Abstract Telomeres progressively shorten with each cell division, leading to critically short telomeres during tumorigenesis. While most cancers activate telomerase to maintain their telomeres, a subset of cancers utilize a recombination-based process known as alternative lengthening of telomeres, or ALT. ALT has been widely characterized mostly in the context of immortalized cells, and characteristic features include ultrabright telomeric foci, colocalization of telomere foci with PML (ALT-associated PML bodies, or APBs), extrachromosomal telomeric DNA (e.g. c-circles), and overall telomere length heterogeneity. While the overall rate of ALT in cancer is low (roughly 5%, overall), the prevalence of ALT is concentrated in certain cancer subtypes, including gliomas. Gliomas employing ALT have a common genomic signature: inactivating mutation of ATRX, a chromatin-modifying protein that functions at the telomeres. In order to study the role of ATRX loss in the biology of ALT in cancer, we have characterized a panel of six human cell lines derived from pediatric high grade glioma, two of which are ALT(+) and lack functional ATRX. Furthermore, in four pediatric and three adult high grade glioma cell lines that endogenously express functional ATRX, we knocked out ATRX using CRISPR/Cas9n and knocked down ATRX expression using a stable shRNA-mediated approach. Knockout or knockdown of ATRX was successful in these seven glioma cell lines. Overall, knockout and/or knockdown of ATRX induced the formation of ultrabright telomeric foci, as assessed by telomere FISH, in two out of seven glioma cell lines. However, the effect of ATRX loss on formation of c-circles and APBs was more variable: the presence of ultrabright telomeric foci was not predictive of the presence of c-circles or ALT-associated PML bodies. These results indicate that ATRX loss, alone, is not necessarily sufficient to activate the ALT pathway in gliomas. Further examination of ALT-competent and ALT-resistant cell lines from our cohort will yield clues as to additional ALT-associated gene expression changes. In addition, our results suggest that previously identified hallmarks of ALT (notably c-circles and APBs) may be more variable in cancer cells compared to immortalized normal cell lines. The ALT pathway is an intriguing target for synthetic lethality in the cancer subtypes that employ it; our hope is to better understand the ALT phenotype in cancer so that we may effectively translate ALT-specific therapeutics to the clinic. Citation Format: Jacqueline A. Brosnan-Cashman, Ming Yuan, Anthony J. Rizzo, Kaylar M. Myers, Walter T. Barry, Christine Davis, David M. Esopi, Dinesh Rakheja, Eric H. Raabe, Charles G. Eberhart, Christopher M. Heaphy, Alan K. Meeker. Context-dependent effects of ATRX loss on telomere biology in glioma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3465. doi:10.1158/1538-7445.AM2017-3465