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Targeting Shelterin Proteins in Cancer

Periodic Reporting for period 2 - SHELTERINS (Targeting Shelterin Proteins in Cancer)

Período documentado: 2022-01-01 hasta 2023-06-30

Telomere biology is also intricately linked with human cancer. To avoid telomere loss associated to cell division, most cancers (95%) reactivate telomerase. In spite of the important role of telomere maintenance for cancer growth, inducing telomerase dysfunction in cancer cells remains an unmet clinical challenge. Indeed, telomerase inhibitors have largely failed in clinical trials of cancer, most likely owing to the fact that the effectivity of telomerase inhibition depends on the telomere length of the cancer cells. Telomerase is not the only telomere component altered in cancer, indicating that alternative or additional approaches are needed to induce telomere maintenance in cancer cells. We explored the idea of targeting shelterin as an anticancer strategy to inducer telomere damage independently of telomere length. We validated the inhibition of the shelterin component TRF1 as an effective therapy in both lung cancer and glioblastoma. We developed chemical inhibitors of TRF1 and found that TRF1 is regulated by key cancer pathways such as PI3K/AKT and RAS. In addition, we and others had identified mutations of the telomere binding protein POT1, a component of shelterin, in several types of human sporadic or familial cancer. These include chronic lymphocytic leukemia (CLL), familial melanoma, Li-Fraumeni like (LFL) syndrome families with cases of cardiac angiosarcoma (CAS), glioma, mantle cell lymphoma, and parathyroid adenoma.

We aimed to unravel the underlying mechanisms by which mutations in shelterin components contribute to, or even drive, tumour development. Our goal is therefore to unveil the role of shelterin mutations in cancer by establishing a comprehensive set of tools, and conduct a fundamental and far-reaching experimental program on the role of such mutations in human cancer. Our specific aims are (i) to generate novel knock-in mouse models to understand the role of POT1 mutations found in human cancer to develop personalized therapeutic strategies based on these alterations, (ii) to generate new knock-in mouse models to understand TRF1 post-translational modifications by multiple cancer pathways to identify new cancer targets based on TRF1/shelterin blockage, and (iii) to dissect the potential role of TRF1 in cancer stem cells.

We are convinced that targeting shelterin will develop into a novel and promising strategy to reverse one of the yet unmet hallmarks of cancer, the maintenance of telomeric integrity. Our proposal is an explicit high-gain research program, since it is one of our aims to develop novel anticancer strategies based on shelterin-mediated telomere protection.
We generated the Ki-Pot1aR117C mouse carrying the LFL mutation with CAS in p53+/+ and p53-/- backgrounds. The Pot1a+/R117C mice shows increased incidence of angiosarcomas to wild-type mice, recapitulating the LFL patients (Martinez, Sanchez-Vazquez et al., Plos Genet., 2022). We also generated the ki model harboring the G95C mutation in human glioma. This mutation is embryonic lethal even in heterozygosis, thus impeding the study of cancer phenotypes in adulthood. We generated another POT1a ki carrying the L259S substitution found in patients with idiopathic pulmonary fibrosis.

We have concluded the generation by CRISPR/Cas9 technology of T330A and T330D homozygous ki mice. These mouse models where the endogenous Trf1 locus is replaced by mutant Trf1 alleles are defective in AKT-mediated phosphorylation. In addition, we have also found that human TRF1 is also subjected to PI3K/AKT-mediated regulation and that the non-phosphorylatable human TRF1 variants display a tumour suppressor phenotype. These results have been published in PloS Genetics (Sánchez-Vázquez et al., Plos Genet., 2021).

We found that mouse TRF1 is also subjected to a post-translational regulation by a non-canonical B-Raf pathway. We identified the domains required for the interaction between BRAF and TRF1. BRAF loss led to the activation of cell-cycle arrest pathways (p21) and the expression of proteins associated with malignant transformation (Sox9 and E-Cadherin) in liver, lung and kidney glomerulus. We also studied the effects of acute ubiquitous BRAFV600E activation in vivo and found that BRAFV600E expression induces DDR that results in a rapid responses of cell cycle and senescence-associated proteins in lung epithelia, revealing the early molecular changes emerging in BRAFV600E-challenged cells during tumorigenesis in vivo. These results have been published in PloS Genetics (Bosso et al., Cell Death Dis., 2022).

To study the role of TRF1 in cancer stem cells we have set up cell lines stably expressing Cas9 and the other screening elements to perform a CRISPR-CAS9 screening. We have also found that during induced pluripotent stem cell generation, TRF1 is required from the very early steps of reprograming.
Targeting shelterin will develop into a novel and promising strategy to reverse one of the yet unmet hallmarks of cancer, the maintenance of telomeric integrity. Our results will lead to the development of novel anticancer strategies based on shelterin-mediated telomere protection.