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BACKGROUND: The phosphoinositide 3-kinase (PI3K) and sonic hedgehog (SHH) pathways play important roles in medulloblastoma (MB) and other pediatric cancers. Aberrant activation of the PI3K pathway has been shown to be an important regulator of cancer cell proliferation, metabolism, protein synthesis and apoptosis. Additionally, mTOR activation contributes to therapy resistance likely in part through SMO-independent activation of the transcription factor GLI. Thus we sought to evaluate the effects of combined PI3K/mTOR inhibition in medulloblastoma and Ewing sarcoma. METHODS: Medulloblastoma (DAOY and D556) and Ewing Sarcoma cell lines (TC71, RDES) were grown in 2-D culture to investigate the effects of pharmacologic inhibition of PI3K and mTOR (using BYL-719, a p110α isoform specific PI3K inhibitor and OSI-027, a catalytic inhibitor of mTOR1/2, respectively) on protein kinase signaling, cell proliferation, colony formation, intracellular localization of GLI, and GLI target gene expression. RESULTS: Of all four class I PI3Ks only the p110α isoform is required for MB cell proliferation and colony formation. Pharmacologic targeting of the p110α isoform of PI3K with BYL-719 synergized with the catalytic mTORC1/2 inhibitor OSI-027, resulting in inhibition of effector pathways, and blocked cell proliferation and colony formation in both medulloblastoma and Ewing Sarcoma. Moreover, dual PI3K/mTOR inhibition resulted in decreased nuclear localization of GLI and reduced GLI target gene expression in both pediatric tumor cell lines. CONCLUSIONS: Inhibition of p110α and mTOR exerts potent antineoplastic effects on cancer cell proliferation and transformation. This effect may be due in part to inhibition of GLI nuclear localization. Thus, besides established roles in inhibition of cancer cell proliferation and protein synthesis, dual inhibition of p110α and mTOR is particularly promising for targeting SHH-driven cancers such as medulloblastoma and Ewing sarcoma.

MB-67. DUAL TARGETING OF PI3K AND mTOR SIGNALING IN MEDULLOBLASTOMA Jessica Clymer1,2, Frank Eckerdt2, Jonathan Bell2, Rishi Lulla1,2, Stewart Goldman1,2, and Leonidas Platanias2,3; Division of Hematology/ Oncology/Stem Cell Transplantation, Department of Pediatrics, Ann & Robert H. Lurie Children’s Hospital, Chicago, IL, USA; Robert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA; Division of Hematology/Oncology, Department of Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA BACKGROUND: Aberrant activation of the PI3K pathway has been shown to play a role in medulloblastoma cell proliferation, while mTOR activation contributes to therapy resistance. Here we sought to evaluate the effects of combined targeting of the PI3K and mTOR pathways in medulloblastoma. METHODS: Medulloblastoma cell lines Daoy and D556 were grown in 2-D cultures to investigate the effects of pharmacologic inhibition of PI3K and mTORon kinase signaling, medulloblastomacell proliferation, colony formation, and apoptosis. After knockdown of p110 isoforms, cells were also grown under stem cell conditions as 3-D neurospheres and subjected to extreme limiting dilution analysis (ELDA) for assessment of stem-like cancer cell growth. RESULTS: Of all class I PI3K isoforms (p110a, p110b, p110d, and p110g), only p110a was essential for medulloblastoma cell proliferation, colony formation and neurosphere growth. Accordingly, pharmacologic targeting of the p110a isoform of PI3K with BYL-719 synergizedwith the mTOR inhibitor OSI-027, as it resulted in inhibition of effector pathways, blocked cell proliferation and colony formation, and increased malignant cell death by apoptosis. Finally,BYL-719and OSI-027 greatly impairedgrowthof stem-likecancer cells as 3-D neurospheres. CONCLUSIONS: The p110a isoform of PI3K exerts crucial roles in medulloblastoma. Inhibition of p110a and mTOR exerts potent antineoplastic effects on medulloblastoma cells and also blocks neurosphere growth. Dual inhibition of p110a and mTOR might be promising for targeting both malignant medulloblastoma cells and the therapy-resistant cancer stem cell population. Neuro-Oncology 18:iii97–iii122, 2016. doi:10.1093/neuonc/now076.63 #The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.