Novel insights into the GCN2 pathway and its targeting. Therapeutic value in cancer and lessons from lung fibrosis development.

Defining the mechanisms that allow cells to adapt to environmental stress is critical for understanding the progression of chronic diseases and identifying relevant drug targets. Among these, activation of the pathway controlled by the eIF2-alpha kinase GCN2 is critical for translational and metabolic reprogramming of the cell in response to various metabolic, proteotoxic, and ribosomal stressors. However, its role has frequently been investigated through the lens of a stress pathway signaling via the eIF2α-activating transcription factor 4 (ATF4) downstream axis, while recent advances in the field have revealed that the GCN2 pathway is more complex than previously thought. Indeed, this kinase can be activated through a variety of mechanisms, phosphorylate substrates other than eIF2α, and regulate cell proliferation in a steady state. This review presents recent findings regarding the fundamental mechanisms underlying GCN2 signaling and function, as well as the development of drugs that modulate its activity. Furthermore, by comparing the literature on GCN2's antagonistic roles in two challenging pathologies, cancer and pulmonary diseases, the benefits, and drawbacks of GCN2 targeting, particularly inhibition, are discussed.

Soft extracellular matrix drives endoplasmic reticulum stress-dependent S quiescence underlying molecular traits of pulmonary basal cells.

Cell culture on soft matrix, either in 2D and 3D, preserves the characteristics of progenitors. However, the mechanism by which the mechanical microenvironment determines progenitor phenotype, and its relevance to human biology, remains poorly described. Here we designed multi-well hydrogel plates with a high degree of physico-chemical uniformity to reliably address the molecular mechanism underlying cell state modification driven by physiological stiffness. Cell cycle, differentiation and metabolic activity could be studied in parallel assays, showing that the soft environment promotes an atypical S-phase quiescence and prevents cell drift, while preserving the differentiation capacities of human bronchoepithelial cells. These softness-sensitive responses are associated with calcium leakage from the endoplasmic reticulum (ER) and defects in proteostasis and enhanced basal ER stress. The analysis of available single cell data of the human lung also showed that this non-conventional state coming from the soft extracellular environment is indeed consistent with molecular feature of pulmonary basal cells. Overall, this study demonstrates that mechanical mimicry in 2D culture supports allows to maintain progenitor cells in a state of high physiological relevance for characterizing the molecular events that govern progenitor biology in human tissues. STATEMENT OF SIGNIFICANCE: This study focuses on the molecular mechanism behind the progenitor state induced by a soft environment. Using innovative hydrogel supports mimicking normal human lung stiffness, the data presented demonstrate that lung mechanics prevent drift while preserving the differentiation capabilities of lung epithelial cells. Furthermore, we show that the cells are positioned in a quiescent state in the atypical S phase. Mechanistically, we demonstrate that this quiescence: i) is driven by calcium leakage from the endoplasmic reticulum (ER) and basal activation of the PERK branch of ER stress signalling, and ii) protects cells from lethal ER stress caused by metabolic stress. Finally, we validate using human single-cell data that these molecular features identified on the soft matrix are found in basal lung cells. Our results reveal original and relevant molecular mechanisms orchestrating cell fate in a soft environment and resistance to exogenous stresses, thus providing new fundamental and clinical insights into basal cell biology.

SUrface SEnsing of Translation (SUnSET), a Method Based on Western Blot Assessing Protein Synthesis Rates in vitro.

As the most energy- and metabolite-consuming process, protein synthesis is under the control of several intrinsic and extrinsic factors that determine its fine-tuning to the cellular microenvironment. Consequently, variations in protein synthesis rates occur under various physiological and pathological conditions, enabling an adaptive response by the cell. For example, global protein synthesis increases upon mitogenic factors to support biomass generation and cell proliferation, while exposure to low concentrations of oxygen or nutrients require translational repression and reprogramming to avoid energy depletion and cell death. To assess fluctuations in protein synthesis rates, radioactive isotopes or radiolabeled amino acids are often used. Although highly sensitive, these techniques involve the use of potentially toxic radioactive compounds and require specific materials and processes for the use and disposal of these molecules. The development of alternative, non-radioactive methods that can be easily and safely implemented in laboratories has therefore been encouraged to avoid handling radioactivity. In this context, the SUrface SEnsing of Translation (SUnSET) method, based on the classical western blot technique, was developed by Schmidt et al. in 2009. The SUnSET is nowadays recognized as a simple alternative to radioactive methods assessing protein synthesis rates. Key features • As a structural analogue of aminoacyl-transfer RNA, puromycin incorporates into the elongating peptide chain. • Detection of puromycin-labeled peptides by western blotting reflects translation rates without the need for radioactive isotopes. • The protocol described here for in vitro applications is derived from the SUnSET method originally published by Schmidt et al. (2009).

Tailoring a specific medical leadership development program for faculty members: the Lyon-Ottawa experience.

The development of leadership skills has been the topic of several position statements over recent decades, and the need of medical leaders for a specific training was emphasized during the COVID-19 crisis, to enable them to adequately collaborate with governments, populations, civic society, organizations, and universities. However, differences persist as to the way such skills are taught, at which step of training, and to whom. From these observations and building on previous experience at the University of Ottawa, a team of medical professors from Lyon (France), Ottawa, and Montreal (Canada) universities decided to develop a specific medical leadership training program dedicated to faculty members taking on leadership responsibilities. This pilot training program was based on a holistic vision of a transformation model for leadership development, the underlying principle of which is that leaders are trained by leaders. All contributors were eminent French and Canadian stakeholders. The model was adapted to French faculty members, following an inner and outer analysis of their specific needs, both contextual and related to their time constraints. This pilot program, which included 10 faculty members from Lyon, was selected to favor interactivity and confidence in older to favor long-term collaborations between them and contribute to institutional changes from the inner; it combined several educational methods mixing interactive plenary sessions and simulation exercises during onescholar year. All the participants completed the program and expressed global satisfaction with it, validating its acceptability by the target. Future work will aim to develop the program, integrate evaluation criteria, and transform it into a graduating training.