Periodic Reporting for period 1 - RTeRloop (Replication and post-replication signalling pathways Regulating R-loop-associated Telomere instability in human cells)
Reporting period: 2021-10-01 to 2023-09-30
Telomeres are crucial chromatin regions located at the ends of linear chromosomes that prevent genome instability and control the cellular replicative life span. Telomeres shorten with each cell division until they reach a critically short length, becoming unprotected and leading to cellular senescence. Unprotected or damaged telomeres constitute a major source of genome instability and massive genome rearrangements characteristic of tumorigenesis, while the activation of telomere maintenance mechanisms ensures cell immortalisation in cancer.
Telomeres are considered "difficult-to-replicate sites" due to their intrinsic characteristics, including the ability to be transcribed into long non-coding RNAs known as TERRAs. TERRAs can hybridise to the DNA template, forming DNA-RNA hybrids and displacing the non-template single-stranded DNA, constituting the so-called R-loops. Unscheduled R-loop formation is known to cause significant hindrances for DNA replication leading to transcription-replication conflicts; thus, contributing to genome instability when accumulated under pathological conditions. However, they also have important physiological regulatory roles, including at telomeres. Many efforts have been made to identify cellular factors that prevent R-loop-associated genome instability, but we have very little knowledge about the mechanisms that regulate TERRA DNA-RNA hybrid metabolism or whether these structures might differentially modulate telomere homeostasis in different cell types or conditions.
Understanding the molecular mechanisms that regulate telomeric R-loops in human cells may help us identify potential targets to combat some telomere-related diseases or certain types of cancers where telomeric dysregulation of these structures promotes DNA damage leading to disease development. A relevant case involves the subset of 10-15% of cancer types that use a telomerase-independent but recombination-dependent pathway, based on break-induced telomere synthesis, to elongate telomeres and achieve immortalisation, known as Alternative Lengthening of Telomeres (ALT). ALT cancers comprise a variety of cancers typically of mesenchymal or neuroendocrine origin, including some types of paediatric cancers, that are very aggressive with poor prognosis and without an established specific treatment. The molecular mechanism of ALT is not fully understood, but recent studies have identified TERRA R-loops as key intermediates that promote ALT recombination. Therefore, the idea of targeting telomeric R-loops has arisen as a promising therapy to specifically target ALT cancer cells without affecting telomerase-positive or primary human cells, where TERRA levels are generally much lower.
This project aims to gain insight into the molecular mechanisms that regulate telomeric DNA-RNA hybrids in normal and cancer cells that use different telomere maintenance mechanisms. By characterising telomeric DNA-RNA hybrids along the cell cycle and focusing on the consequences of the deregulation of new factors implicated in telomeric R-loop homeostasis, we seek to conduct a detailed analysis of R-loop control at telomeres. This will provide potential clues for targeting R-loop-associated malignancies.
Telomeres are considered "difficult-to-replicate sites" due to their intrinsic characteristics, including the ability to be transcribed into long non-coding RNAs known as TERRAs. TERRAs can hybridise to the DNA template, forming DNA-RNA hybrids and displacing the non-template single-stranded DNA, constituting the so-called R-loops. Unscheduled R-loop formation is known to cause significant hindrances for DNA replication leading to transcription-replication conflicts; thus, contributing to genome instability when accumulated under pathological conditions. However, they also have important physiological regulatory roles, including at telomeres. Many efforts have been made to identify cellular factors that prevent R-loop-associated genome instability, but we have very little knowledge about the mechanisms that regulate TERRA DNA-RNA hybrid metabolism or whether these structures might differentially modulate telomere homeostasis in different cell types or conditions.
Understanding the molecular mechanisms that regulate telomeric R-loops in human cells may help us identify potential targets to combat some telomere-related diseases or certain types of cancers where telomeric dysregulation of these structures promotes DNA damage leading to disease development. A relevant case involves the subset of 10-15% of cancer types that use a telomerase-independent but recombination-dependent pathway, based on break-induced telomere synthesis, to elongate telomeres and achieve immortalisation, known as Alternative Lengthening of Telomeres (ALT). ALT cancers comprise a variety of cancers typically of mesenchymal or neuroendocrine origin, including some types of paediatric cancers, that are very aggressive with poor prognosis and without an established specific treatment. The molecular mechanism of ALT is not fully understood, but recent studies have identified TERRA R-loops as key intermediates that promote ALT recombination. Therefore, the idea of targeting telomeric R-loops has arisen as a promising therapy to specifically target ALT cancer cells without affecting telomerase-positive or primary human cells, where TERRA levels are generally much lower.
This project aims to gain insight into the molecular mechanisms that regulate telomeric DNA-RNA hybrids in normal and cancer cells that use different telomere maintenance mechanisms. By characterising telomeric DNA-RNA hybrids along the cell cycle and focusing on the consequences of the deregulation of new factors implicated in telomeric R-loop homeostasis, we seek to conduct a detailed analysis of R-loop control at telomeres. This will provide potential clues for targeting R-loop-associated malignancies.
Throughout the project, a comprehensive analysis of the differential regulation of telomeric DNA-RNA hybrids helps us gain insight into the molecular mechanisms and key factors that control telomeric R-loop levels and their implications in telomere integrity. Interestingly, as observed in the rest of the genome and despite the differential regulation of TERRA RNA levels, DNA-RNA hybrids accumulate along the cell cycle in normal and cancer cells that use different telomere maintenance mechanisms. Molecular and genetic analysis combined with the design of specific approaches for the modulation of DNA-RNA hybrids suggest that a variety of factors may process these structures along the cell cycle, having different consequences for telomere replication and stability.
Unscheduled dysregulation of telomeric DNA-RNA hybrids leads to telomere replication stress, which may represent a significant source of telomere damage, commonly associated with tumorigenesis and aging-related diseases. While RNA processing factors might directly act through these structures to modulate their stability, key factors of the DNA Damage Response may orchestrate a cellular response to resolve the conflict. Comparative studies using data from unbiased proteomics analysis of isolated chromatin from telomeres help identify potential RNA processing and chromatin factors that, by acting through different mechanisms on telomeric DNA-RNA hybrid homeostasis, play a crucial role in preserving telomere stability. Discovering new factors that specifically control R-loop-associated telomere stability could be exploited for designing new treatments that selectively target telomeres in specific types of cancer cells, thereby avoiding the undesirable and common side effects of current antitumor treatments.
The findings of this project have been disseminated within the scientific community through presentations at seminars and conferences, and will be available upon publication in peer-reviewed journals.
Unscheduled dysregulation of telomeric DNA-RNA hybrids leads to telomere replication stress, which may represent a significant source of telomere damage, commonly associated with tumorigenesis and aging-related diseases. While RNA processing factors might directly act through these structures to modulate their stability, key factors of the DNA Damage Response may orchestrate a cellular response to resolve the conflict. Comparative studies using data from unbiased proteomics analysis of isolated chromatin from telomeres help identify potential RNA processing and chromatin factors that, by acting through different mechanisms on telomeric DNA-RNA hybrid homeostasis, play a crucial role in preserving telomere stability. Discovering new factors that specifically control R-loop-associated telomere stability could be exploited for designing new treatments that selectively target telomeres in specific types of cancer cells, thereby avoiding the undesirable and common side effects of current antitumor treatments.
The findings of this project have been disseminated within the scientific community through presentations at seminars and conferences, and will be available upon publication in peer-reviewed journals.
The project outcomes allow us to gain insight into the relationship between telomeric DNA-RNA hybrids and telomere homeostasis, thus highlighting the regulation of telomeric DNA-RNA hybrids as a key process in preventing telomere instability. Controlling this phenomenon is essential in cancer cells that use different mechanisms to achieve immortality, especially in ALT-type tumours and human syndromes that provoke the deregulation of telomere transcription. These results will be highly relevant not only to the fundamental biology of telomeres but also to the translational field of biomedicine, as they will contribute to increasing our knowledge about the mechanisms of resistance to current anti-tumour therapies and the aging process. Similarly, this study will open new avenues for the design of molecular targets in the treatment of ALT cancers and R-loop-linked malignancies, which could be implemented in the future to aid in the development of new protocols for more personalised medicine.