Decoding the mechanisms of cancer and stem cell immortality

Telomeres are the repetitive sequences at the ends of linear chromosomes. Thekey functions of telomeres are to protect the cells from losing genomic information and toprevent chromosome ends from being repaired by the double strand break repairmachinery. To counteract loss of telomeric DNA, cells can express a reversetranscriptase, telomerase, that synthesizes telomeric repeats de novo. In humans,telomerase activity is mostly restricted to germ and stem cells, so the telomeres of mostsomatic cells progressively shorten with each cell division. Once telomeres becomecritically short, they are recognized as sites of DNA damage and cells cease to proliferate.By this mechanism, telomere shortening functions as a tumor suppression mechanism.TERT, the protein component of telomerase, becomes silenced once stem cellsdifferentiate. However, in 90% of cancer cells, TERT is transcriptionally re-activated.Thus, telomerase regulation is crucial for our understanding of telomere length regulationin stem cell maintenance and tumorigenesis. To understand how telomerase acts ontelomeres, I attempted to endogenously tag telomerase. To do this I inserted epitope tagsat the endogenous TERT locus in hESCs using genome editing. However, I found that allthe tested tags cause defects in telomere maintenance, which was previously notappreciated in experiments using exogenous overexpression.Recently, point mutations in the TERT promoter were identified as the mostfrequent non-coding mutations in cancer. To elucidate the role of TERT promotermutations (TPMs) in tumorigenesis, I genetically engineered these TPMs into humanembryonic stem cells (hESCs) using genome editing. Using the resulting isogenic hESClines, I demonstrated that TPMs lead to a failure of TERT silencing upon differentiationfrom stem into somatic cells. To understand role of TPMs in tumorigenesis, I monitoredlong-term telomere maintenance and proliferation in human fibroblasts engineered tocarry TPMs. I found that TPMs immortalize cells but do not prevent telomere shorteningand telomere fusions. In vitro, around the time when telomere fusions occurred, TERTexpression was gradually increased. Thus, TPMs are required, but not sufficient, forcancer cell immortality and contribute to tumorigenesis in two steps. First, TPMs expandproliferation capacity of a cell by elongating only the shortest telomeres but do notprevent overall telomere shortening. In the second step, TPMs fuel tumorigenesis by notfully suppressing genomic instability. In order for cells to immortalize they need toupregulate TERT during this second step