Abstract B14: Establishment and characterization of in vitro models of alternative lengthening of telomeres (ALT) in pediatric high-grade glioma

Abstract

Abstract Telomeres consist of many kilobases of repeated TTAGGG sequences at the ends of chromosomes, protected by a sequence-specific protein cap. Telomeres progressively shorten with each cell division and ultimately become critically short; due to their extensive proliferation, cancer cells must find a way to counteract this telomere loss. While most cancers utilize telomerase to maintain their telomere length, about 5% of cancers use a telomerase-independent telomere maintenance strategy, termed alternative lengthening of telomeres (ALT). While, overall, ALT is rare in cancer (~5-10% of cases), this telomere maintenance mechanism is enriched in pediatric high-grade glioma (pHGG). Previous work in our laboratory suggests that nearly half of pHGG utilize ALT. To date, therapeutic options are largely ineffective for children with HGG, reflected in the five-year survival rate, which is less than 33%. Our goal is to better harness ALT as a clinical marker in pHGG, specifically by the identification of drugs that target ALT-positive cancers. In order to study ALT in this context, we obtained and characterized a panel of six pHGG cell lines. Two of these six pHGG cell lines displayed features of ALT, including the presence of ALT-associated PML bodies and extrachromosomal telomeric DNA in the form of c-circles. Furthermore, these lines lacked measurable telomerase activity. It is well established that ATRX is commonly mutated in ALT-positive cancers, including pHGG. Interestingly, only one of these two ALT-positive pHGG cell lines displayed total loss of ATRX; the second cell line has an in-frame deletion in the ATRX gene, which may provide insight into the mechanism of how ATRX acts to suppress ALT. In addition, we have generated ATRX knockout cell lines from the four ALT-negative pHGG cells identified in this panel. Despite the strong link between ATRX loss and ALT in clinical samples, only one cell line displayed ALT characteristics after ATRX knockout. Comparison of the ALT-competent cell line to the ALT-resistant cell lines will yield important information about additional genetic or epigenetic events that allow ALT to occur. In conclusion, we have established in vitro models of ALT in pHGG cell lines based on endogenous ALT positivity and induction of ALT-like features based on ATRX modulation. These models will be invaluable resources as we strive to understand the molecular characteristics of ALT and translate these findings to better therapies for children with pHGG. Citation Format: Jacqueline A. Brosnan-Cashman, Mindy K. Graham, Anthony J. Rizzo, Kaylar Myers, Rebecca Zhang, Ezgi Göger, Reza Zarinshenas, Christine Davis, Ming Yuan, Dinesh Rakheja, Eric H. Raabe, Charles G. Eberhart, Christopher M. Heaphy, Alan K. Meeker. Establishment and characterization of in vitro models of alternative lengthening of telomeres (ALT) in pediatric high-grade glioma [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr B14.