HG-02 * ATRX LOSS PROMOTES TUMOR GROWTH AND IMPAIRS GENETIC STABILITY IN GLIOBLASTOMA

Abstract

Pediatric glioblastoma (GBM) remains one of the most difficult childhood tumors to treat. ATRX is a histone chaperone protein that is mutated primarily in pediatric patients with GBM and younger adults with secondary GBM. No previous animal model has demonstrated the effect of ATRX loss on GBM formation. We cloned an ATRX knockdown sequence into a Sleeping Beauty (SB) transposase-responsive plasmid (shATRX) for insertion into host genomic DNA. Glioblastomas were induced by injecting plasmids encoding SB transposase/luciferase, shp53 and NRAS, with or without shATRX, into the lateral ventricle of neonatal mice. Tumors in both groups (with or without shATRX) showed histological hallmarks of human GBM. The loss of ATRX was localized only within tumors generated with the shATRX plasmid and not in the adjacent cortex. Notably, loss of ATRX significantly reduced median survival (p = 0.0032). Loss of ATRX in mouse GBM impaired genetic stability using multiple assays: (1) ATRX-deficient tumors were more likely to develop microsatellite instability, a hallmark of impaired DNA-damage repair; (2) ATRX-deficient tumors displayed evidence of telomeric lengthening, and (3) ATRX loss impaired non-homologous end-joining (NHEJ) utilizing a GFP reporter plasmid system. Analysis of human pediatric (St. Jude's/Washington University) and adult (UCSF) glioma datasets confirmed increased number of somatic nucleotide mutations in tumors with ATRX mutation. Finally, treatment of primary mouse GBM cell cultures showed that ATRX-deficient tumor cells were significantly more sensitive to certain DNA damaging agents, with greater evidence of double-stranded DNA breakage. In addition, mice with ATRX-deficient GBM treated with whole brain irradiation showed reduced early tumor growth by luminescence. This data demonstrates the role of ATRX in GBM tumor progression, treatment response and loss of tumor genetic stability, and provides a platform for future development of targeted therapy for pediatric and young adult patients with ATRX-deficient GBM.