Abstract 980: Encoding immortality: Transcriptional control of telomerase in stem cells in vivo

One of the invariant features of human cancer is unlimited proliferation, a hallmark conferred by telomerase in 90% tumors. Somatic mutations in the telomerase reverse transcriptase (TERT) gene promoter are highly recurrent in human cancers. Telomerase is also critically important in human stem cells, as evidenced by mutations in telomerase, which contribute to degenerative diseases. Despite the importance of telomerase in tissue maintenance, the identity of telomerase-positive cells has remained elusive, owing to low levels of the core telomerase components. The ability to isolate TERT-positive cells in vivo would significantly advance our understanding of telomerase regulation, tissue function and carcinogenesis. To address these issues, we created knock-in transcriptional reporters of TERT expression by replacing the TERT open reading frame with the red fluorescent protein, TdTomato. Among mouse tissues, telomerase activity is most strongly expressed in testis, a tissue in which resident stem cells fuel the continuous generation of male gametes. In human sperm, telomere lengths are preserved with age, although how this is achieved, in contrast to the age-dependent telomere shortening seen in somatic tissues, remains unresolved. Using TERTTdTomato/+ knock-in reporter mice, we found that only a rare subset of cells in mouse testis expresses high levels of TERT. By double immunostaining, these TERTHigh cells were synonymous with undifferentiated spermatogonia, the primitive cell population in which male germline stem cells reside. By FACS of the germ cells in testis, TERTHigh cells and TERTLow cells represent discrete populations that were further studied using additional markers. The undifferentiated spermatogonia in the TERTHigh population were further fractionated into GFRalpha+ and GFRalpha- populations. Cells in the TERTLow population were nearly all cKit+, consistent with their identification as differentiated spermatogonia. We characterized these populations in molecular and functional terms. Using RNAseq, we established a hierarchy among these populations according to which the TERTHigh GFRalpha1+ cells give rise to TERTHigh GFRalpha1- cells, which in turn yield TERTLow cKit+ cells. Surprisingly, in transplantation studies, TERTHigh GFRalpha1+ cells and TERTHigh GFRalpha1- cells possess comparable stem cell activity. These data suggest the existence of stem cell plasticity according to which cells in either primitive population retain stem cell potential. In contrast, TERTLow cKit+ cells fail to reconstitute spermatogenesis in transplantation experiments and therefore lack stem cell activity. These studies reveal marked transcriptional regulation of telomerase in vivo and show a strong concordance between stemness and telomerase levels in rare subsets of tissue stem cells in vivo. These findings indicate the existence of innate signaling pathways controlling TERT expression over a surprising dynamic range. Citation Format: Matthew Pech, Alina Garbuzov, Meena Sukhwani, Berenice Benayoun, Shengda Lin, Anne Brunet, Kyle Orwig, Steven E. Artandi. Encoding immortality: Transcriptional control of telomerase in stem cells in vivo. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 980. doi:10.1158/1538-7445.AM2015-980