Abstract 3412: Functional knockout of ATRX or DAXX permits the alternative lengthening of telomeres (ALT) mechanism in prostate cancer cells
Authors: Graham Mindy K. 
Published: July 2017

A key hallmark of cancer is unlimited replication, which requires cancer cells to evade replicative senescence induced by short telomeres. The majority of cancers overcome this critical replication barrier by upregulating the telomerase enzyme, a telomere-specific reverse transcriptase. However, a subset of cancers lack telomerase, and telomeres are maintained by employing the Alternative Lengthening of Telomeres (ALT) pathway, which is dependent on homologous recombination. Across a variety of tumor types, our laboratory and others have reported a robust correlation between ALT and recurrent cancer-associated somatic inactivating mutations in the ATRX-DAXX chromatin remodeling complex. In a previous comprehensive cancer survey of ALT, we reported that ALT was highly prevalent in some tumor types (e.g. astrocytomas, sarcomas, pancreatic neuroendocrine tumors), but we did not observe any ALT-positive primary prostate cancer (out of 1,176 analyzed). However, we subsequently found a subset of metastatic prostate cancers that harbor ALT, suggesting that mutations giving rise to ALT in this disease are unique to metastatic prostate cancer.

Here, we have created the first prostate cancer cell lines exhibiting ALT, with the explicit purpose of molecularly characterizing ALT in prostate cancer and identifying promising therapeutic targets for men with ALT-positive lethal metastatic prostate cancer. Inactivating mutations in either ATRX or DAXX using the CRISPR/Cas9n system were introduced into the genetically well-characterized, telomerase-positive (ALT-negative) prostate cancer cell lines, LAPC-4 and CWR22Rv1. Resulting mutant subclones were compared to their parental (or empty vector) counterparts. In these new models, abolishing ATRX expression was sufficient to induce the ALT phenotype in both LAPC-4 and CWR22Rv1, as assessed by multiple biomarkers of ALT, including the presence of bright telomeric FISH foci, ALT-associated PML bodies (ABPs), and extrachromosomal telomere c-circles. Interestingly, abolishing DAXX expression induced ALT in only a subset (3/5) of LAPC-4 DAXX KO clones, and a subset (4/19) of CWR22Rv1 DAXX KO clones.

We have successfully activated the ALT telomere maintenance phenotype in two prostate cancer cell lines through CRISPR-mediated targeted gene deletion. We are currently utilizing these isogenic cell lines to further characterize and elucidate the underlying biology of cancers harboring ALT, with the goal of pharmacologically targeting the molecular features unique to the ALT phenotype (e.g. ATR inhibition). The identification of ALT-specific drugs may pave the way for the development of new targeted treatments for the subset of men with ALT-positive lethal metastatic prostate cancer, and more broadly, other ALT-positive cancers. ALT is easily detected in clinical tissue samples, and thus would serve as a predictive biomarker for personalized medicine.

Citation Format: Mindy K. Graham. Functional knockout of ATRX or DAXX permits the alternative lengthening of telomeres (ALT) mechanism in prostate cancer 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 3412. doi:10.1158/1538-7445.AM2017-3412