Inhibition of GSK3B signaling in pediatric brain tumors

Advances in genomic profiling have enabled increasingly precise molecular classification of pediatric embryonal brain tumors. However, the five-year overall survival rate for atypical teratoid/rhabdoid tumors (ATRT) and embryonal tumors with multilayered rosettes (ETMR) remains below 30%, despite intensive multimodal therapies. 9-ING-41 is a potent GSK3B inhibitor that has been shown to cross the blood-brain barrier and have biological activity against patient-derived intracranial xenograft models of other brain tumors, as well as good tolerability in recent Phase I human clinical trials. Previously we demonstrated that 9-ING-41 decreases ETMR and ATRT cell viability through apoptosis and increases p53 signaling, however the exact mechanism is unknown. The primary objective of this study was to determine whether GSK3B inhibition using 9-ING-41 could effectively suppress ATRT and ETMR growth by modulating p53 signaling pathway. ATRT and ETMR cell lines were grown for 24 hours prior to 9-ING-41 treatment in DMEM and Neurocult medium, respectively. Cell viability was assessed after 72hrs of treatment using Cell Titer Glo 2.0. Cells treated with IC50 determined dosing for 24-72 hours were collected for Western blot analysis of key proteins responsible for apoptosis and p53 signaling molecules. Cells were harvested for RNA isolation using Qiagen RNeasy kit. Transcriptomic profiles were established by RNA seq. Neurosphere assays were done by seeding two cells per well in a 96-well plate followed by treatment with 9-ING-41. In vivo xenograft models were established by orthotopic injection of ATRT 2141 cells into the mice brain through intra cranial stereotaxic system followed by treatment with either vehicle or 70mg/Kg 9-ING-41 by I/P injection for 9 weeks. In vitro analyses using multiple ATRT and ETMR cell lines revealed a significant therapeutic response to GSK3B inhibitor within clinically relevant dosing ranges. The IC50 of 9-ING-41 in BT183, CHLA02-ATRT, ATRT-2187, ATRT 2141 are 145.5, 481.2, 503.1, and 528.3nM respectively. Whole-exome and RNA sequencing of treated cells demonstrated upregulation of p53 tumor suppressor genes and downregulation of Shh pathway genes, supporting a tumor-suppressive mechanism. Protein expression analyses of p53 negative regulator further confirmed that 9-ING-41 treatment enhanced the activation of apoptotic pathways through p53 signaling pathway. In vivo studies showed a significant improvement in overall survival in mice treated with 9-ING-41 increased survival from 35 days to 55 days (P < 0.0001). Mechanistic evaluation of tumor tissues revealed increased p53 expression and marked downregulation of Ki-67, indicating reduced proliferative activity.