A screen-informed candidate gene approach identifies a large human telomere maintenance network. B. Holohan, W. Wright, J. Shay Cell Biology, UT Southwestern Medical Center, Dallas, TX.

   Telomeres are nucleoprotein structures on the ends of chromosomes that shield the termini of linear chromosomes from recognition by the DNA double strand break repair machinery. Because of the end replication problem, telomeres shorten with every cell division, thus telomere shortening is an inherent limitation in the number of times that a cell can divide before triggering a DNA damage response. Germ cells and certain stem cells utilize telomerase, a ribonucleoprotein reverse transcriptase, to partially maintain their telomere lengths. This allows them to have extended proliferative potential, but telomeres still get progressively shorter with increased age. In contrast, roughly 90% of human tumors use telomerase to maintain their telomeres, and they can fully maintain their telomere lengths although most cancer cells have very short telomeres. Since telomerase is not expressed in most somatic cells but is expressed in the vast majority of cancer cells, telomerase is a near-universal target for cancer therapy. In order to identify ways to target telomerase for cancer therapy, we constructed a mutant telomerase RNA (TERC) component that templates non-canonical telomere repeats when utilized by telomerase. These mutant repeats are toxic via telomere upcapping and activation of the DNA double strand break repair machinery, causing senescence, genomic catastrophe or apoptosis. The introduction of the mutant TERC into telomerase silent cells had no effect on cell growth. However, using cells with an inducible telomerase (TERT) allowed us to utilize shRNA tools to identify positive regulators of telomerase; shRNAs that reduced the incorporation of mutant sequences upon telomerase induction would allow cell survival. Using this system and a screen-informed candidate gene approach, we have identified and validated 50 new genes that induce telomere shortening in human cells upon knockdown. Based on the upstream regulators of some of the genes identified, we examined the effects of Perifosine, an AKT inhibitor, on telomere biology in vitro and in human xenograft studies. Perifosine induced telomere shortening in a majority; of tumor cell lines investigated and reduced telomere length and tumor size in xenografts after prolonged exposure. Our work has revealed a large and surprisingly malleable telomerase regulation network that suggests a number of existing drugs may be useful telomerase modulators.

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