Medulloblastoma (MB) is the most common malignant brain tumor in children, accounting for nearly 20 percent of all childhood brain tumors. New treatment strategies are needed to improve patient survival outcomes and to reduce adverse effects of current therapy. The phosphatidylinositol-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) intracellular signaling pathway plays a key role in cellular metabolism, proliferation, survival and angiogenesis, and is often constitutively activated in human cancers, providing unique opportunities for anticancer therapeutic intervention. The aim of this study was to evaluate the pre-clinical activity of BKM120, a selective pan-class I PI3K inhibitor, on MB cell lines and primary samples. IC50 values of BKM120 in the twelve MB cell lines tested ranged from 0.279 to 4.38 μM as determined by cell viability assay. IncuCyte ZOOM Live-Cell Imaging system was used for kinetic monitoring of cytotoxicity of BKM120 and apoptosis in MB cells. BKM120 exhibited cytotoxicity in MB cells in a dose and time-dependent manner by inhibiting activation of downstream signaling molecules AKT and mTOR, and activating caspase-mediated apoptotic pathways. Furthermore, BKM120 decreased cellular glycolytic metabolic activity in MB cell lines in a dose-dependent manner demonstrated by ATP level per cell. In MB xenograft mouse study, DAOY cells were implanted in the flank of nude mice and treated with vehicle, BKM120 at 30 mg/kg and 60 mg/kg via oral gavage daily. BKM120 significantly suppressed tumor growth and prolonged mouse survival. These findings help to establish a basis for clinical trials of BKM120, which could be a novel therapy for the treatment of medulloblastoma patients.

Background: Medulloblastoma (MB) is the most common malignant brain tumor in children with poor survival outcome. New treatment strategies are needed for control of MB. The phosphatidylinositol-3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) intracellular signaling pathway plays a key role in cellular metabolism, proliferation, survival and angiogenesis. This pathway is often constitutively activated in human tumor cells, providing unique opportunities for anticancer therapeutic intervention. BKM120 (Buparlisib) is an oral pan-class I PI3K inhibitor that targets all 4 isoforms of class I PI3K. BKM120 is currently being clinically evaluated for the treatment of different adult cancers including breast cancer, glioblastoma, prostate cancer, advanced non-small cell lung cancer, and colorectal cancer. In this study, we screened our MB established and patient primary cell lines by genomic profiling analysis, and validated the targeted therapy both in vitro and in vivo in xenograft mouse model. Methods: RNA expression profiling analysis was performed with Affymetrix GeneChip U133 Plus 2.0 genome wide expression cDNA microarray. Analysis was done using R/Bioconductor packages and Partek Genomics Suite. Eleven MB cell lines were treated with increasing concentrations (0-4 μM) of BKM120 for 48 hours. CellTiter-Glo Luminescent Cell Viability Assay was used to determine cell viability. IC50 values were calculated with a four-parameter variable-slope dose response curve using GraphPad Prism v.5 software. IncuCyte ZOOM Live-Cell Imaging system was used for kinetic monitoring of cytotoxicity of BKM120 and apoptosis in MB cells. Western blot analysis was used to measure phospho-Akt, phospho-mTOR, and cleaved caspase 3 protein levels. ATP level per cell was measured using CyQuant fluorescent DNA assay combined with CellTiter-Glo luminescent cell viability assay. Xenograft study was performed with DAOY cells implanted in the flank of nude mice and treated with vehicle, BKM120 at 30 mg/kg and 60 mg/kg via oral gavage daily. Results: BKM120 exhibited cytotoxicity in MB cells in a dose-dependent manner by inhibiting activation of downstream signaling molecules Akt and mTOR, and activating apoptotic pathways and inducing cell death in the eleven cell lines tested. IC50s of BKM120 in the MB cell lines ranged from 0.456 to 2.9 μM determined by cell viability assay. Furthermore, BKM120 decreased cellular glycolytic metabolic activity in MB cell lines in a dose-dependent manner. In MB xenograft mouse study, BKM120 significantly suppressed tumor growth and prolonged mouse survival at 30 mg/kg and 60 mg/kg. Conclusion: This study indicates that BKM120 promotes apoptosis and suppresses medulloblastoma tumor growth both in vitro and in vivo. Additional investigation of BKM120 for the treatment of pediatric medulloblastoma is warranted. Citation Format: Ping Zhao, Jacob Hall, Austin Voydanoff, Mary Durston, Elizabeth VanSickle, Abhinav B. Nagulapally, Jeffery Bond, Giselle Saulnier Sholler. BKM120 promotes apoptosis and suppresses tumor growth in medulloblastoma by targeting the phosphoinositide 3-kinase pathway. [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 3197.

Background: Neuroblastoma (NB) is an aggressive childhood cancer that arises from neural crest cells of the sympathetic nervous system. These cells contain cathecholamines which may be a therapeutic target. Catechol-O-methyltransferase (COMT) metabolizes catechol-containing compounds, including dopamine. COMT inhibitors, including tolcapone, are used as an adjunctive treatment for Parkinson's disease, as they increase dopamine within cells. Increase in dopamine and other catecholamines within cells may result in cell toxicity. Methods: Cell viability was measured using Calcein AM fluorescent assay at tolcapone doses 1.5625 μM - 200 μM, both alone and in combination with oxaliplatin at doses 1.5 μM - 6 μM. Western blot analysis was used to measure cleaved poly ADP ribose polymerase (PARP) and cleaved and full caspase-3 levels. ATP level per cell was measured using CyQuant fluorescent DNA assay combined with the Cell Titer GLO luminescent cell viability assay. Expression of COMT was confirmed using microarray analysis and immunofluorescence. Reactive oxygen species (ROS) levels were measured using DCFDA - Cellular Reactive Oxygen Species Detection Assay Kit. IncuCyte ZOOM machine was used in conjugation with SYTOX Green dye to measure cell death and CellPlayer™ Kinetic Caspase-3/7 Apoptosis Assay Reagent to measure apoptosis over 48 hours. Microarray analysis using U133+ RNA expression profiles were used to evaluate gene expression changes after cells were treated with tolcapone for 12 hours. Results: Tolcapone IC50 values ranged from 13.33 μM to 156 μM in seven different NB cell lines. Concentrations of 25 μM and 50 μM increased levels of cleaved PARP and cleaved caspase-3 over 48, 72, and 96 hours treated. Intracellular ATP decreased significantly with concentrations ranging from 12.5-200 μM treated over 48 hours and ROS levels increased significantly with concentrations ranging from 12.5-200 μM treated over 24 hours. IncuCyte Zoom analysis displayed dose dependent levels of cell death and presence of cleaved caspase-3 and 7 with increasing concentrations ranging from 1.5625 μM -200 μM. The combination of tolcapone at 25 μM and oxaliplatin at 3.5 μM show synergy in cell viability assays. Conclusion: There is no curative therapy for relapsed/refractory NB patients. The preclinical evidence suggests that patients with overexpression of COMT may respond to tolcapone via catecholamine induced ROS and cytotoxicity, especially when combined with oxaliplatin. Therefore, tolcapone and oxaliplatin may be a potential new therapy for children with NB with plans to be evaluated in a Phase I/II trial. Citation Format: David E. Hayes, Ping Zhao, Austin Voydanoff, Abhinav Nagulapally, Jeff Bond, Giselle Sholler. Tolcapone, a catechol-O-methyltransferase inhibitor, alone and in combination with oxaliplatin induces cell death in neuroblastoma. [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 1621. doi:10.1158/1538-7445.AM2015-1621

Background: A key component of high risk neuroblastoma (NB) therapy involves cis-retinoic acid (RA) for differentiation of minimal residual disease. DFMO induces differentiation and inhibition of tumor formation through the targeting of cancer stem cell (CSC) pathways via reversal of the Lin28/Let7 axis. Preventatitve DFMO therapy is currently in a phase II clinical trial at the end of therapy and in a Pilot study in combination with RA and ch14:18 antibody. We hypothesize that the combination of cis-RA and DFMO will induce greater differentiation, inhibition of tumor formation, and reduction of cell proliferation of NB. Methods: NB cell lines SMSKCNR, BE2C and CHLA90 were incubated in 96 well plates for 24 and 48 hours with low doses of DFMO (2.5 and 5 mM), RA (5 and 10 μM), and the four combinations of dosages. A Calcein AM Cell Viability Assay and BrdU Cell Proliferation Assay Kit were used to determine cell viability and cell proliferation, respectively. Western blot analysis was used to measure protein levels of CSC and differentiation markers. A neurosphere assay was used to assess inhibition of CSCs and tumor formation within wells. Cells were plated 2 cells/well in 96 well plates, drugged with single agents, cominbation, or DMSO and the percentage of wells per plate that formed neurospheres was determined after 1 and 2 weeks. IncuCyte ZOOM Live-Cell Imaging system was used for kinetic monitoring of neurite length to assess differentiation of NB cells. Results: Low dose RA and DFMO combination treatment (2.5-5mM DFMO and 5-10 μM RA) resulted in decreased cell viability as demonstrated through calcein AM. DFMO and RA combination treatments reduced cell viability by 60-71%, 75-78%, and 83-91%, in Be2C, CHLA90, and SMSR cells, respectively. BrdU incorporation demonstrated a reduction in cell proliferation at 48 hours of was 69-70.%, 60.2-64.5%, 62.7-71.1% in Be2c, CHLA90, and SMSR, respectively . Western blot analysis showed that DFMO, RA, and their combination reduced the CSC and increased the differentiation markers at 48 hours compared to control. The combination treatment also decreased tumor formation; the relative reduction in neurosphere formation at 2 weeks was 34.5% with 2.5 mM DFMO, 48% with 5 µM RA, and 73.2% with combination treatment. Lastly, differentiation was shown by neurite length increased by a factor of 1.4-1.6 and 5, in SMSR and BE2C cells, respectively with combination treatment. Conclusion: This study indicates that the combination retinoic acid and DFMO effectively cause a decrease in cell viability with a reduction in cell proliferation. Further, the combination results in differentiation of NB cells as well as targeting of CSC pathways and inhibition of tumor formation. Preventative DFMO therapy has been initiatied with RA in a pilot study for the treatment of high-risk neuroblastoma patients. Citation Format: Austin Voydanoff, Ping Zhao, Abhinav Nagulapally, Jeff Bond, Giselle L. Sholler. Retinoic acid and DFMO induce differentiation and inhibit tumor formation in neuroblastoma [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 1934. doi:10.1158/1538-7445.AM2017-1934