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International audience; Telomeres ensure genome integrity and are maintained by telomerase. During replicative senescence, telomerase is inactivated, telomere sequences progressively shorten and set the limit for cell proliferation. When telomeres shorten, they are thought to lose their protective caps at a critical short length, which activates the DNA damage response and recruits DNA damage repair activities that would degrade, fuse, or recombine dysfunctional telomeres. However, the structure(s) of short and dysfunctional telomeres, which respectively trigger permanent replicative senescence or potentially promote genome instability, remain unclear.To define the structure of telomeres at the point of dysfunction and the fate of cells carrying them, we have developed in Saccharomyces cerevisiae a system called FinalCut to induce a single telomere of defined length in cells in which we can conditionally inactivate telomerase. This allows structural analysis of this telomere and, combined with the use of our microfluidic system to track consecutive cell cycles from telomerase inactivation to cell death, we can achieve single telomere and single cell resolution. Our results show that cells reach senescence earlier and more synchronously, as the shortest telomere is set at a shorter length. This is in agreement with the first telomere reaching a short length being sufficient to trigger senescence in budding yeast. Below a certain threshold, the shorter telomere becomes unstable and degrades. However, we can define a length for which a short telomere appears to be stable, and the cells stop dividing at the same time. Combined with a mathematical model of senescence, our results suggest that the probability of a telomere signaling senescence increases as it shortens, but at low frequencies. When a telomere reaches a critical length, it can be stably maintained in cells, while activating the DNA damage checkpoint, without causing obvious fusions or degradation. The structure of this critically short telomere will be discussed.

International audience; Progress in understanding telomere replication and its relationship to senescence has been hampered by the intrinsic variations in telomeres and the stochastic nature of senescence onset. The work in our lab relies on our ability to circumvent and experimentally dissect the causes of this heterogeneity. Our strategy is to manipulate and track single telomeres in individual cells and experimentally evaluate the effects on proliferation potential of single cell lines to build a mathematical model of senescence.We therefore set up a microfluidics-based live cell imaging assay to study replicative senescence in single Saccharomyces cerevisiae cell lineages after telomerase inactivation. We found that most lineages undergo an abrupt and irreversible transition consistent with a mathematical model in which the first telomere reaching a short critical length triggers the onset of senescence. Other cells exhibit transient checkpoint-dependent cell cycle delays followed by normal cell cycles before senescence. We have now shown that these two pathways to senescence correspond to two kinetically and mechanistically distinct, age-dependent processes underlying non- terminal and terminal senescence arrest.Taking advantage of our novel FinalCut system, we also investigated the proliferation potential of telomerase-negative cells with critically short telomeres of different defined lengths. Our results are consistent with a model whereby a small portion of cells may enter senescence in a probabilistic manner as the length of the shortest telomere decreases. Our results thus directly demonstrate that the length of the shortest telomere is a major determinant of entry into senescence, but a hidden parameter may also contribute to the onset of senescence upon telomerase inactivation. However, when the shortest telomere reaches a critical length, all cells immediately arrest. In addition, we found that at this critical length, the shortest telomere can be stably maintained without signs of fusions or degradations. This suggests that telomeres can be both critically short and functional with respect to telomere protection.

International audience; Telomeres ensure genome integrity and are maintained by telomerase. During replicative senescence, telomerase is inactivated, telomere sequences progressively shorten, and set the limit for cell proliferation. As telomeres shorten, they are thought to lose their protective caps at a critical short length, activating the DNA damage response and recruiting DNA damage repair activities that would degrade, fuse, or recombine dysfunctional telomeres. However, the structure(s) of short and dysfunctional telomeres, which respectively trigger permanent replicative senescence or potentially promote genome instability, remain unclear. To define the structure of telomeres at the point of dysfunction and the fate of cells carrying them, we developed a system called FinalCut to induce a single telomere of defined length in cells in which we can conditionally inactivate telomerase. This allows structural analysis of this telomere, and combined with the use of our microfluidic system to track consecutive cell cycles from telomerase inactivation to cell death, we can achieve single telomere and single cell resolution. Our results show that the loss of telomere stability is progressive as the telomere shortens, and that telomeres of a given length can be protected or unprotected in separate cells. We conclude that telomere length is not the sole determinant of the switch between functional and dysfunctional telomeres. We therefore propose that the minimum length of telomeres to maintain cell viability follows a probabilistic, rather than deterministic, model.

Introduction: In the JAVELIN Lung 200 trial, avelumab (anti-programmed death-ligand 1 [PD-L1] antibody) did not significantly prolong overall survival (OS) versus docetaxel in patients with platinum-treated PD-L1+ NSCLC. We report greater than 2-year follow-up data. Methods: Patients with stage IIIB or IV or recurrent NSCLC with disease progression after platinum-doublet chemotherapy were randomized 1:1 to avelumab 10 mg/kg every 2 weeks or docetaxel 75 mg/m(2) every 3 weeks. The primary end point was OS in patients with PD-L1+ tumors (greater than or equal to 1% tumor cell expression; IHC 73-10 pharmDx assay). Results: Of 792 patients, 529 had PD-L1+ tumors (264 versus 265 in the avelumab versus docetaxel arms, respectively). As of March 4, 2019, median duration of follow-up for OS in the PD L1+ population was 35.4 months in the avelumab arm and 34.7 months in the docetaxel arm; study treatment was ongoing in 25 (9.5%) versus 0 patients, respectively. In the PD-L1+ population, 2-year OS rates (95% confidence interval [CI]) with avelumab versus docetaxel were 29.9% (24.5%-35.5%) versus 20.5% (15.6%-25.8%); in greater than or equal to 50% PD L1+ subgroups, 2-year OS rates were 36.4% (29.1%-43.7%) versus 17.7% (11.8%-24.7%) and in the greater than or equal to 80% subgroup were 40.2% (31.3%-49.0%) versus 20.3% (12.9%-28.8%), respectively. Median duration of response (investigator assessed) was 19.1 months (95% CI: 10.8-34.8) versus 5.7 months (95% CI: 4.1-8.3). Safety profiles for both arms were consistent with the primary analysis. Conclusions: Although the JAVELIN Lung 200 primary analysis (reported previously) revealed that avelumab did not significantly prolong OS versus docetaxel in patients with platinum-treated PD-L1+ NSCLC, posthoc analyses at 2 years of follow-up revealed that 2-year OS rates were doubled with avelumab in subgroups with higher PD-L1 expression (greater than or equal to 50% and greater than or equal to 80%). (C) 2021 International Association for the Study of Lung Cancer. Published by Elsevier Inc., EMD Serono Research & Development Institute, Inc., an affiliate of Merck KGaA, Darmstadt, Germany; Pfizer; Merck KGaA, LL This study was sponsored by EMD Serono Research & Development Institute, Inc., an affiliate of Merck KGaA, Darmstadt, Germany, as part of an alliance between Merck KGaA and Pfizer. Employees of the sponsor are coauthors of this manuscript and contributed to the design, execution, and interpretation of the analyses being reported, writing the report, and the decision to submit the article for publication, along with the other coauthors. The authors thank the patients and their families; the investigators, coinvestigators, and study teams at each participating center and at Merck KGaA, Darmstadt, Germany, and EMD Serono Research & Development Institute, Inc., Billerica, MA, an affiliate of Merck KGaA, Darmstadt, Germany, and Quintiles (Durham, NC) . Medical writing support was provided by Abhijith Thippeswamy of ClinicalThinking and funded by Merck KGaA and Pfizer.

Telomere length in the yeast Saccharomyces cerevisiae is under stringent genetic control such that a narrow length distribution of TG₁₋₃ repeats is observed. Previous studies have shown that Rap1p, which binds to the double-stranded telomeric repeats, plays a role in regulating repeat length: point mutations in the Rap 1 p C-terminus often result in a higher average telomere length and deletion of this region causes extreme telomere elongation. We have investigated further the role of Rap1p in this process. Our results suggest that telomere length is regulated by a negative feedback mechanism that can sense the number of Rap1p molecules bound at the chromosome end. This length regulatory mechanism requires two other proteins, Rif1p and Rif2p, that interact with each other and with the Rap1p C-terminus. Although the same C-terminal domain of Rap1p is also involved in the initiation of telomere position effect (telomeric transcriptional silencing), this Rap1p function appears to be separate from, and indeed antagonistic to, its role in telomere length regulation.