Abstract 995: Loss of ATRX decreases survival and improves response to DNA damaging agents in a novel mouse model of glioblastoma

Abstract

Abstract Pediatric glioblastoma (GBM) remains one of the most difficult childhood tumors to treat, and most patients will die within the first two years of receiving this diagnosis. ATRX is a histone chaperone protein that is mutated primarily in adolescent GBMs. No previous animal model has demonstrated the effect of ATRX loss on GBM formation. In this study, we determined the contribution of ATRX knockdown to GBM formation and treatment response in a novel mouse model of GBM. Using the second-generation shRNA-mir library, we cloned an ATRX knockdown sequence into a plasmid with flanking sequences recognized by the Sleeping Beauty (SB) transposase for insertion into host genomic DNA. Glioblastomas were induced in mice using the SB transposase system injecting plasmids encoding luciferase, shp53 and NRAS, with or without shATRX, into the ventricle of neonatal mice. Uptake of plasmid DNA as well as development of intracranial tumors was monitored by bioluminescence. When animals showed symptoms of tumor burden they were euthanized and brains were processed for histological evaluation or placed in culture with neural stem cell media (with EGF and FGF supplementation). Tumors in both groups (with or without shATRX) showed histological hallmarks of human grade IV glioblastoma. The loss of ATRX was confirmed by IHC, and was specifically localized within tumors generated with the shATRX plasmid and not in the tumors generated with shp53 and NRAS alone, nor in the adjacent normal cortex. Notably, loss of ATRX reduced median survival of mice by 43% (p=0.012). Tissue was analyzed by FISH telomere probe as ATRX loss in human tumors is associated with alternative lengthening of telomeres (ALT). ATRX-deficient tumors were significantly more likely to show chromosomal aneuploidy (p=0.015) by telomere FISH. Cell lines generated from ATRX-deficient tumors were confirmed to have reduction of ATRX expression. Tumor cell lines (with and without ATRX loss) were plated, treated at 24 hours with intervention or control, and analyzed for viability at 72 hours. ATRX-deficient tumor cells were significantly (p≤0.005) more sensitive to DNA damaging agents, including: (1) 5-FU, (2) doxorubicin, (3) UV irradiation, and (4) adenoviral thymidine kinase with ganciclovir; with the notable exception of temozolomide (p=0.86), which is the standard of care for treatment of pediatric GBM. Loss of ATRX in a mouse model hastens glioblastoma formation and decreases survival. In addition, loss of ATRX leads to aneuploidy and improved response to DNA damaging agents, providing possible targeted therapies for tumors with this mutation. This mouse model prospectively validates ATRX as a tumor suppressor in pediatric GBM for the first time in an animal model; and provides a platform for analysis of relevant pathways and development of potential novel therapies. Supported by NIH/NINDS grants to MGC and PRL. Citation Format: Carl Koschmann, Alexandra Calinescu, Marta Dzaman, Rosie Lemons, Daniel Thomas, Maria G. Castro, Pedro R. Lowenstein. Loss of ATRX decreases survival and improves response to DNA damaging agents in a novel mouse model of glioblastoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 995. doi:10.1158/1538-7445.AM2014-995