Telomere disorders including dyskeratosis congenita (DC) manifest in stem cell failure in highly proliferative tissues such as the skin, blood, and gut, as well as fibrosis in organs including lung and liver. While hematopoietic manifestations can be treated with bone marrow transplant, therapeutic strategies are limited for other affected organs. In a collaboration supported by Team Telomere, the Lengner and Johnson labs set out to develop human induced pluripotent stem cell (iPSC)-based platforms to model DC phenotypes in the intestine, lung, and liver. We utilize CRISPR/Cas9-enabled genome editing to correct or introduce DC mutations in to iPSCs, then direct the differentiation of the resulting pairs of isogenic cell lines into intestinal, lung, or liver epithelial cell types. We then utilize these models to identify molecular aberrations in cultures harboring DC mutations relative to their isogenic controls, and ultimately test whether small molecules targeting pathways affected by DC mutations influence phenotypes related to cell/tissue function as well as telomere length and capping.

In a series of studies, we found that the Wnt pathway exists in a positive feed-forward loop with telomere fidelity in the epithelia of both the lung and intestine. Wnt activity is critical for the proliferative self-renewal of stem and progenitor cells in these tissues, and when telomeres become dysfunctional, the resulting DNA damage signals to inhibit Wnt activity. This results in loss of stem cell function, a mechanism which presumably evolved for tumor suppression during normal ageing, but one that becomes pathogenic in DC. Interestingly, while DC patients have decreased telomerase activity, there remains some residual activity. Because of the feed-forward relationship between Wnt and telomerase (including both telomerase enzymatic activity and telomere capping), we reasoned that pharmacological stimulation of Wnt might elevate telomerase activity in DC cultures sufficiently to restore telomere capping and suppress DC phenotypes. Indeed, in both the intestinal epithelium and the lung, Wnt agonists were able to largely inhibit DC phenotypes and promote re-capping of telomeres1–3.

In contrast to the lung and intestine, we found that Wnt pathway activity was not dramatically affected by DC mutations in liver models, and Wnt agonists did not reverse DC phenotypes in this system. Rather, we found that liver phenotypes resulting from telomere dysfunction in hepatocytes, were driven largely by aberrant activation of the AKT/mTORC pathway. Here, we employed various pharmacological inhibitors targeting components of this pathway and found that AKT inhibitors were sufficient to suppress DC phenotypes and restore telomere capping in cultures harboring DC mutations. This includes non-cell-autonomous phenotypes in the liver stroma driven by telomere dysfunction in the parenchymal hepatocytes4.
Taken together, these studies provide unprecedented insight into the tissue-specific consequences of DC mutations in human models and offer conceptual strategies for treatment. However, unanswered questions remain. Particularly, we have begun to view DC as a developmental disorder, and patient symptoms may not manifest until cellular phenotypes are quite advanced. Thus, while our findings offer promising strategies for potentially preventing onset of these symptoms, it is unknown whether these strategies will be efficacious in reversing advanced phenotypes, particularly the fibrosis observed in the liver and lung.

Our pilot funding from Team Telomere enabled this work and led to the receipt of NIH funding of this collaborative project through the NIA (R21) and NIDDK (R01), for which we are deeply grateful. Moreover, working with and getting to know the Team Telomere community has been immensely gratifying and we hope to continue pushing this work forward.