Telomerase and telomere length regulation in human disease

Telomere length shows a very wide distribution, from short to long, in the human population. Only at the extremes of the distribution are long or short telomeres associated with severe pathologies, such as cancer or bone marrow failure respectively. This variable constitutional telomere length provides a framework for the proliferative capacity of the cells of an individual. It thereby establishes the balance of enabling and limiting cell divisions required for tissue homeostasis. On the molecular level the key regulators of telomere length are the telomere binding protein complex shelterin and the telomere elongating enzyme telomerase./nTelomerase is expressed in embryonic and adult stem cells but downregulated upon differentiation. During tumorigenesis pre-malignant cells stabilize telomeres and acquire the capacity to proliferate indefinitely by various mechanisms. 90% of all cancers are immortalized by telomerase expression. Promoter mutations of the telomerase reverse transcriptase gene (TERT) are the most common non-coding mutations in cancer. However, the timing and consequences of TERT promoter mutations (TPMs) have not been fully established. I used a combination of melanoma patient samples and human embryonic stem cell (hESC) models to show that TPMs can occur before the loss of DNA damage checkpoints and do not prevent telomere shortening. In vitro, cells with TPMs proliferate for a prolonged time with ever shorter and partially deprotected telomeres. Thus, TPMs do not prevent bulk telomere shortening but extend cellular lifespan by healing the shortest telomeres for sustained proliferation. Over time cells with TPMs can gradually upregulate telomerase to levels sufficient to sustain all telomeres and thereby become immortalized as a bulk cell population./nThe finding that TPMs occur very early during tumorigenesis of several cancers and provide a selective advantage for overcoming replicative senescence, led me to further investigate the role of telomere shortening as a tumor suppressive mechanism. However, pre-cancerous melanocytic neoplasms – dysplastic nevi – are widely believed to arrest due to oncogene induced senescence caused by the acquisition of an initial driver mutation. To assess when and how TPMs confer a selective advantage to melanocytes, I analyzed dysplastic nevi for melanoma-associated mutations and telomere length. I found that TPMs were detected with a higher frequency in older patients, indicating that not all melanocytes were arrested and that later in life telomere length becomes limiting for proliferation. The analysis of telomere length revealed a dichotomy: melanocytes of samples with TPMs had relatively shorter telomeres than TERT promoter wild-type samples. In contrast, BRAF p.V600E-positive nevi had longer telomeres than nevi with other driver mutations and none of the BRAF p.V600E nevi harbored a TPM. This indicates that melanocytic growth can be restricted by either replicative senescence or oncogene induced senescence in a driver mutation dependent manner./nTelomere shortening acts as a tumor suppressor mechanism by restricting proliferation but in order to replenish tissues for homeostasis cells have to be able to undergo a sufficient number of cell divisions. In the short telomere disease Dyskeratosis congenita (DC) very short constitutional telomeres lead to severe pathologies of regenerating tissues like the bone marrow, lung and liver. 11 to 24% of DC cases are caused by heterozygous, autosomal dominant mutations in the shelterin component TIN2. We established a human embryonic stem cell (hESC) model system of TIN2 DC missense mutations, which recapitulates the short telomere patient phenotype. In hESCs the DC mutation leads to telomere shortening in a telomerase-dependent manner, while maintaining telomere protection. As a consequence, the differentiated daughter cells have a reduced proliferative capacity. Disruption of one allele of TINF2 restores telomere length and proliferative capacity. We conclude that TIN2-DC mutations exert their disease effect in the telomerase-positive stem cell compartments of the human body, such as hematopoietic stem cells, which could provide an attractive target for therapeutic intervention in the future.

Advisor: Hockemeyer, Dirk