Decottignies, Anabellede Lhoneux, GuillaumeGuillaumede Lhoneux2025-05-142025-05-142025-05-142021https://hdl.handle.net/2078.2/23680Telomeres are specialized protective structures present at chromosome ends. Replicative immortality, defined by the ability of cells to maintain telomere length over successive divisions, characterizes the majority of cancer cells. In most cancer cells, telomerase is reactivated but immortalization is sometimes achieved through activation of an alternative telomere maintenance mechanism (TMM) termed ALT (Alternative Lengthening of Telomeres), based on homologous recombination events between telomeric sequences. ALT positive (ALT+) cells display distinct features including telomere length heterogeneity, the presence of extrachromosomal telomeric circles, including C-circles, and ALT-associated PML bodies (APBs), which are characterized by the colocalization of telomeres with promyelocytic leukaemia (PML) nuclear bodies. ALT activation is frequent in tumours deriving from mesenchymal tissues which display high prevalence in paediatric tumours. Being absent from normal somatic cells, ALT represents an attractive and specific target for cancer therapy. Our laboratory has proposed a model in which TSPYL5 acts, only in ALT+ cells, as a stabilizer of the key shelterin component POT1 through USP7 deubiquitinase inhibition to keep ALT+ cells alive. Based on this model, the TSPYL5/USP7 interaction could be a promising novel target for ALT+-specific cancer therapy. My master’s thesis aims first, at mapping the TSPYL5/USP7 interaction domain, second, at establishing new ALT+ paediatric tumour cell lines and, third, at developing ALT+ xenograft models in mice. For the first part, we overexpressed full length TSPYL5 or truncated forms in U2OS/ALT+ cells and performed co-immunoprecipitation assays. We demonstrated that both N-terminal and middle part of TSPYL5 interacted with endogenous USP7 whereas the C-terminal part showed no interaction, suggesting TSPYL5 contains at least two autonomous interaction domains for USP7. For the second part, with the aim of characterizing the TMM of paediatric tumour samples of Saint-Luc hospital (Brussels), we first set up the different ALT marker assays using tumours coming from the xenografts of LB857/ALT+ and HT1080/TEL+ cell lines. We confirmed the efficiency of both the C-circle and the native FISH assay. Finally, we confirmed the ability of LB857/myxoid sarcoma/ALT+ cells to form a tumour one month after subcutaneous injection into NOD scid gamma (NSG) immunodeficient mice.paediatric cancerstelomeresALTernative mechanism of telomere maintenancetargeted therapiesTSPYL5text::thesis::master thesisthesis:31032