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.

No abstract

Background: Embryonal tumors of the central nervous system continue to present therapeutic challenges with high rates of relapse and poor prognosis at advanced stages. Medulloblastoma (MB) accounts for over 15% of pediatric brain tumors, with a five-year survival rate of 62%. Neuroblastoma (NB) is the most common extracranial solid tumor in children, accounting for 15% of pediatric cancer deaths. Among many pathways that contribute to MB and NB potency, several histone deacetylases (HDACs) have been shown to prevent cell differentiation. In cells expressing wt-p53, HDAC inhibitors induce nuclear relocalization of p53 to the nucleus to induce expression of the cell cycle inhibitor p21/Waf1/Cip1. As most NB tumors express wt-p53, HDAC inhibitors are promising candidates for therapy. Panobinostat has been shown to induce differentiation, cell cycle arrest, and apoptosis in NB cells. This effect is partially due to down-regulation of CHK1, a pathway by which cancer cells can develop resistance to conventional chemotherapeutic drugs. This study proposes panobinostat coupled with conventional chemotherapeutics such as doxorubicin, etoposide, and velcade, as an effective target against the HDAC pathway in MB and NB. Methods: Established MB and NB cell lines and patient derived cell lines (MB: DAOY, D283, D341, 384MED, 458MED, 487MED, 556MED, 581MED, 721MED; NB: BE(2)-C, CHLA-90, SMS-KCNR, SH-SY5Y, MGT8-117-08, BIO-036-08) were used to quantify panobinostat's effects. Cell viability was measured using Calcein AM fluorescent assay at doses of 0.39-50 nM. Isobologram analysis of panobinostat in combination with doxorubicin, etoposide, and velcade were generated using Calcein AM. Western blot was used to measure HIF1 alpha, CHK1, and acetyl H4, Caspase 3 and PARP levels. ATP level per cell was measured using CyQuant fluorescent DNA assay with CellTiter-Glo luminescent cell viability assay. Results: Cell viability assays show cytotoxicity of panobinostat in MB and NB cell lines, with IC50 values from 2-10 nM in MB cells, 5-20 nM in established NB cell lines and 23-91 nM in patient derived NB lines. ATP/cell activity was inhibited in MB and NB cells following treatment with panobinostat alone. Isobologram experiments suggest synergistic cytotoxicity of panobinostat in combination with doxorubicin, etoposide, and velcade in NB cell lines BE(2)-C and SMS-KCNR. Western blot analysis indicates caspase-mediated apoptosis occurs, with inhibition of overexpressed HDAC proteins among multiple classes in MB and NB cells. Conclusions: This study indicates that panobinostat targets the HDAC inhibition pathway of MB and NB cells. Additionally, panobinostat synergizes with doxorubicin, etoposide, and velcade in inducing apoptosis in MB and NB cells. This study provides rationale for initiation of a clinical trial in treating MB and NB patients with panobinostat in combination with conventional chemotherapeutics. Citation Format: Amanda B. Witte, Mary Durston, Ping Zhao, Abhinav B. Nagulapally, Jeffrey Bond, Giselle L. Saulnier Sholler. HDAC inhibitor panobinostat as a selective agent, synergizes with chemotherapeutics in medulloblastoma and neuroblastoma. [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 3198.

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.