our MYCN pHGG and human MYCN pHGG cells. Inhibition of both pathways in our MYCN pHGG model and human MYCN cells were synergistic, leading to significant decreases in cell growth and MYCN signaling.

More than 60% of supratentorial ependymomas harbor a ZFTA–RELA (ZRfus) gene fusion (formerly C11orf95–RELA). To study the biology of ZRfus, we developed an autochthonous mouse tumor model using in utero electroporation (IUE) of the embryonic mouse brain. Integrative epigenomic and transcriptomic mapping was performed on IUE-driven ZRfus tumors by CUT&RUN, chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin sequencing, and RNA sequencing and compared with human ZRfus-driven ependymoma. In addition to direct canonical NFκB pathway activation, ZRfus dictates a neoplastic transcriptional program and binds to thousands of unique sites across the genome that are enriched with PLAGL family transcription factor (TF) motifs. ZRfus activates gene expression programs through recruitment of transcriptional coactivators (Brd4, Ep300, Cbp, Pol2) that are amenable to pharmacologic inhibition. Downstream ZRfus target genes converge on developmental programs marked by PLAGL TF proteins, and activate neoplastic programs enriched in Mapk, focal adhesion, and gene imprinting networks. Significance: Ependymomas are aggressive brain tumors. Although drivers of supratentorial ependymoma (ZFTA- and YAP1-associated gene fusions) have been discovered, their functions remain unclear. Our study investigates the biology of ZFTA–RELA-driven ependymoma, specifically mechanisms of transcriptional deregulation and direct downstream gene networks that may be leveraged for potential therapeutic testing. This article is highlighted in the In This Issue feature, p. 2113

Diffuse intrinsic pontine glioma (DIPG) is a surgically unresectable and devasting tumour in children. To date, there are no effective chemotherapeutics despite a myriad of clinical trials. The intact blood-brain barrier (BBB) is likely responsible for the limited clinical response to chemotherapy. MRI-guided focused ultrasound (MRgFUS) is a promising non-invasive method for treating CNS tumours. Moreover, MRgFUS allows for the temporary and repeated disruption of the BBB. Our group previously reported the feasibility of temporary BBB opening within the normal murine brainstem using MRgFUS following intravenous (IV) administration of microbubbles. In the current study, we set out to test the effectiveness of targeted chemotherapy when paired with MRgFUS in murine models of DIPG. Doxorubicin was selected from a drug screen consisting of conventional chemotherapeutics tested on patient-derived cell lines. We studied the RCAS/Tv-a model where RCAS-Cre, RCAS-PDGFB, and RCAS-H3.3K27M were used to drive tumourigenesis upon injection in the pons. We also used orthotopically injected SU-DIPG-6 and SU-DIPG-17 xenografts which demonstrated a diffusely infiltrative tumour growth pattern similar to human DIPG. In our study, SU-DIPG-17 xenografts were more representative of human DIPG with an intact BBB. Following IV administration of doxorubicin, MRgFUS-treated animals exhibited a 4-fold higher concentration of drug within the SU-DIPG-17 brainstem tumours compared to controls. Moreover, the volumetric tumour growth rate was significantly suppressed in MRgFUS-treated animals whose tumours also exhibited decreased Ki-67 expression. Herein, we provide evidence for the ability of MRgFUS to enhance drug delivery in a mouse model of DIPG. These data provide critical support for clinical trials investigating MRgFUS-mediated BBB opening, which may ameliorate DIPG chemotherapeutic approaches in children.

Patient-derived xenografts retain the genotype of the parent tumors more readily than tumor cells maintained in culture. The two previously reported clival chordoma xenografts were derived from recurrent tumors after radiation. To study the genetics of clival chordoma in the absence of prior radiation exposure we established a patient-derived xenograft at primary resection of a clival chordoma. Epicranial grafting of clival chordoma collected during surgery was performed. Tumor growth was established in a nonobese diabetic/severe combined immunodeficiency mouse and tumors have been passaged serially for seven generations. Physaliferous cell architecture was shown in the regenerated tumors, which stained positive for Brachyury, cytokeratin, and S100 protein. The tumors showed bone invasion. Single-nucleotide polymorphism analysis of the tumor xenograft was compared with the parental tumor. Copy number gain of the T gene (brachyury) and heterozygous loss of cyclin dependent kinase inhibitor 2A (CDKN2A) was observed. Heterozygous loss of the tumor-suppressor fragile histidine triad (FHIT) gene also was observed, although protein expression was preserved. Accumulation of copy number losses and gains as well as increased growth rate was observed over three generations. The patient-derived xenograft reproduces the phenotype of clival chordoma. This model can be used in the future to study chordoma biology and to assess novel treatments.

Atypical Teratoid/Rhabdoid Tumours (ATRT) are highly malignant neoplasms arising primarily in the CNS of children. They are defined by loss of function mutations in smarcb1, a gene serving a vital role in neurogenesis and differentiation. In order to recapitulate ATRT in the mouse, we used a Cre-Lox recombination system to conditionally knockout smarcb1 in specific cell compartments. Loss of smarcb1 in BLBP-expressing cells of the developing brain led to severe neurologic defects. Mice exhibited seizures, ataxia, and median 12-day survival. Histological analysis revealed severe thinning of the cerebral cortex and cerebellum. Temporally-targeted smarcb1 loss in BLBP/Nestin-expressing embryonic compartments did not result in tumour formation. Similarly, BLBP-expressing, smarcb1-deficient neural stem/progenitor cells (NSC/NPCs) were isolated and allografted but did not form tumours. These cells demonstrated decreased proliferation, higher apoptosis, and upregulation of p53, CDKN1A, and CDKN2A. In contrast, ubiquitous smarcb1 loss at the earlier embryonic day 6.5 produced widespread tumorigenicity in the forebrain, hindbrain, skullbase, and spine; each with unique phenotypes, survival, and morphology. We employed a clinically-relevant Nanostring gene-panel screen to stratify tumours into genetically distinct subgroups. Our findings indicate that smarcb1 plays an important role in CNS development. Loss of smarcb1 in NSC/NPCs is lethal, and its developmental context influences cell fate. Targeted smarcb1 loss likely plays a tumorigenic role at an earlier developmental stage than previously determined, in a diverse array of primitive stem cells. These data support the generation of a murine ATRT model capable of producing distinct tumour entities that recapitulate the human disease.

Pediatric glioblastoma (pGBM) are incurable brain tumors with overall poor prognosis and response to treatments due to molecular and epigenetic heterogeneity. In particular, the MYCN subtype of pGBM are a highly aggressive form of GBM with a dismal median survival of only 14 months. Furthermore, this subtype is enriched with loss of the tumor suppressor genes TP53 and PTEN, leading to aberrantly active PI3K-AKT signaling pathway and DNA-checkpoint abnormalities. Here, we report the generation of a novel syngeneic mouse model that recapitulates the features of the MYCN subtype of pGBM. We isolated Sox2-Cre neural stem cells from C57BL/6 mice and transduced inverted retroviral-cassettes of the murine Mycn oncogene simultaneously with shRNA targeting tumor suppressor genes p53 and Pten. Retroviral-cassettes are flanked by tandem LoxP sites arranged so that Cre recombinase expression inverts the cassettes in frame allowing for MYCN protein expression and loss of the P53/PTEN proteins. Transgene activation is accompanied with selectable cell surface markers and fluorescent tags enabling for fluorescent activated cell sorting (FACS) of the desired cell populations. Neural stem cells with MYCN protein expression and concurrent silencing of P53 and PTEN protein (NPP cells) result in significantly increased proliferation and activation of PI3K-AKT pathway as compared to control neural stem cells and have. Injection of NPP cells into the forebrain of immune competent C57BL/6 mice result in the formation of invasive high-grade gliomas with a lethal phenotype at ~50 days post injection. Using several next generation brain penetrant small molecule inhibitors of the PI3K-AKT pathway, we show inhibition of tumorigenesis in vitro. Moreover, we have identified several novel mechanisms of PI3KAKT treatment resistance and are currently identifying therapies that may overcome this resistance through RNA seq analysis. In summary, well defined genetic drivers of GBM can lead to informed mouse model generation to test promising therapies.

To improve therapeutic responses in glioma patients, new combination therapies that exploit a mechanistic understanding of the inevitable emergence of drug resistance are needed. Intra-tumoral heterogeneity enables a low barrier to resistance in individual glioma patients. We reasoned that targeting two or more fundamental processes that gliomas are particularly dependent upon could result in pleiotropic effects that would reduce the diversity of resistant subpopulations allowing convergence to a more robust therapeutic strategy. In contrast to the cytostatic responses observed with each drug alone, the combination of the HDAC inhibitor panobinostat and the proteasome inhibitor bortezomib synergistically induced apoptosis of adult and pediatric glioma cell lines at clinically achievable doses. Resistance that developed was examined using RNA sequencing and pharmacological screening of resistant versus drug naive cells. Quinolinic acid phosphoribosyltransferase (QPRT), the rate-determining enzyme for de novo synthesis of nicotinamide adenine dinucleotide (NAD+) from tryptophan, exhibited particularly high differential gene expression in resistant U87 cells and protein expression in all resistant lines tested. Reducing QPRT expression reversed resistance, suggesting that QPRT is a selective and targetable dependency for the panobinostat-bortezomib resistance phenotype. Pharmacological inhibition of either NAD+ biosynthesis or processes such as DNA repair that consume NAD+ or their simultaneous inhibition with drug combinations, specifically enhanced apoptosis in treatment-resistant cells. Concomitantly, de novo vulnerabilities to known drugs were observed.

Most medulloblastoma protocols worldwide include vincristine during radiation and chemotherapy. A significant dose-limiting toxicity is peripheral neuropathy; however, there is a paucity of data to support the view that omission of vincristine does not impact survival. Herein we report two adolescent patients with Group 4 and SHH medulloblastoma, where vinblastine successfully replaced vincristine with resolution of their peripheral neuropathy. We furthermore show vinblastine is highly active in vitro and demonstrates equivalent antitumoral activity compared to vincristine. Substitution of vincristine with vinblastine in future studies should be considered for all patients with medulloblastoma, particularly those with hereditary neuropathy, severe vincristine toxicity, and adults.

First-line cancer therapies such as alkylating agents and radiation have limited survival benefits for Glioblastoma (GBM) patients. Current research strongly supports the notion that inhibition of aberrant tumor metabolism holds promise as a therapeutic strategy when used in combination with radiation and chemotherapy. Hexokinase 2 (HK2) has been shown to be a key driver of altered metabolism in GBM, and presents an attractive therapeutic target. To date, no study has fully assessed the therapeutic value of targeting HK2 as a mechanism to sensitize cells to standard therapy, namely in the form of radiation and temozolomide (TMZ). Using cell lines and primary cultures of GBM, we showed that inducible knockdown of HK2 altered tumor metabolism, which could not be recapitulated by HK1 or HK3 loss. HK2 loss diminished both in vivo tumor vasculature as well as growth within orthotopic intracranial xenograft models of GBMs, and the survival benefit was additive with radiation and TMZ. Radio-sensitization following inhibition of HK2 was mediated by increased DNA damage, and could be rescued through constitutive activation of ERK signaling. This study supports HK2 as a potentially effective therapeutic target in GBM.

Proliferating cancer cells are characterized by high rates of glycolysis, lactate production, and altered mitochondrial metabolism. This metabolic reprogramming provides important metabolites for proliferation of tumor cells, including glioblastoma. These biological processes, however, generate oxidative stress that must be balanced through detoxification of reactive oxygen species (ROS). Using an unbiased retroviral loss-of-function screen in nontransformed human astrocytes, we demonstrate that mitochondrial PTEN-induced kinase 1 (PINK1) is a regulator of the Warburg effect and negative regulator of glioblastoma growth. We report that loss of PINK1 contributes to the Warburg effect through ROS-dependent stabilization of hypoxia-inducible factor-1A and reduced pyruvate kinase muscle isozyme 2 activity, both key regulators of aerobic glycolysis. Mechanistically, PINK1 suppresses ROS and tumor growth through FOXO3a, a master regulator of oxidative stress and superoxide dismutase 2. These findings highlight the importance of PINK1 and ROS balance in normal and tumor cells. PINK1 loss was observed in a significant number of human brain tumors including glioblastoma (n > 900) and correlated with poor patient survival. PINK1 overexpression attenuates in vivo glioblastoma growth in orthotopic mouse xenograft models and a transgenic glioblastoma model in Drosophila. Cancer Res; 76(16); 4708–19. ©2016 AACR.

Diffuse intrinsic pontine glioma (DIPG) is a surgically unresectable and devasting tumor in children. To date, there have been no effective chemotherapeutics despite a myriad of clinical trials. The intact blood-brain barrier (BBB) in part is responsible for the limited clinical response to chemotherapy. MRI guided focused ultrasound (MRgFUS) is a promising non-invasive tissue ablative method for CNS tumors. Moreover, MRgFUS allows for the temporary disruption of BBB. Our first objective was to determine the feasibility and safety of temporary BBB disruption within the brainstem using MRgFUS following intravenous (IV) administration of microbubbles in vivo. Our second objective was to select effective chemotherapeutics against DIPG cell lines, and to examine their therapeutic effects with MRgFUS in a mouse model of DIPG which exhibits an intact BBB. The non-invasive opening of the BBB was determined in the brainstem of normal rodents using physiological monitoring and histological analysis. Doxorubicin was selected from a drug screen consisting of conventional chemotherapeutics using SU-DIPG4 and SU-DIPG17 cell lines. We established SU-DIPG17 xenografts which demonstrated diffusely infiltrative tumor growth similar to human DIPG. By LC-MS/MS analysis, MRgFUS led to a 4-fold increase in doxorubicin concentrations within the brainstem tumors following IV administration when compared to IV administration alone, We demonstrated feasibility and safety of MRgFUS in the rodent brainstem and have shown that MRgFUS increases doxorubicin uptake in the brainstem of a rodent model of DIPG. These preclinical data will be helpful in designing clinical trials of BBB disruption using MRgFUS for DIPG in children.

Diffuse midline gliomas (DMGs) are incurable brain tumors with an aggressive onset. Apart from irradiation, there are currently no effective therapies available for patients with DMG, who have a median survival time of less than one year. Most DMG cells harbor mutations in genes encoding histone H3 (H3K27M) proteins, resulting in a global reduction of H3K27 trimethylation and activation of oncogenic signaling pathways. Here we show that the H3K27M mutations contribute to RAS pathway signaling, which is augmented by additional RAS activators including PDGFRA. H3K27M mutation led to increased expression of receptor tyrosine kinases (RTK). A RAS pathway functional screen identified ERK5, but not ERK1/2, as a RAS pathway effector important for DMG growth. Suppression of ERK5 decreased DMG cell proliferation and induced apoptosis in vitro and in vivo. In addition, depletion or inhibition of ERK5 significantly increased survival of mice intracranially engrafted with DMG cells. Mechanistically, ERK5 directly stabilized the proto-oncogene MYC at the protein level. Additionally, persistent ERK5 depletion does not result in complete growth inhibition and therefore we set out to determine potential adaptation or resistance mechanisms in response to ERK5 loss. Using RNA-sequencing and Immunoprecipitation (IP) mass spectrometry (IP-MS), we have identified several positive and negative feedbacks involved in ERK5 that are also targetable. These findings identify the H3K27M mutation as an enhancer of RAS activation in DMG with ERK5 and ERK5 regulated networks immediately actionable pathways.

INTRODUCTION Pediatric High-Grade Gliomas (PHGG), which include Diffuse Midline Gliomas (DMG), are a leading cause of brain tumor death in children. Our recent work has identified extracellular signal-regulated kinase 5 (ERK5) as a critical mediator of cell survival in DMG, as ERK5 knockdown decreases cell proliferation and extends survival time in orthotopic xenograft mice. Further investigation into the structure of ERK5 shows that it has a kinase domain and, unlike other ERKs, a transactivation domain, and both are important for promoting cell proliferation. HYPOTHESIS AND METHODS We hypothesize that identifying interactors and substrates of ERK5 could identify clinically actionable proteins and provide a more mechanistic insight into ERK5 in the progression of PHGGS. To determine protein–protein associations (PPAs), we employed the proximity-dependent biotin identification (BioID2) method and generated inducible ERK5-BioID2 and ERK2-BioID2 constructs to overcome barriers of conventional screening methods for PPAs. ERK2, similar in structure but much more studied than ERK5, was used as a comparison. Using DIPG lines as proof of principle, we performed streptavidin pull down assays for putative biotinylated ERK PPA and followed with mass spectrometry to identify the ERK2 and ERK5 interactomes. RESULTS Using data-dependent acquisition (DDA), we identified several unique and common interactors of ERK5 compared to ERK2. Through STRING network and pathway analysis, we identified a novel function of ERK5 with respect to membrane trafficking and vesicle transport. Identification of interactors with ERK5 may lead to effective therapeutic combinations. Our current work is focused on validating these interactions and the function of ERK5 in these biological processes. CONCLUSIONS Currently, ERK5 is not as understood as ERK1 or ERK2. Identification of interactors and substrates of ERK5 will further our understanding of PHGG biology, and may lead to identifying druggable targets and pathways.

Pediatric High-Grade Gliomas (PHGG), which include Diffuse Midline Gliomas (DMG), are a leading cause of brain tumor death in children. Our recent work has identified extracellular signal-regulated kinase 5 (ERK5) as a critical mediator of cell survival in PHGG. Suppression of ERK5 genetically or pharmacologically leads to decreased cell proliferation and increased apoptosis both in vitro and in vivo in multiple PHGG and H3K27M mutant DMG cell lines. Mechanistically, we show that ERK5 directly stabilizes the proto-oncogene MYC at the protein level, providing rationale to clinically target ERK5. ERK5 contains both a kinase domain (KD) and a transactivation domain (TAD), unlike all other ERKs. Unexpectedly, we found that our ERK5 depleted cells could be partially rescued by an ERK5 kinase domain dead (ERK5-KDD) but TAD intact construct. Additionally, persistent ERK5 depletion does not result in complete growth inhibition and therefore we set out to determine potential adaptation or resistance mechanisms in response to ERK5 loss. To address this, we performed RNA sequencing of DMG cells, comparing control cells to ERK5 knockdown cells, and performed gene-ontology (GO) pathway analysis to identify transcriptional changes that occur in response to ERK5 depletion. We identified 105 differentially expressed genes, and GO analysis identified alternative receptor tyrosine kinase (RTK) gene-expression as one of the top biological processes upregulated in response to ERK5 loss. We validated our top targets at the RNA and the protein level. Our top targets were Erb-B2 Receptor Tyrosine Kinase 4 (ERBB4) and Discoidin Domain Receptor Tyrosine Kinase 2 (DDR2), both clinically actionable targets. Our future work will focus on functional validation of these RTKs as potential resistance mechanisms to ERK5 loss. Identification of resistance mechanisms to ERK5 loss will have both biological and translational relevance and may lead to effective therapeutic combinations.

Pediatric high-grade astrocytomas (pHGA) and diffuse intrinsic pontine gliomas (DIPG) are devastating malignancies for which no effective therapies exist. We investigated the therapeutic potential of PARP1 inhibition in preclinical models of pHGA and DIPG. PARP1 levels were characterized in pHGA and DIPG patient samples and tumor-derived cell lines. The effects of PARP inhibitors veliparib, olaparib, and niraparib as monotherapy or as radiosensitizers on cell viability, DNA damage, and PARP1 activity were evaluated in a panel of pHGA and DIPG cell lines. Survival benefit of niraparib was examined in an orthotopic xenograft model of pHGA. About 85% of pHGAs and 76% of DIPG tissue microarray samples expressed PARP1. Six of 8 primary cell lines highly expressed PARP1. Interestingly, across multiple cell lines, some PARP1 protein expression was required for response to PARP inhibition; however, there was no correlation between protein level or PARP1 activity and sensitivity to PARP inhibitors. Niraparib was the most effective at reducing cell viability and proliferation (MTT and Ki67). Niraparib induced DNA damage (γH2AX foci) and induced growth arrest. Pretreatment of pHGA cells with a sublethal dose of niraparib (1 μmol/L) before 2 Gy of ionizing radiation (IR) decreased the rate of DNA damage repair, colony growth, and relative cell number. Niraparib (50 mg/kg) inhibited PARP1 activity in vivo and extended survival of mice with orthotopic pHGA xenografts, when administered before IR (20 Gy, fractionated), relative to control mice (40 vs. 25 days). Our data provide in vitro and in vivo evidence that niraparib may be an effective radiosensitizer for pHGA and DIPG. Mol Cancer Ther; 14(11); 2560–8. ©2015 AACR.

Magnetic Resonance Image-guided Focused Ultrasound (MRgFUS) has been used to achieve transient blood brain barrier (BBB) opening without tissue injury. Delivery of a targeted ultrasonic wave causes an interaction between administered microbubbles and the capillary bed resulting in enhanced vessel permeability. The use of MRgFUS in the brainstem has not previously been shown but could provide value in the treatment of tumours such as Diffuse Intrinsic Pontine Glioma (DIPG) where the intact BBB has contributed to the limited success of chemotherapy. Our primary objective was to determine whether the use of MRgFUS in this eloquent brain region could be performed without histological injury and functional deficits. Our secondary objective was to select an effective chemotherapeutic against patient derived DIPG cell lines and demonstrate enhanced brainstem delivery when combined with MRgFUS in vivo. \ud \ud \ud \ud Female Sprague Dawley rats were randomised to one of four groups: 1) Microbubble administration but no MRgFUS treatment; 2) MRgFUS only; 3) MRgFUS + microbubbles; and 4) MRgFUS + microbubbles + cisplatin. Physiological assessment was performed by monitoring of heart and respiratory rates. Motor function and co-ordination were evaluated by Rotarod and grip strength testing. Histological analysis for haemorrhage (H & E), neuronal nuclei (NeuN) and apoptosis (cleaved Caspase-3) was also performed. A drug screen of eight chemotherapy agents was conducted in three patient-derived DIPG cell lines (SU-DIPG IV, SU-DIPG XIII and SU-DIPG XVII). Doxorubicin was identified as an effective agent. NOD/SCID/GAMMA (NSG) mice were subsequently administered with 5 mg/kg of intravenous doxorubicin at the time of one of the following: 1) Microbubbles but no MRgFUS; 2) MRgFUS only; 3) MRgFUS + microbubbles and 4) no intervention. Brain specimens were extracted at 2 h and doxorubicin quantification was conducted using liquid chromatography mass spectrometry (LC/MS). \ud \ud \ud \ud BBB opening was confirmed by contrast enhancement on T1-weighted MR imaging and positive Evans blue staining of the brainstem. Normal cardiorespiratory parameters were preserved. Grip strength and Rotarod testing demonstrating no decline in performance across all groups. Histological analysis showed no evidence of haemorrhage, neuronal loss or increased apoptosis. \ud \ud \ud \ud Doxorubicin demonstrated cytotoxicity against all three cell lines and is known to have poor BBB permeability. Quantities measured in the brainstem of NSG mice were highest in the group receiving MRgFUS and microbubbles (431.5 ng/g). This was significantly higher than in mice who received no intervention (7.6 ng/g). \ud \ud \ud \ud Our data demonstrates both the preservation of histological and functional integrity of the brainstem following MRgFUS for BBB opening and the ability to significantly enhance drug delivery to the region, giving promise to the treatment of brainstem-specific conditions.

Patient-derived xenografts (PDXs) retain the genotype of the parental tumors more readily than tumor cells maintained in culture. In order to study the genetics of clival chordoma in the absence of prior radiation we sought to establish a PDX following the primary resection of a clival chordoma. Epicranial grafting of a primary clival chordoma was performed in NOD/SCID mice. Tumors have been passaged serially in mice for 7 generations. Physaliferous cell architecture was demonstrated in the regenerated tumors, which stained positive for Brachyury, Cytokeratin, and S100. The tumors demonstrated bone invasion. Single-nucleotide polymorphism analysis of the tumor xenograft was compared to the parental tumor. We identified homozygous amplification of the T-gene (brachury) and heterozygous loss of CDKN2A. We also identified heterozygous loss of the tumor suppressor FHIT gene, although protein expression was preserved. Accumulation of copy number losses and gains over three generations showed for the first time that there is inherent genomic instability in chordoma. In conclusion, this PDX reproduces the phenotype of clival chordoma. This study is the first to show chromosomal genomic instability in a chordoma that is serially propagated through multiple generations. The genomic instability observed in chordoma may serve as the biological basis for resistance to chemotherapy as is observed for other forms of cancer.

Pediatric ependymomas are highly recurrent tumors resistant to conventional chemotherapy. Telomerase, a ribonucleoprotein critical in permitting limitless replication, has been found to be critically important for the maintenance of tumor-initiating cells (TICs). These TICs are chemoresistant, repopulate the tumor from which they are identified, and are drivers of recurrence in numerous cancers. In this study, telomerase enzymatic activity was directly measured and inhibited to assess the therapeutic potential of targeting telomerase. Telomerase repeat amplification protocol (TRAP) (n = 36) and C-circle assay/telomere FISH/ATRX staining (n = 76) were performed on primary ependymomas to determine the prevalence and prognostic potential of telomerase activity or alternative lengthening of telomeres (ALT) as telomere maintenance mechanisms, respectively. Imetelstat, a phase 2 telomerase inhibitor, was used to elucidate the effect of telomerase inhibition on proliferation and tumorigenicity in established cell lines (BXD-1425EPN, R254), a primary TIC line (E520) and xenograft models of pediatric ependymoma. Over 60 % of pediatric ependymomas were found to rely on telomerase activity to maintain telomeres, while no ependymomas showed evidence of ALT. Children with telomerase-active tumors had reduced 5-year progression-free survival (29 ± 11 vs 64 ± 18 %; p = 0.03) and overall survival (58 ± 12 vs 83 ± 15 %; p = 0.05) rates compared to those with tumors lacking telomerase activity. Imetelstat inhibited proliferation and self-renewal by shortening telomeres and inducing senescence in vitro. In vivo, Imetelstat significantly reduced subcutaneous xenograft growth by 40 % (p = 0.03) and completely abolished the tumorigenicity of pediatric ependymoma TICs in an orthotopic xenograft model. Telomerase inhibition represents a promising therapeutic approach for telomerase-active pediatric ependymomas found to characterize high-risk ependymomas. Electronic supplementary material The online version of this article (doi:10.1007/s00401-014-1327-6) contains supplementary material, which is available to authorized users.

Glioblastoma (GBM) is the most common primary brain tumor in adults. Despite current advances in therapy consisting of surgery followed by chemotherapy and radiation, the overall survival rate still remains poor. Therapeutic failures are partly attributable to the highly infiltrative nature of tumor adjacent to normal brain parenchyma. Recently, evidence is mounting to suggest that actin cytoskeleton dynamics are critical components of the cell invasion process. Drebrin is an actin-binding protein involved in the regulation of actin filament organization, and plays a significant role in cell motility; however, the role of drebrin in glioma cell invasiveness has not yet been fully elucidated. Therefore, this study was aimed to clarify the role of drebrin in glioma cell morphology and cell motility. Here we show that drebrin is expressed in glioma cell lines and in operative specimens of GBM. We demonstrate that stable overexpression of drebrin in U87 cells leads to alterations in cell morphology, and induces increased invasiveness in vitro while knockdown of drebrin in U87 cells by small interfering RNA (siRNA) decreases invasion and migration. In addition, we show that depletion of drebrin by siRNA alters glioma cell morphology in A172 GBM cell line. Our results suggest that drebrin contributes to the maintenance of cell shape, and may play an important role in glioma cell motility.

We recently reported that atypical teratoid rhabdoid tumors (ATRTs) comprise at least two transcriptional subtypes with different clinical outcomes; however, the mechanisms underlying therapeutic heterogeneity remained unclear. In this study, we analyzed 191 primary ATRTs and 10 ATRT cell lines to define the genomic and epigenomic landscape of ATRTs and identify subgroup-specific therapeutic targets. We found ATRTs segregated into three epigenetic subgroups with distinct genomic profiles, SMARCB1 genotypes, and chromatin landscape that correlated with differential cellular responses to a panel of signaling and epigenetic inhibitors. Significantly, we discovered that differential methylation of a PDGFRB-associated enhancer confers specific sensitivity of group 2 ATRT cells to dasatinib and nilotinib, and suggest that these are promising therapies for this highly lethal ATRT subtype.