Abstract 3464: CRISPR-mediated inactivation of ATRX and DAXX in pancreatic neuroendocrine tumor cell lines
Authors: Rizzo Anthony J. , Brosnan-Cashman Jacqueline A. , Graham Mindy K. , Meeker Alan K. , Heaphy Christopher M. 
Published: July 2017

Cancer cells must find a way to subvert replicative senescence in order to achieve cellular immortality. While most malignancies (&gt; 90 %) overcome this critical barrier by reactivating the telomerase enzyme, a telomere-specific reverse transcriptase; other cancers (5 - 10 %) utilize a telomerase-independent pathway of telomere maintenance, referred to as Alternative Lengthening of Telomeres (ALT). ALT is thought to utilize homologous recombination and DNA damage repair (DDR) machinery to maintain the chromosome ends. Importantly, this mechanism appears to be cancer-specific and dependent on alterations in chromatin dynamics at the telomeres, almost invariably facilitated by inactivating mutations in the genes, ATRX and DAXX. The protein products of these genes are responsible for the deposition of histone variant H3.3 at repetitive regions of DNA (e.g. telomeres) and when altered, the affected cells tend to exhibit the ALT phenotype. Pancreatic neuroendocrine tumors (PanNETs) are an example of a tumor type that often employs the ALT pathway with coincident somatic mutations in either the ATRX or DAXX genes. PanNETs are the second most common pancreatic malignancy, with only 40% of affected patients surviving past 10 years. Recently, we and others have demonstrated that in primary PanNETs, ALT is an independent prognostic marker, associated with aggressive clinicopathologic behavior and reduced recurrence-free survival. In addition, current data suggest that ALT-positive cancer cells are sensitive to inhibition of the DNA-damage mediator, ATR. In light of these findings, there seems to be an opportunity to develop ALT specific therapeutic modalities that would have considerable clinical utility. Models of ALT-positive PanNET pathology are needed to further progress in this direction, unfortunately, there are currently no ALT-positive PanNET cell lines. BON1 and QGP1, the two well characterized PanNET cell lines that are available, are both ALT-negative and retain expression of telomerase, ATRX, and DAXX. In an attempt recapitulate the ALT phenotype in a PanNET cell line, we derived a panel of isogenic cell lines from both BON1 and QGP1 by utilizing the CRISPR-Cas9 nickase system to target ATRX and DAXX. In both cell lines, we have successfully established 5 independent subclones with inactivating mutations in either ATRX or DAXX, validated by Sanger sequencing. We are currently characterizing these novel subclones for the hallmarks of ALT, including the presence of ultrabright telomeric foci, dramatic telomere length heterogeneity, and the presence of extrachromosomal telomeric DNA (e.g. c-circles). Once validated, we plan to investigate whether these ATRX or DAXX-deficient subclones are more sensitive to DDR pathway inhibition (i.e. ATR, ATM, CHK1 and CHK2) pharmacologically or with RNA interference in order to identify new therapeutic strategies for PanNETs.

Citation Format: Anthony J. Rizzo, Jacqueline A. Brosnan-Cashman, Mindy K. Graham, Alan K. Meeker, Christopher M. Heaphy. CRISPR-mediated inactivation of ATRX and DAXX in pancreatic neuroendocrine tumor cell lines [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 3464. doi:10.1158/1538-7445.AM2017-3464