Rumo à erradicação precisa dos cancros ALT: explorando ferramentas inovadoras para estudar a dinâmica e regulação dos R-loops nas extremidades dos cromossomas

The ALTRLOOP project comprises three primary objectives: Objective 1) Advancing scientific knowledge within and beyond the telomere biology field. Our aim is to provide a comprehensive characterization of telR-loop metabolism and the ALT mechanism, while identifying promising targets and tools for cancer therapy. Our research plan is organized into 6 scientific tasks. Task 1 involves establishing novel ALT reporter cell lines to visualize telR-loops by exploiting a genetically encoded R-loop sensor able to detect telR-loops in live and fixed cells with unprecedented specificity. Tasks 2 and 3 focus on combining molecular and cellular biology with advanced microscopy techniques applied to our reporter cell lines to determine the timing of telR-loop generation and dismantling throughout the cell cycle, and investigate the influence of telomere length on their levels with single telomere resolution. Task 4 centers on further enhancing our reporter cell lines by tagging key factors of the ALT machinery (e.g., POLD3, RAD52, XPF, and Mus81) with fluorescent proteins. These new cell lines will be utilized in Task 5 to elucidate the molecular crosstalk between telR-loops and ALT. All experiments will be conducted in both normal cells and cells deficient in RNaseH1 or FANCM activities using siRNAs or small molecule inhibitors. The final scientific task of the project (Task 6) will identify and characterize compounds modulating telR-loops through a small molecule screening campaign based on our reporter cell lines and libraries of DNA and RNA binding compounds. Drugs increasing or decreasing telR-loop levels will be further characterized in panels of ALT and non-ALT cells lines for their ability to affect telomere stability and, most importantly, viability. Small molecules showing promising cellular activity will then be further tested in ALT xenograft murine models through collaboration with TessellateBIO, a precision oncology company cofounded by Claus M. Azzalin in 2020. Objective 2) Improving the well-being of cancer patients by providing novel therapeutic opportunities. The ALTRLOOP project framework positions us well to discover novel molecular interactions that can be targeted for ALT cancer therapy. Moreover, our screening campaigns will likely identify small molecules affecting ALT cancer cell viability. The Tech Transfer Office of iMM will ensure the implementation of optimal intellectual property and valorization strategies to maximize social and economic impact. Our partnership with TessellateBIO and established collaborations between our institute and leading companies in the oncology field will help expedite the future translation of laboratory discoveries into clinical applications. Objective 3) Promoting data dissemination to stakeholders and scientific literacy. Our findings will be presented at international and national conferences, and our work will be published in preprint repositories and open-access formats to ensure widespread accessibility. We plan to engage with the public through various outreach activities, including multimedia interviews, science clubs for pediatric cancer patients and their families, and hands-on activities within the Lisbon area.