Cancer-associated TERT promoter mutations abrogate telomerase silencing.

Mutations in the human telomerase reverse transcriptase (TERT) promoter are the most frequent non-coding mutations in cancer, but their molecular mechanism in tumorigenesis has not been established. We used genome editing of human pluripotent stem cells with physiological telomerase expression to elucidate the mechanism by which these mutations contribute to human disease. Surprisingly, telomerase-expressing embryonic stem cells engineered to carry any of the three most frequent TERT promoter mutations showed only a modest increase in TERT transcription with no impact on telomerase activity. However, upon differentiation into somatic cells, which normally silence telomerase, cells with TERT promoter mutations failed to silence TERT expression, resulting in increased telomerase activity and aberrantly long telomeres. Thus, TERT promoter mutations are sufficient to overcome the proliferative barrier imposed by telomere shortening without additional tumor-selected mutations. These data establish that TERT promoter mutations can promote immortalization and tumorigenesis of incipient cancer cells

Human Intestinal Tissue with Adult Stem Cell Properties Derived from Pluripotent Stem Cells

Genetically engineered human pluripotent stem cells (hPSCs) have been proposed as a source for transplantation therapies and are rapidly becoming valuable tools for human disease modeling. However, many applications are limited due to the lack of robust differentiation paradigms that allow for the isolation of defined functional tissues. Here, using an endogenous LGR5-GFP reporter, we derived adult stem cells from hPSCs that gave rise to functional human intestinal tissue comprising all major cell types of the intestine. Histological and functional analyses revealed that such human organoid cultures could be derived with high purity and with a composition and morphology similar to those of cultures obtained from human biopsies. Importantly, hPSC-derived organoids responded to the canonical signaling pathways that control self-renewal and differentiation in the adult human intestinal stem cell compartment. This adult stem cell system provides a platform for studying human intestinal disease in vitro using genetically engineered hPSCs

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

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

Cancer-associated TERT promoter mutations abrogate telomerase silencing.

Mutations in the human telomerase reverse transcriptase (TERT) promoter are the most frequent non-coding mutations in cancer, but their molecular mechanism in tumorigenesis has not been established. We used genome editing of human pluripotent stem cells with physiological telomerase expression to elucidate the mechanism by which these mutations contribute to human disease. Surprisingly, telomerase-expressing embryonic stem cells engineered to carry any of the three most frequent TERT promoter mutations showed only a modest increase in TERT transcription with no impact on telomerase activity. However, upon differentiation into somatic cells, which normally silence telomerase, cells with TERT promoter mutations failed to silence TERT expression, resulting in increased telomerase activity and aberrantly long telomeres. Thus, TERT promoter mutations are sufficient to overcome the proliferative barrier imposed by telomere shortening without additional tumor-selected mutations. These data establish that TERT promoter mutations can promote immortalization and tumorigenesis of incipient cancer cells

Endogenous Telomerase Reverse Transcriptase N-Terminal Tagging Affects Human Telomerase Function at Telomeres In Vivo

Telomerase action at telomeres is essential for the immortal phenotype of stem cells and the aberrant proliferative potential of cancer cells. Insufficient telomere maintenance can cause stem cell and tissue failure syndromes, while increased telomerase levels are associated with tumorigenesis. Both pathologies can arise from only small perturbation of telomerase function. To analyze telomerase at its low endogenous expression level, we genetically engineered human pluripotent stem cells (hPSCs) to express various N-terminal fusion proteins of the telomerase reverse transcriptase from its endogenous locus. Using this approach, we found that these modifications can perturb telomerase function in hPSCs and cancer cells, resulting in telomere length defects. Biochemical analysis suggests that this defect is multileveled, including changes in expression and activity. These findings highlight the unknown complexity of telomerase structural requirements for expression and function in vivo

Endogenous Telomerase Reverse Transcriptase N-Terminal Tagging Affects Human Telomerase Function at Telomeres In Vivo

Telomerase action at telomeres is essential for the immortal phenotype of stem cells and the aberrant proliferative potential of cancer cells. Insufficient telomere maintenance can cause stem cell and tissue failure syndromes, while increased telomerase levels are associated with tumorigenesis. Both pathologies can arise from only small perturbation of telomerase function. To analyze telomerase at its low endogenous expression level, we genetically engineered human pluripotent stem cells (hPSCs) to express various N-terminal fusion proteins of the telomerase reverse transcriptase from its endogenous locus. Using this approach, we found that these modifications can perturb telomerase function in hPSCs and cancer cells, resulting in telomere length defects. Biochemical analysis suggests that this defect is multileveled, including changes in expression and activity. These findings highlight the unknown complexity of telomerase structural requirements for expression and function in vivo