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Projeto de investigação
Telomeric R-loop-mediated recombination in the alternative lengthening of telomeres pathway: towards designing novel therapeutic avenues for cancer therapy
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FANCM limits ALT activity by restricting telomeric replication stress induced by deregulated BLM and R-loops
Publication . Silva, Bruno; Pentz, Richard; Figueira, Ana Margarida; Arora, Rajika; Lee, Yong Woo; Hodson, Charlotte; Wischnewski, Harry; Deans, Andrew J.; Azzalin, Claus Maria
Telomerase negative immortal cancer cells elongate telomeres through the Alternative Lengthening of Telomeres (ALT) pathway. While sustained telomeric replicative stress is required to maintain ALT, it might also lead to cell death when excessive. Here, we show that the ATPase/translocase activity of FANCM keeps telomeric replicative stress in check specifically in ALT cells. When FANCM is depleted in ALT cells, telomeres become dysfunctional, and cells stop proliferating and die. FANCM depletion also increases ALT-associated marks and de novo synthesis of telomeric DNA. Depletion of the BLM helicase reduces the telomeric replication stress and cell proliferation defects induced by FANCM inactivation. Finally, FANCM unwinds telomeric R-loops in vitro and suppresses their accumulation in cells. Overexpression of RNaseH1 completely abolishes the replication stress remaining in cells codepleted for FANCM and BLM. Thus, FANCM allows controlled ALT activity and ALT cell proliferation by limiting the toxicity of uncontrolled BLM and telomeric R-loops.
ALTernative functions for human FANCM at telomeres
Publication . Domingues Silva, Ana Beatriz; Silva, Bruno; Azzalin, Claus Maria
The human FANCM ATPase/translocase is involved in various cellular pathways including DNA damage repair, replication fork remodeling and R-loop resolution. Recently, reports from three independent laboratories have disclosed a previously unappreciated role for FANCM in telomerase-negative human cancer cells that maintain their telomeres through the Alternative Lengthening of Telomeres (ALT) pathway. In ALT cells, FANCM limits telomeric replication stress and damage, and, in turn, ALT activity by suppressing accumulation of telomeric R-loops and by regulating the action of the BLM helicase. As a consequence, FANCM inactivation leads to exaggerated ALT activity and ultimately cell death. The studies reviewed here not only unveil a novel function for human FANCM, but also point to this enzyme as a promising target for anti-ALT cancer therapy.
TERRA transcription destabilizes telomere integrity to initiate break-induced replication in human ALT cells
Publication . Silva, Bruno; Arora, Rajika; Bione, Silvia; Azzalin, Claus Maria
Alternative Lengthening of Telomeres (ALT) is a Break-Induced Replication (BIR)-based mechanism elongating telomeres in a subset of human cancer cells. While the notion that spontaneous DNA damage at telomeres is required to initiate ALT, the molecular triggers of this physiological telomere instability are largely unknown. We previously proposed that the telomeric long noncoding RNA TERRA may represent one such trigger; however, given the lack of tools to suppress TERRA transcription in cells, our hypothesis remained speculative. We have developed Transcription Activator-Like Effectors able to rapidly inhibit TERRA transcription from multiple chromosome ends in an ALT cell line. TERRA transcription inhibition decreases marks of DNA replication stress and DNA damage at telomeres and impairs ALT activity and telomere length maintenance. We conclude that TERRA transcription actively destabilizes telomere integrity in ALT cells, thereby triggering BIR and promoting telomere elongation. Our data point to TERRA transcription manipulation as a potentially useful target for therapy.
RNA–DNA hybrids support recombination-based telomere maintenance in fission yeast
Publication . Hu, Yan; Bennett, Henrietta W; Liu, Na; Moravec, Martin; Williams, Jessica F; Azzalin, Claus Maria; King, Megan C
A subset of cancers rely on telomerase-independent mechanisms to maintain their chromosome ends. The predominant "alternative lengthening of telomeres" pathway appears dependent on homology-directed repair (HDR) to maintain telomeric DNA. However, the molecular changes needed for cells to productively engage in telomeric HDR are poorly understood. To gain new insights into this transition, we monitored the state of telomeres during serial culture of fission yeast (Schizosaccharomyces pombe) lacking the telomerase recruitment factor Ccq1. Rad52 is loaded onto critically short telomeres shortly after germination despite continued telomere erosion, suggesting that recruitment of recombination factors is not sufficient to maintain telomeres in the absence of telomerase function. Instead, survivor formation coincides with the derepression of telomeric repeat-containing RNA (TERRA). In this context, degradation of TERRA associated with the telomere in the form of R-loops drives a severe growth crisis, ultimately leading to a novel type of survivor with linear chromosomes and altered cytological telomere characteristics, including the loss of the shelterin component Rap1 (but not the TRF1/TRF2 ortholog, Taz1) from the telomere. We demonstrate that deletion of Rap1 is protective in this context, preventing the growth crisis that is otherwise triggered by degradation of telomeric R-loops in survivors with linear chromosomes. These findings suggest that upregulation of telomere-engaged TERRA, or altered recruitment of shelterin components, can support telomerase-independent telomere maintenance.
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Fundação para a Ciência e a Tecnologia
Programa de financiamento
3599-PPCDT
Número da atribuição
PTDC/MED-ONC/28282/2017