Abstract 1831: 9-ING-41, a glycogen synthase kinase 3B inhibitor targets TP53 in pediatric brain tumors ATRT and ETMR

Abstract

Abstract Purpose: The study aims to determine the activity of the blood brain barrier permeable glycogen synthase kinase 3B (GSK3B) inhibitor, 9-ING-41, in pediatric brain tumors: embryonal tumor with multilayered rosettes (ETMR) and atypical rhabdoid tumor (ATRT). Methods: ETMR cell line, BT183, was grown in ultralow attachment flasks and ATRT cell lines: ATRT-7316-2187, ATRT-787197, and CHLA02 were grown in suspension flasks. Cells were treated with 9-ING-41 at dose range (0-20µM). After 24 and 48 hours of 9-ING-41 cells were analyzed by western blot. After 72 hours of 9-ING-41 cell viability was assessed with Cell Titer Glo 2.0 and IC50s were analyzed by GraphPad. RNA seq was performed on cells after 12, 24, and 48 hours of 9-ING-41 treatment. Total RNA was isolated with miRNeasy Mini Kit. RNA integrity was evaluated by an Agilent 2100 Bioanalyzer profile. rRNA was selectively depleted by employing RiboZero Globin. Next generation sequencing libraries were prepared using an Illumina TruSeq Stranded Total RNA kits. Read mapping was performed using STAR in BaseSpace. DESeq2 was used for normalization and differential expression analysis. Ingenuity Pathway Analysis was performed. Results: The IC50s of 9-ING-41 in the cell lines BT183, CHLA02-ATRT, ATRT-787197, and ATRT-2187 are 145.5nM, 481.2nM, 503.1nM, and 528.3nM, respectively. In BT183 cells treated with 150nM 9-ING-41 for 48 hours, XIAP decreased significantly by 55% and cleaved caspase 3 increased significantly by 29-fold, quantified by densitometry. In addition, western blot showed a decrease in LIN28A and an increase in TP53 protein levels. RNA-seq data show an increase in the p53 signaling pathway in the ETMR BT183 cell line with z-scores of 2.53 and 3 (p=3.73x10-13 and 2.35x10-14) after 12 and 24 hours of 150nM 9-ING-41, respectively. In ATRT-787197 cells p53 signaling increased with z-scores of 1.0 and 2.236 (p=2.62x10-8 and 2.89x10-5) after 24 and 48 hours of 400nM 9-ING-41, respectively. RNA seq data also show the ability of 9-ING-41 to significantly increase the expression of CDK inhibitor, CDKN1A, and pro-apoptotic genes BBC3, DRAM1, and BAX in ETMR and ATRT while significantly decreasing stemness associated genes: SMO and GLI1 in ETMR. Conclusions: 9-ING-41 decreases ETMR and ATRT cell viability at clinically relevant concentrations and these doses increase the p53 pathway and induce apoptosis while reducing the expression of stemness associated genes. Citation Format: Jason L. Haw, Elizabeth M. Moore, Kimberly McKinney, Karl Dykema, Abhinav Nagulapally, Nury Steuerwald, Giselle S. Sholler. 9-ING-41, a glycogen synthase kinase 3B inhibitor targets TP53 in pediatric brain tumors ATRT and ETMR [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 1831.