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STAT3, IL-6 and inflammatory response pathway signaling associated gene expression is upregulated in Group A EPN.

Cell cycle analysis of acute leukemia cells treated with olaparib and AZD1775. A. The indicated cell lines were treated with DMSO (vehicle), olaparib (2 uM), and/or AZD1775 (200 nM) for 48 hr then fixed and stained with propidium iodide. Results are shown as mean {plus minus} SEM from a minimum of two independent experiments. B. .MV4;11 cells were treated with DMSO (vehicle), olaparib (2 uM), and/or AZD1775 (200 nM) for 48 hr then fixed and stained with propidium iodide. Representative plots are displayed. C. Jurkat cells were treated with DMSO (vehicle), olaparib (2 uM), and/or AZD1775 (200 nM) for 48 hr then fixed and stained with propidium iodide. Representative plots are displayed.

Inhibition of endogenous P53 in OCI-AML3 cells does not enhance the combinatorial activity of AZD1775 and olaparib. A-C. OCI-AML3 parental (A), MiG (B), and MiG-DDp53 (C) cells were treated with DMSO (vehicle control), olaparib (2 uM), and/or AZD1775 (200 nM) for 72 hr. Viable cell counts are normalized to cells receiving no treatment (NT). Results are shown as mean {plus minus} SEM from three independent experiments. *, P < 0.05. **, P < 0.01. ***, P < 0.001.

Combined AZD1775 and olaparib treatment is tolerated in vivo. A and B. Mice injected with a murine AML cell line were treated as described in the text. Complete blood counts (A) and weights (B) were obtained 10 days after leukemia transfer. Results are displayed as mean {plus minus} SEM from 2 independent experiments. *, P < 0.05. **, P < 0.01.

Experimentally determined STAT3 upregulated genes.

AZD1775 enhances the anti-proliferative effect of olaparib in AML patient samples with high baseline apoptosis or limited colony-forming ability. A and B. AML patient samples were treated with DMSO (vehicle control), olaparib (2 uM), and/or AZD1775 (200 nM) for 72 hr and analyzed for cell viability (A) and apoptosis induction (B). Live cell counts are normalized to cells receiving no treatment (NT). Results are shown as mean {plus minus} SEM of 2 technical replicates.

Ependymoma (EPN) in childhood is a brain tumor with substantial mortality. Inflammatory response has been identified as a molecular signature of high-risk Group A EPN. To better understand the biology of this phenotype and aid therapeutic development, transcriptomic data from Group A and B EPN patient tumor samples, and additional malignant and normal brain data, were analyzed to identify the mechanism underlying EPN Group A inflammation. Enrichment of IL6 and STAT3 pathway genes were found to distinguish Group A EPN from Group B EPN and other brain tumors, implicating an IL6 activation of STAT3 mechanism. EPN tumor cell growth was shown to be dependent on STAT3 activity, as demonstrated using shRNA knockdown and pharmacologic inhibition of STAT3 that blocked proliferation and induced apoptosis. The inflammatory factors secreted by EPN tumor cells were shown to reprogram myeloid cells, and this paracrine effect was characterized by a significant increase in pSTAT3 and IL8 secretion. Myeloid polarization was shown to be dependent on tumor secretion of IL6, and these effects could be reversed using IL6-neutralizing antibody or IL6 receptor–targeted therapeutic antibody tocilizumab. Polarized myeloid cell production of IL8 drove unpolarized myeloid cells to upregulate CD163 and to produce a number of proinflammatory cytokines. Collectively, these findings indicate that constitutive IL6/STAT3 pathway activation is important in driving tumor growth and inflammatory cross-talk with myeloid cells within the Group A EPN microenvironment. Effective design of Group A–targeted therapy for children with EPN may require reversal of this potentially immunosuppressive and protumor pathway. Cancer Immunol Res; 3(10); 1165–74. ©2015 AACR.

FDA panel: Details of drug screen library of 97 FDA approved anticancer active compounds. FDA drug targets: Genes corresponding to known protein targets of compounds in FDA approved oncology drug screen library, corresponding Affymetrix probeset IDs and literature source. GSEA of axitinib Tx: Axitinib-induced enrichment and depletion of MSigDB "Hallmark" genesets in transcriptomic profiles of axitinib-treated versus untreated controls.

IL-6 is the driving cytokine for immune suppressive phenotype observed in 811 CM monocytes.

Summary of p-NF-KB and NF-KB values and ratios from Figure 1f, h and i

S3I-201 effects on cell cycle EPN cell line 811 and apoptosis in EPN cell line 723.

Children with ependymoma (EPN) are cured in less than 50% of cases, with little improvement in outcome over the last several decades. Chemotherapy has not affected survival in EPN, due in part to a lack of preclinical models that has precluded comprehensive drug testing. We recently developed two human EPN cell lines harboring high-risk phenotypes which provided us with an opportunity to execute translational studies. EPN and other pediatric brain tumor cell lines were subject to a large-scale comparative drug screen of FDA-approved oncology drugs for rapid clinical application. The results of this in vitro study were combined with in silico prediction of drug sensitivity to identify EPN-selective compounds, which were validated by dose curve and time course modeling. Mechanisms of EPN-selective antitumor effect were further investigated using transcriptome and proteome analyses. We identified three classes of oncology drugs that showed EPN-selective antitumor effect, namely, (i) fluorinated pyrimidines (5-fluorouracil, carmofur, and floxuridine), (ii) retinoids (bexarotene, tretinoin and isotretinoin), and (iii) a subset of small-molecule multireceptor tyrosine kinase inhibitors (axitinib, imatinib, and pazopanib). Axitinib's antitumor mechanism in EPN cell lines involved inhibition of PDGFRα and PDGFRβ and was associated with reduced mitosis-related gene expression and cellular senescence. The clinically available, EPN-selective oncology drugs identified by our study have the potential to critically inform design of upcoming clinical studies in EPN, in particular for those children with recurrent EPN who are in the greatest need of novel therapeutic approaches. Mol Cancer Ther; 17(9); 1984–94. ©2018 AACR.

FLT3-ITD does not enhance the combinatorial activity of AZD1775 and olaparib. The 32D cell line was transduced with FLT3-ITD or empty vector and treated with olaparib at the indicated doses and/or AZD1775 (100nM) in triplicate for 72 hours. Proliferation and viability were assessed by Cell Titer Glow, per the manufacturer's instructions, and normalized to the value of untreated (UT) cells. Data the mean {plus minus} SEM from 3 independent experiments.

Single agent treatment with olaparib or AZD1775 reduces cell viability and enhances apoptosis in AML and ALL cell lines. A. MV4;11, Molm13, Jurkat, and REH cells were treated with 0.25 uM- 20 uM olaparib for 72 hr. Percent of viable cells relative to cells receiving no treatment (NT) are shown. The IC50 value of each cell line is displayed. B. MV4;11, Molm13, Jurkat, and REH cells were treated with 0.25 uM- 20 uM olaparib for 72 hr and stained with Annexin V-PE. The EC50 value of each cell line is displayed. C. MV4;11, Molm13, Jurkat, and REH cells were treated with 25 nM - 2000 nM AZD1775 for 72 hr. Percent of viable cells relative to cells receiving no treatment (NT) are shown. The IC50 value of each cell line is displayed. D. MV4;11, Molm13, Jurkat, and REH cells were treated with 25 nM - 2000 nM AZD1775 for 72 hr and stained with Annexin V-PE. The EC50 value of each cell line is displayed.

Apoptosis time courses for EPN cell lines 811 and 928 treated with floxuridine, bexarotene, and axitinib all at 1µM, as measured by live cell imaging (IncuCyte) ofcaspase 3/7 activation (green objects/mm2). Idarubicin (1µM) provides a positive control for apoptosis and untreated cells serve as a negative control.

Ratio of Group A EPN IL6/STAT3 pathway genes in tumor versus the myeloid compartments.

NCI approved anti-cancer agents in combination drug screen with selinexor

Consensus NMF of 81 primary ependymoma samples at k = 3 delineates two posterior fossa subgroups in the study cohort.

Supplemental Figure Legends

Supplemental Figures 1 - 6

AML and ALL cell lines display elevated homologous recombination in response to olaparib treatment. A-E. MV;411 (A), Molm13 (B), Jurkat (C), REH (D), and OCI-AML3 (E) cells received no treatment (NT) or were treated with olaparib (2 uM) for 72 hr then fixed and stained with anti-Rad51 (primary antibody), Alexo Fluor 488 conjugated secondary antibody, and DAPI. Representative images are shown. Rad51 foci per cell were quantified and are displayed to the right of the images. Results are shown and mean {plus minus} SEM calculated from a minimum of 50 cells per condition. ***, P < 0.001. ****, P < 0.0001.

Supplemental Methods

Cell line characteristics and selinexor in vitro IC50 levels

Expression of EPN-selective drug targets in primary PFA patient samples versus cerebellum. Gene expression for (A) RARB, (B) PDGFRB, (C) VEGFR2 (KDR) and (D) VEGFC in twelve PFA patient samples and 4 pediatric cerebellum samples. Red bars represent mean and standard deviation. P-values = student's t-test.

High-grade glioma (HGG) is the leading cause of cancer-related death among children. Selinexor, an orally bioavailable, reversible inhibitor of the nuclear export protein, exportin 1, is in clinical trials for a range of cancers, including HGG. It inhibits the NF-κB pathway and strongly induces the expression of nerve growth factor receptor (NGFR) in preclinical cancer models. We hypothesized that selinexor inhibits NF-κB via upregulation of NGFR. In HGG cells, sensitivity to selinexor correlated with increased induction of cell surface NGFR expression. Knocking down NGFR in HGG cells increased proliferation, anchorage-independent growth, stemness markers, and levels of transcriptionally available nuclear NF-κB not bound to IκB-α, while decreasing apoptosis and sensitivity to selinexor. Increasing IκB-α levels in NGFR knockdown cells restored sensitivity to selinexor. Overexpression of NGFR using cDNA reduced levels of free nuclear NF-κB, decreased stemness markers, and increased markers of cellular differentiation. In all HGG lines tested, selinexor decreased phosphorylation of NF-κB at serine 536 (a site associated with increased transcription of proliferative and inflammatory genes). Because resistance to selinexor monotherapy occurred in our in vivo model, we screened selinexor with a panel of FDA-approved anticancer agents. Bortezomib, a proteasome inhibitor that inhibits the NF-κB pathway through a different mechanism than selinexor, showed synergy with selinexor against HGG in vitro. Our results help elucidate selinexor's mechanism of action and identify NGFR as a potential biomarker of its effect in HGG and in addition suggest a combination therapy strategy for these challenging tumors.

Purpose:Tumor relapse after radiotherapy is a major hurdle in treating pediatric H3K27M-mutant diffuse midline gliomas (DMG). Radiotherapy-induced stress increases association of BCL2 family of proteins with BH3 pro-apoptotic activators preventing apoptosis. We hypothesized that inhibition of radiotherapy-induced BCL2 with a clinically relevant inhibitor, venetoclax, will block BCL2 activity leading to increased apoptosis. BCL2 has never been implicated in DMG as a radiotherapy-induced resistant mechanism.Experimental Design:We performed an integrated genomic analysis to determine genes responsible for radioresistance and a targeted drug screen to identify drugs that synergize with radiation in DMG. Effect of venetoclax on radiation-naïve and 6 Gy radiation on cells was evaluated by studying cell death, changes in BCL2 phosphorylation, reactive oxygen species (ROS), and apoptosis, as well as BCL2 association with BH3 apoptosis initiators. The efficacy of combining venetoclax with radiation was evaluated in vivo using orthotopic xenograft models.Results:BCL2 was identified as a key regulator of tumor growth after radiation in DMGs. Radiation sensitizes DMGs to venetoclax treatment independent of p53 status. Venetoclax as a monotherapy was not cytotoxic to DMG cells. Postradiation venetoclax treatment significantly increased cell death, reduced BCL2–BIM association, and augmented mitochondrial ROS leading to increased apoptosis. Combining venetoclax with radiotherapy significantly enhanced the survival of mice with DMG tumors.Conclusions:This study shows that venetoclax impedes the antiapoptotic function of radiation-induced BCL2 in DMG, leading to increased apoptosis. Results from these preclinical studies demonstrate the potential use of the BCL2 inhibitor venetoclax combined with radiotherapy for pediatric DMG.

Supplementary Figure from Venetoclax Cooperates with Ionizing Radiation to Attenuate Diffuse Midline Glioma Tumor Growth

Supplementary Table from Venetoclax Cooperates with Ionizing Radiation to Attenuate Diffuse Midline Glioma Tumor Growth

Supplementary Figures S1-S6 and Table S1

Purpose: Tumor relapse after radiotherapy is a major hurdle in treating pediatric H3K27M-mutant diffuse midline gliomas (DMG). Radiotherapy-induced stress increases association of BCL2 family of proteins with BH3 pro-apoptotic activators preventing apoptosis. We hypothesized that inhibition of radiotherapy-induced BCL2 with a clinically relevant inhibitor, venetoclax, will block BCL2 activity leading to increased apoptosis. BCL2 has never been implicated in DMG as a radiotherapy-induced resistant mechanism. Experimental Design: We performed an integrated genomic analysis to determine genes responsible for radioresistance and a targeted drug screen to identify drugs that synergize with radiation in DMG. Effect of venetoclax on radiation-naïve and 6 Gy radiation on cells was evaluated by studying cell death, changes in BCL2 phosphorylation, reactive oxygen species (ROS), and apoptosis, as well as BCL2 association with BH3 apoptosis initiators. The efficacy of combining venetoclax with radiation was evaluated in vivo using orthotopic xenograft models. Results: BCL2 was identified as a key regulator of tumor growth after radiation in DMGs. Radiation sensitizes DMGs to venetoclax treatment independent of p53 status. Venetoclax as a monotherapy was not cytotoxic to DMG cells. Postradiation venetoclax treatment significantly increased cell death, reduced BCL2–BIM association, and augmented mitochondrial ROS leading to increased apoptosis. Combining venetoclax with radiotherapy significantly enhanced the survival of mice with DMG tumors. Conclusions: This study shows that venetoclax impedes the antiapoptotic function of radiation-induced BCL2 in DMG, leading to increased apoptosis. Results from these preclinical studies demonstrate the potential use of the BCL2 inhibitor venetoclax combined with radiotherapy for pediatric DMG.

High-grade glioma (HGG) is the leading cause of cancer-related death among children. Selinexor, an orally bioavailable, reversible inhibitor of the nuclear export protein, exportin 1, is in clinical trials for a range of cancers, including HGG. It inhibits the NF-κB pathway and strongly induces the expression of nerve growth factor receptor (NGFR) in preclinical cancer models. We hypothesized that selinexor inhibits NF-κB via upregulation of NGFR. In HGG cells, sensitivity to selinexor correlated with increased induction of cell surface NGFR expression. Knocking down NGFR in HGG cells increased proliferation, anchorage-independent growth, stemness markers, and levels of transcriptionally available nuclear NF-κB not bound to IκB-α, while decreasing apoptosis and sensitivity to selinexor. Increasing IκB-α levels in NGFR knockdown cells restored sensitivity to selinexor. Overexpression of NGFR using cDNA reduced levels of free nuclear NF-κB, decreased stemness markers, and increased markers of cellular differentiation. In all HGG lines tested, selinexor decreased phosphorylation of NF-κB at serine 536 (a site associated with increased transcription of proliferative and inflammatory genes). Because resistance to selinexor monotherapy occurred in our in vivo model, we screened selinexor with a panel of FDA-approved anticancer agents. Bortezomib, a proteasome inhibitor that inhibits the NF-κB pathway through a different mechanism than selinexor, showed synergy with selinexor against HGG in vitro. Our results help elucidate selinexor's mechanism of action and identify NGFR as a potential biomarker of its effect in HGG and in addition suggest a combination therapy strategy for these challenging tumors.

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. The most aggressive MB subtype Group 3, is characterized by Myc amplifications. However, targeting Myc has proven unsuccessful, necessitating alternative therapies for MB. The histone methyltransferase, enhancer of zeste homolog 2 (EZH2), is well-known for its role in catalyzing the tri-methylation of H3K27 (H3K27me3). Recent studies have demonstrated that EZH2 is overexpressed in Group 3 MB and that Myc and EZH2 regulate the expression of the B7 homolog 3 (B7-H3). As we previously showed the oncogenic functions of B7-H3 in Group 3 MB, this study aims to investigate the efficacy of EZH2 inhibition to reduce B7-H3 levels in an effort to improve treatment options and patient survival. Datamining studies conducted using MB patient datasets showed a positive correlation between EZH2 and B7-H3 transcripts. Additionally, we showed that inhibition of EZH2 using EPZ005687 in Myc-amplified MB cell lines downregulated B7-H3 and Myc expression. Furthermore, targeting EZH2 through shRNA and pharmacological inhibition reduced the viability and augmented apoptosis, as indicated by LDH, MTT, clonogenic, and FACS assays. We additionally tested the consequences of EZH2 inhibition on the C17.2, murine neural stem cells, and C17.2 NSC transformed with EZH2 oncogene (C17.2_EZH2OX). Furthermore, the C17.2_EZH2OXcells were susceptible to EPZ005687 treatment compared to C17.2 control cells, as demonstrated by MTT and FACS. Collectively, our study highlights the therapeutic benefits of targeting EZH2 in Myc-amplified MB cell survival. Citation Format: Katherine Shishido, Sujatha Venkataraman, Rajeev Vibhakar, Kiran Velpula, Swapna Asuthkar. EZH2 inhibition targets B7-H3 in Myc amplified medulloblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5667.

B7-H3 (CD276), an immune checkpoint member of the B7/CD28 family, plays a key role in the repression of the immune response by cancer cells. B7-H3 is overexpressed on tumor and tumor-associated cells, making it an interesting therapeutic target. B7-H3 also appears to play a role outside of immune evasion, contributing to the progression of cancer via invasion/migration, angiogenesis, and metastasis. Our previous data has confirmed the presence of B7-H3 in highly aggressive Myc+ medulloblastoma (MB) tumors. Data-mining studies revealed that B7-H3 is correlated with Myc expression in Group 3 and 4 MBs, and is associated with worse patient outcomes. miR-29, a tumor suppressor, has been shown downregulating the expression of B7-H3 in various cancers. We have shown that miR-29 can reduce the angiogenesis abilities of D283 and D425 cells by downregulating B7-H3 in vitro. Subsequent RNA-sequencing analysis of miR-29-transfected D283 cells revealed upregulation of STAT1 activity. Immunoblot analysis and real-time PCR of D283 and D425 cells demonstrated increased expression of p-STAT1, when transfected with miR-29 plasmid. Treatment with all-trans retinoic acid induced p-STAT1 expression, with a concomitant decrease in the expression of B7-H3 and MMP-9. This preliminary data revealed a novel role for STAT1 in potentially regulating B7-H3, and consequently, angiogenesis and metastasis in MB. To date, the role of STAT1 in MB progression has not been fully investigated, and so our results provide a new avenue for developing potential treatments for aggressive MB tumors. Citation Format: Ian J. Purvis, Janardhan Avilala, Maheedhara R. Guda, Sujatha Venkataraman, Rajeev Vibhakar, Andrew J. Tsung, Kiran K. Velpula, Swapna Asuthkar. STAT1 regulates B7H3 mediated angiogenesis in medulloblastoma [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 202.

B7-H3 (CD276), an immune checkpoint member of the B7/CD28 family, plays a key role in the repression of the immune response by cancer cells. B7-H3 is overexpressed exclusively on tumor and tumor-associated cells, making it an interesting therapeutic target. B7-H3 also appears to play a role outside of immune evasion, contributing to the progression of cancer via invasion, migration, and angiogenesis. Our preliminary data have confirmed the presence of B7-H3 in highly aggressive MYC+ medulloblastoma (MB) tumors. Data-mining studies revealed that B7-H3 is highly expressed across the subgroups of MB, and is correlated with MYC expression in Group 3 and 4 MBs. Recently, miR-29, a tumor-suppressor miRNA, was shown downregulating B7-H3 expression by targeting the 3' UTR of B7-H3 mRNA. Immunoblot analysis of D283 and D458 cells demonstrated reduced expression of both cellular and soluble form of B7-H3 when transfected with miR-29 plasmid. Our preliminary data have shown a novel method of targeting B7-H3 expression by miR-29 in MB cells, inhibiting the ability of MB cells to induce angiogenesis. Because both miR-29 and B7-H3 form an inversely related axis that plays a role in tumor angiogenesis, inhibition of B7-H3 by miR-29 may have potential value in developing novel antiangiogenic treatments in MB. Citation Format: Ian Purvis, Maheedhara R. Guda, Sujatha Venkataraman, Rajeev Vibhakar, Kiran K. Velpula, Swapna Asuthkar. miR-29 expression regulates B7-H3 mediated angiogenesis in medullablastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 512.

Promoter hypermethylation and histone deacetylation are common epigenetic mechanisms implicated in the transcriptional silencing of tumor suppressor genes in human cancer. We treated two immortalized glioma cell lines, T98 and U87, and 10 patient-derived primary glioma cell lines with trichostatin A (TSA), a histone deacetylase inhibitor, or 5-aza-2′-deoxycytidine (5-AzaC), a DNA methyltransferase inhibitor, to comprehensively identify the cohort of genes reactivated through the pharmacologic reversal of these distinct but related epigenetic processes. Whole-genome microarray analysis identified genes induced by TSA (653) or 5-AzaC treatment (170). We selected a subset of reactivated genes that were markedly induced (greater than two-fold) after treatment with either TSA or 5-AzaC in a majority of glioma cell lines but not in cultured normal astrocytes. We then characterized the degree of promoter methylation and transcriptional silencing of selected genes in histologically confirmed human tumor and nontumor brain specimens. We identified two novel brain expressed genes, BEX1 and BEX2, which were silenced in all tumor specimens and exhibited extensive promoter hypermethylation. Viral-mediated reexpression of either BEX1 or BEX2 led to increased sensitivity to chemotherapy-induced apoptosis and potent tumor suppressor effects in vitro and in a xenograft mouse model. Using an integrated approach, we have established a novel platform for the genome-wide screening of epigenetically silenced genes in malignant glioma. This experimental paradigm provides a powerful new method for the identification of epigenetically silenced genes with potential function as tumor suppressors, biomarkers for disease diagnosis and detection, and therapeutically reversible modulators of critical regulatory pathways important in glioma pathogenesis. (Cancer Res 2006; 66(13): 6665-74)

Background: Pediatric high-grade gliomas (HGG) and diffuse intrinsic pontine gliomas (DIPG) account for the majority of pediatric brain tumor deaths and respond poorly to chemotherapy. Selinexor, a nuclear export inhibitor, is effective against HGG and DIPG in in vitro and in vivo models, but resistance to treatment develops. We previously identified the NF-κB pathway as a likely mediator of selinexor’s activity in these tumors. NF-κB transcriptional activity is regulated by an inhibitor, IKB-α, whose levels are in turn regulated by ubiquitination and proteasomal degradation. IKB-α is a client of exportin-1 (XPO1); its nuclear levels are increased by selinexor treatment, leading to inhibition of NF-κB. We subsequently identified proteasome inhibitors as potentially synergistic with selinexor in HGG and DIPG through a screen of all FDA-approved chemotherapy agents. Proteasome inhibition has also been shown to synergize with selinexor treatment in multiple myeloma and osteosarcoma. Methods: We treated HGG cell lines (BT245 and GBM1) and DIPG cell lines (DIPG4, DIPG7 and SF7761) for five days with selinexor in combination with each of three proteasome inhibitors, bortezomib, carfilzomib and marizomib, and assayed cell viability at the conclusion of treatment. In each experiment, cells were treated with selinexor, a proteasome inhibitor, and a combination of the two drugs at several constant ratios. IC50 values were computed for each drug acting alone, and the combination index (CI) of the two drugs acting together was computed using the Chou-Talalay method. We also treated SF7761 cells with a combination of radiation (8Gy), selinexor, and a proteasome inhibitor. Results: The proteasome inhibitors had widely varying IC50 values in the cell lines treated, ranging from 1nM to 5µM. The CI for the combination of selinexor and each proteasome inhibitor was consistently less than 1 (indicating a synergistic relationship) in the cell lines tested. We found that radiation and proteasome inhibition had an antagonistic relationship (CI>1), radiation and selinexor a synergistic relationship (CI Conclusions: Selinexor and proteasome inhibitors show promise as a combination therapy for HGG and DIPG. We are conducting in vivo experiments to further explore this combination for subsequent clinical trial use. Citation Format: John DeSisto, Patrick Flannery, Trinayan Kashyap, Rakeb Lemma, Shelby Mestnik, Andrew Kung, Rajeev Vibhakar, Yosef Landesman, Adam Green. Synergistic effects of the XPO1 inhibitor selinexor with proteasome inhibitors in pediatric high-grade glioma and diffuse intrinsic pontine glioma [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 1946. doi:10.1158/1538-7445.AM2017-1946

Medulloblastoma is the most common primary brain tumor in children. Current treatment for medulloblastoma includes surgical resection, radiation and cytotoxic chemotherapy. Although this approach has improved survival rates, the high doses of chemotherapy required to circumvent drug resistance mechanisms and result in clinical efficacy often give rise to lasting neurocognitive defects, stunted growth, deafness, and even secondary tumors. Therefore, synergistic drug combinations that maintain clinical efficacy, but allow dose reductions of cytotoxic agents limiting their adverse effects would be an attractive approach for patients with medulloblastoma. We identified Wee1 kinase as a new molecular target for medulloblastoma from an integrated genomic analysis using pathway analysis of gene expression and a kinome-wide siRNA screen of medulloblastoma cells and tissue. Wee1 participates in the G2-M checkpoint to prevent mitosis in the presence of DNA damage and therefore may play a role in drug resistance to DNA alkylating agents, such as cisplatin. Our data indicate that Wee1 prevents DNA damage-induced cell death by cisplatin and that the known Wee1 inhibitor AZD1775 (previously MK1775) displays synergistic activity with cisplatin. However, AZD1775 is known to have nanomolar activity with at least 8 other kinases and there is limited structure-activity relationship (SAR) data for AZD1775 as it was identified as a Wee1 inhibitor from a high-throughput screen. Therefore, we developed a small series of AZD1775 derivatives to establish a SAR and further examine the effects of Wee1 inhibition in medulloblastoma. The compounds, that inhibited Wee1 activity in a TR-FRET assay in the same nanomolar range as AZD1775, had significantly reduced single-agent cytotoxicity and displayed synergistic activity with cisplatin at lower concentrations than AZD1775 in medulloblastoma cells. Recently, it has been reported that AZD1775 has limited ability to diffuse across the blood-brain barrier (BBB) and this would limit its effectiveness in brain tumors. We have performed cell permeability and BBB permeability assays using AZD1775 and our lead Wee1 inhibitor and our studies support that AZD1775 has poor BBB penetration. Our studies have now transitioned into in vivo systems to determine the tissue distribution and pharmacokinetics of our lead Wee1 inhibitor compared with AZD1775. In addition, our studies will determine the effect of our lead Wee1 inhibitor and AZD1775 on tumor growth as single agents and in combination with cisplatin in medulloblastoma in vivo models. Citation Format: Christopher J. Matheson, Sujatha Venkataraman, Vladimir Amani, Peter Harris, Donald S. Backos, Nicholas K. Foreman, Rajeev Vibhakar, Philip Reigan. A Wee1 inhibitor analog of AZD1775 demonstrates synergy with cisplatin with reduced single-agent toxicity in medulloblastoma. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C195.

Background: Medulloblastoma is the most common malignant brain tumor in children and remains a therapeutic challenge due to its significant therapy-related morbidity. Checkpoint kinase 1 (CHK1) is highly expressed in many cancers and regulates critical steps in mitotic progression and DNA-damage response. Activation of CHK1 pathway promotes treatment resistance in tumor cells. Recent studies suggest that targeting CHK1 with a small molecule inhibitor, to sensitize tumors to a variety of DNA-damaging agents, is a promising approach to tumor therapy. Methods: The expression of CHK1 mRNA in medulloblastoma patient samples and cell lines were examined using microarray analysis and qRT-PCR respectively. Western blot analysis was conducted on medulloblastoma cell lines to analyze expression level of CHK1 protein. The effect of AZD7762, a small molecule inhibitor of CHK1, on colony forming ability of medulloblastoma cell lines in combination with cisplatin was examined using colony formation assay and methylcellulose assay. The impact of AZD7762 in combination with cisplatin on tumor cell proliferation was further evaluated in real-time using xCELLigence. The effect of AZD7762 on cell viability was assessed using Guava ViaCount assay. Furthermore, the effects on key signaling pathways were analyzed using immunofluorescence, senescence assay, and apoptosis assay. Results: Analysis of gene expression and western blot experiments revealed that CHK1 mRNA and protein levels are over expressed in all medulloblastoma patient samples and in cell lines when compared to normal pediatric cerebellum. High CHK1 expression correlates with adverse outcomes in a large cohort of medulloblastoma patients. Inhibition of CHK1 by a low nanomolar concentration of AZD7762 potently inhibited cell growth, suppressed the colony-forming ability, and increased cellular apoptosis of medulloblastoma cells. Furthermore, AZD7762 pretreatment sensitized medulloblastoma cells to cisplatin synergistically. Conclusions: CHK1 expression is a prognostic marker in medulloblastoma and targeting CHK1 with a small molecule inhibitor, in combination with cisplatin, is an attractive strategy in treatment of medulloblastoma that warrants further investigation. Citation Format: Monil Shah, Eric Prince, Sujatha Venkataraman, Ilango Balakrishnan, Irina Alimova, Peter Harris, Marc Remeke, Michael D. Taylor, Michael H. Handler, Nicholas K. Foreman, Rajeev Vibhakar. Checkpoint kinase 1 inhibition suppresses cell growth and enhances cisplatin sensitivity in medulloblastoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 496. doi:10.1158/1538-7445.AM2015-496

Overexpression of the polycomb group gene Enhancer of Zeste 2 (EZH2) occurs in many tumors including prostate cancer, breast cancer and brain tumors such as Glioblastoma multiform and Medulloblastoma. Recent evidence suggests it may also have a role in rhabdoid tumors. Atypical teratoid rhabdoid tumor (ATRT) is a rare high-grade brain tumor with very poor survival that occurs most commonly in children. AT/RT tumors are characterized by deletion of the chromatin associated protein SMARCB1. Given the role of EZH2 in regulating epigenetic changes we chose to investigate the role of EZH2 in AT/RT. Here we show that targeted disruption of EZH2 by RNAi or pharmacologic means strongly impairs ATRT cell growth, induces apoptosis and potently sensitizes these cells to radiation. Using functional analysis of transcription factor activity we found the Cyclin D-E2F1 axis to be repressed upon EZH2 depletion in ATRT cells. Taken together our observations provide evidence that EZH2 regulates cell cycle progression by targeting the G1-S transition machinery and may be an important new therapeutic target particularly in combination with radiation in ATRT. 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 2483. doi:1538-7445.AM2012-2483

Medulloblastoma is the most common type of malignant brain tumor that afflicts children. Although cisplatin chemotherapy and radiation have been the cornerstones of medulloblastoma intervention for over 20 years, the outcomes of these highly cytotoxic treatments are far from optimal, and there is increasing evidence that they cause long-term problems such as neurocognitive deficits and secondary tumors, even in patients with a good initial response. Thus, there is a critical need for more effective therapies to combat this disease. To approach this problem we investigated protein kinase function in medulloblastoma using RNAi combined with genomic methods. First, we performed a kinome-wide siRNA screen and identified a cohort of genes that mediates medulloblastoma cell viability. These include a signaling node of kinases that are key components of the G2-M transition including, Aurora Kinase A, Aurora Kinase B, Plk1, Wee1, Nek2 and Mps1. Clearly the G2-M transition is critical for medulloblastoma cells, so kinases required for the process are potentially important therapeutic targets. We subsequently validated PLK1 and WEE 1 as bona fide therapeutic targets. Small molecule inhibitors of WEE 1 and PLK1 potently inhibited medulloblastoma cell growth and sensitized cells to radiation. In conclusion, using siRNA screens we identified novel therapeutic targets for medulloblastoma therapy Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C83.

EZH2 (enhancer of zeste homologue2) is a polycomb group protein that mediates repression of gene transcription through its Histone 3 Lysine 27methyltransferase activity. EZH2 is highly expressed in many cancers including metastatic prostate cancer, breast cancer, and glioblastoma multiform. EZH2 expression is associated with tumor cell proliferation, invasive growth and aggressive cancer phenotypes. The fact that EZH2 increases tumor cell proliferation and aggressive subgroups in several cancers may be of practical interest because the polycomb proteins have been suggested as candidates for targeted therapy. We found EZH2 expression to be increased in medulloblastoma when compared to normal cerebellum. Inhibition of EZH2 by RNAi in medulloblastoma cells resulted in suppression of cell growth. To further examine this phenotype we used a global histone methylation inhibitor, 3-Deazaneplanocin A (DZNep). DZNep preferentially inhibits H3 Lys27me via EZH2. DZNep showed a dose dependent, anti-proliferative and anti-clonogenic activity in medulloblastoma cells. Synergetic effect was shown in cells transfected with shEZH2 and treated with DZNEP. Furthermore DZNep treatment decreased EZH2 protein levels. We found that DZNep induced differentiation of mouse neural stem cells even when they are grown in proliferation medium. Most critically transformation of these neural stem cells was significantly attenuated by shEZH2 and DZNep. Our results suggest that EZH2 is a target for medulloblastoma treatment and that DZNep is an excellent candidate for further testing as a therapeutic candidate. 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 3446. doi:10.1158/1538-7445.AM2011-3446

MicroRNAs are small non-coding RNAs with gene expression regulation at posttranscriptional and transcriptional level. MicroRNAs are widely known to play a key role in carcinogenesis. We investigated the role of microRNAs in medulloblastoma, the most common malignant brain tumor of childhood. We found that microRNA 520b is over expressed in medulloblastoma compared to normal cerebellum. We next examined the biological significance of microRNA 520b. Transfection of medulloblastoma cells (DAOY and ONS76) with microRNA 520b significantly increased cell proliferation and enhanced anchorage-independent growth in soft agar compared to parental cells. Conversely, over expression of anti-microRNA 520b in medulloblastoma cells decreases cell proliferation and colony formation in soft agar. Bio-informatics analysis revealed several chromatin-remodeling proteins as potential targets of microRNA 520b. One of these, SUV39H1 is a histone methyl transferase that methylates histone 3 lysine 27 and suppresses activation of associated promoters. It is known that histone methylation is a major determinant for the formation of active and inactive regions of the genome and is crucial for the proper programming of the genome during development. We found that inhibition of microRNA 520b results in increased expression of SUV39H1 and subsequent methylation of Histone 3 Lys27 in medulloblastoma cells. SUV39H1 is known to be a tumor suppressor and is mutated in a subset of medulloblastoma. These data indicate that microRNA 520b can function as an oncogene in medulloblastoma by regulating histone modifications. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2092.