Genetic factors play an important role in the pathogenesis of Paget’s Disease of Bone (PDB). The most important predisposing gene is SQSTM1 which is mutated in about 10% of patients, additionally common variants at seven other loci have also been shown to predispose to PDB as well as environmental factors which are also important in the pathogenesis of PDB. Little research has been conducted on the relationship between the genetic variants that predispose to PDB and disease severity. Similarly, only limited information exists on the role that gene-environment interactions play in the pathogenesis of PDB or its severity. The aim of the present thesis was to explore these issues in participants of the Paget’s Disease Randomised Trial of Intensive versus Symptomatic Management study (PRISM) and other study cohorts. In chapter 3, I investigate the relationship between SQSTM1 mutation status, disease severity and clinical outcome in 737 patients from the PRISM study. Mutations of SQSTM1 were detected in 80/737 (10.9%) patients. Mutation carriers had an earlier age at diagnosis; a greater number of affected bones and more commonly had required orthopaedic surgery and bisphosphonate therapy than those without mutations. Quality of life was significantly reduced in carriers and during the study; fractures were more common although most of these occurred in unaffected bone. This study demonstrates that SQSTM1 mutations are strongly associated with disease severity and complications of PDB. In chapter 4, I study associations between common genetic variants identified by genome wide association (GWAS), clinical severity and extent of PDB, alone and in combination with SQSTM1 mutations. This showed that these common variants were also associated with severity and extent of PDB in PRISM, but with weaker effects than SQSTM1 mutations. The findings were replicated in a multinational study involving 1940 subjects from centres in Italy, Spain and Australia. In all cohorts the GWAS risk alleles acted in an additive manner with SQSTM1 mutations to regulate disease severity and extent. By combining information from SQSTM1 status and the new risk alleles, however, we are able to develop a genetic risk score which delineated three distinct groups with markedly differing effects on disease extent and severity. In chapter 5, I study associations between PDB, severity and extent in relation to circulating levels of IgG antibodies against various viruses including Rubella, respiratory syncytial virus, distemper, varicella zoster virus, measles and mumps. We found little evidence of an interaction between viral antibody titres and SQSTM1 in predicting disease severity with the notable exception of mumps virus where subjects with the highest levels of antibodies that were SQSTM1 positive had in increased age at diagnosis than the other genotype / viral antibody groups. Overall the studies do provide no support for the notion that patients with PDB have an abnormal antibody response to paramyxovirus or have had previous infections with these viruses more frequently than controls. This of course does not exclude the possibility that PDB patients might have a clinically occult slow virus infection which is not accompanied by an abnormality in the immune response. . This raises the possibility that genetic testing may be of value in identifying individuals at risk of developing severe disease and those at risk of complications. I also demonstrate that PBD patients have abnormalities in circulating antibodies to various viruses suggesting that the disease may be associated with disturbance in the response of the immune system to infectious agents but further investigation is required. This, perhaps, could explain the changes in the severity and prevalence of PDB that have been observed over recent years in several countries.

L’autophagie, processus catabolique lysosomal de recyclage de constituants cellulaires, est essentielle à la survie, à la différenciation et au maintien de l’homéostasie cellulaire. Ce processus est fréquemment impliqué dans la survie et la chimiorésistance des tumeurs. La leucémie aiguë promyélocytaire (LAP) est caractérisée par un blocage de la différenciation de la lignée hématopoïétique au stade promyélocytaire. Le traitement des LAP a considérablement progressé depuis l’administration aux patients de doses pharmacologiques d’acide rétinoïque tout-trans (ATRA), un puissant agent de différenciation. L’objectif de ma thèse a consisté à étudier la régulation de l’autophagie au cours de l’induction de différenciation des cellules de LAP par l’ATRA et de rechercher son implication éventuelle dans les mécanismes d’action de ce traitement et les modes d’échappement observés. Lors de mon travail de thèse, j’ai mis en évidence une activation de l’autophagie lors de l’induction de la différenciation granulocytaire des cellules de LAP par l’ATRA. J’ai montré que cette réponse était associée à une inhibition de la voie mTOR et une induction de l’expression des protéines BECLIN 1 et p62/SQSTM1. De façon intéressante, les cellules de LAP résistantes à la maturation par l’ATRA ne sont pas capables d’induire l’expression de p62/SQSTM1 en réponse à l’ATRA. De même, l’expression de p62/SQSTM1 dans les blastes des patients atteints de leucémie aiguë myéloïde est plus faible que celle des granulocytes de sujets sains. L’ensemble de ces données indique que l’expression de p62/SQSTM1 est réprimée dans les phénotypes immatures des cellules myéloïdes mais au contraire induite dans les cellules leucémiques qui s’engagent vers une différenciation terminale (granulocytes/neutrophiles). Enfin, j’ai démontré que les protéines BECLIN 1 et p62/SQSTM1 sont essentielles à la survie de cellules de LAP matures mais non pas à l’engagement de ces cellules vers la différenciation granulocytaire. Ainsi, ces résultats suggèrent qu’en permettant la survie des cellules de LAP différenciées, p62/SQSTM1 et BECLIN 1 pourraient contribuer au développement des résistances à l’ATRA et/ou à l’induction des complications associées à ce traitement tel que le syndrome de différenciation. / Autophagy, a lysosomal process used by the cell to degrade and recycle cytoplasmic constituents, is essential for cell survival, differentiation and the maintenance of cellular homeostasis. Autophagy is often involved in cell survival and resistance to anti-tumor therapy. Acute promyelocytic leukemia (APL) results from a blockade of granulocyte differentiation at the promyelocytic stage. All-trans retinoic acid (ATRA), a potent differentiation agent, has been shown to induce clinical remission in APL patients. The aim of our study was to investigate the regulation and roles of autophagy during ATRA-induced APL cells maturation into neutrophils/granulocytes with the ultimate objective to identify critical mechanisms involved in chemoresistance of APL patients. During my thesis, I demonstrated that autophagy is upregulated during the course of ATRA-induced neutrophil/granulocyte differentiation of APL cells. This response is associated with inhibition of mTOR activity and upregulation of both BECLIN 1 and p62/SQSTM1 proteins. Interestingly, induction of p62/SQSTM1 by ATRA was impaired in maturation-resistant NB4 cells but is re-activated when differentiation was restored in these cells. Accordingly, primary blast cells of AML patients exhibited significantly lower p62/SQSTM1 mRNA levels than did granulocytes from healthy donors. Together, these results highlight that p62/SQSTM1 expression level is repressed in immature myeloid cells compared to mature ones. Moreover, I demonstrated that BECLIN 1 and p62/SQSTM1 proteins are essential for the survival of myeloid cells that undergo differentiation but have no crucial effect on the granulocytic differentiation. This finding may help to elucidate the mechanisms involved in ATRA resistance of APL patients, and in the ATRA syndrome caused by an accumulation of mature APL cells.

Malgré les avancées récentes, le cancer reste la première cause de mortalité en France avec ~ 150000 décès recensés par an (INCa 2012). Notamment, le cancer broncho-pulmonaire est l’un des plus agressifs, avec 29100 décès en 2011. La survie à cinq ans de seulement 10% des patients atteints des cancers du poumon non à petites cellules (NSCLC, 80% des cancers bronchiques), pose un problème d’ordre scientifique, médical et de santé publique. Il est communément admis qu’une sous-population de la tumeur acquiert en raison d’une instabilité génétique de nouvelles propriétés agressives nécessaires à sa dissémination, sa prolifération et une plus grande résistance aux chimiothérapies. Une meilleure compréhension de ces propriétés tumorales constitue un enjeu majeur pour prévenir à terme cette progression maligne. Nous avons axé notre recherche sur la macro-autophagie, un processus catabolique lysosomal essentiel à l’homéostasie cellulaire et sur un de ses substrats SQSTM1 (séquestosome ou p62). Au moment où j’ai initié ma thèse, une relation étroite entre l’autophagie, SQSTM1 et la progression tumorale venait d’être mise en lumière. Plusieurs membres de la machinerie autophagique sont fréquemment mutés/déletés dans les cancers. De façon paradoxale, l’autophagie peut également permettre la survie des cellules tumorales face à l’hypoxie, la carence nutritionnelle ou les chimiothérapies. / Despite recent advances, cancer remains the leading cause of death in France with~150,000 annual deaths (INCA, 2012). Notably, lung cancer is one of the most aggressive, with 29 100 deaths in 2011. The five-year survival is only 10 % of patients with lung cancer non-small cell (NSCLC, 80% of lung cancers), so it’s a medical/scientific challenge, and public health problem. Due to genetic instability, it’s commonly accepted that a subpopulation of tumor acquires new aggressive properties for its dissemination, its proliferation and greater resistance to chemotherapy. A better understanding of these tumor properties is a major issue to prevent this malignant progression. We focused our research on macro-autophagy, a lysosomal catabolic process essential for cellular homeostasis, and alos on one of its substrates SQSTM1 (Sequestosome or p62). When I started my PhD, a close relationship between autophagy, SQSTM1 and tumor progression had been highlighted. Several members of the autophagic machinery are frequently mutated/deleted in cancers. Paradoxically, autophagy can also allow the survival of tumor cells under hypoxia, nutritional deficiency or chemotherapy. How could we understand these conflicting functions, considering that the only autophagy substrates known are long half-lives proteins, damaged organelles and scaffold SQSTM1? Thus, SQSTM1 is essential for RasV12 driven oncogenesis (A Duran et al, Cancer Cell 2008).

Yes / Topoisomerase1 (TOP1)-mediated chromosomal breaks are endogenous sources of DNA damage that affect neuronal genome stability. Whether TOP1 DNA breaks are sources of genomic instability in Huntington's disease (HD) is unknown. Here, we report defective 53BP1 recruitment in multiple HD cell models, including striatal neurons derived from HD patients. Defective 53BP1 recruitment is due to reduced H2A ubiquitination caused by the limited RNF168 activity. The reduced availability of RNF168 is caused by an increased interaction with p62, a protein involved in selective autophagy. Depletion of p62 or disruption of the interaction between RNAF168 and p62 was sufficient to restore 53BP1 enrichment and subsequent DNA repair in HD models, providing new opportunities for therapeutic interventions. These findings are reminiscent to what was described for p62 accumulation caused by C9orf72 expansion in ALS/FTD and suggest a common mechanism by which protein aggregation perturb DNA repair signaling. / This work is funded by a Welcome Trust Investigator Award (103844), a Lister Institute of Preventative Medicine Fellowship (137661) and a UKIERI grant (DST/INT/UK/P-147/2016) to S.F.E.- K. JG is additionally funded by a Clinical PhD Fellowship from the Pathological Society of Great Britain and Ireland and the Jean Shanks Foundation (JSPS-CPHD-2018-01).

L’hypothalamus est une structure cérébrale ayant un rôle clé dans la régulation de la prise alimentaire. Parmi les différentes populations neuronales qui le composent, les neurones produisant la pro-opiomélanocortine (POMC) sont classiquement connus pour diminuer la prise alimentaire et le poids corporel via la libération de neuropeptides produits par le clivage de POMC. Notre étude, grâce à l’utilisation d’approches génétiques, pharmacologiques, électrophysiologiques et moléculaires, remet en question les notions classiques sur la fonction des neurones à POMC dans la balance énergétique, en démontrant qu’il existe deux sous-populations fonctionnellement distinctes de neurones à POMC, qui augmentent ou diminuent la prise alimentaire en fonction du neurotransmetteur qu’elles libèrent, l’acide γ-aminobutyrique (GABA) ou le glutamate. Une troisième population capable de produire aussi bien du GABA que du glutamate a également été identifiée. La régulation des neurones à POMC GABAergiques et glutamatergiques dépend de la voie de la cible de la rapamycine chez les mammifères (mTORC1), qui fonctionne comme un détecteur d’énergie cellulaire, et du système endocannabinoïde (ECS), qui régule la libération de neurotransmetteurs. De plus, nous avons également démontré, via l’utilisation de souris mutantes conditionnelles, l’importance de la protéine p62 ou séquestrome 1 (p62/SQSTM1), qui régule l’activité de mTORC1 et l’autophagie, dans les neurones à POMC dans la régulation de l’homéostasie énergétique. Nos données offrent un nouvel aperçu sur les mécanismes moléculaires impliqués dans la régulation de la balance énergétique. / The hypothalamus is a brain structure with a key role in the regulation of food intake. Among the different neuronal populations of which it is composed, pro-opiomelanocortin (POMC) neurons are classically known to decrease food intake and body weight through the release of neuropeptides produced by the cleavage of POMC. Our study, through the use of genetic, pharmacological, electrophysiological and molecular approaches, challenges conventional notions about POMC neuron function in energy balance by showing that there are two functionally distinct POMC neuronal sub-populations, which increase or decrease food intake depending on which neurotransmitter they release, γ-aminobutyric acid (GABA) or glutamate. A third population capable of producing both GABA and glutamate has also been identified. The regulation of POMC GABAergic and glutamatergic neurons depends on the mechanistic target of rapamycin complex 1 (mTORC1) pathway, which functions as a cellular energy sensor, and the endocannabinoid system (ECS), which regulates neurotransmitters release. In addition, we have also demonstrated through the use of a conditional knockout mice, the importance of the protein p62 or sequestrome 1 (p62/SQSTM1), which regulates mTORC1 activity and autophagy, in POMC neurons for the regulation of energy homeostasis. Our data provide new insights on the molecular mechanisms involved in the regulation of energy balance.

Apoptosis (Type I) and autophagy (Type II) represent two major forms of programmed cell death. Numerous anticancer agents employed in standard chemotherapy or novel targeted therapy induce both apoptosis and autophagy. Of note, a cytoprotective autophagic response often counteracts apoptosis triggered by such agents, potentially contributing to drug-resistance. Mechanistically, autophagy and apoptosis share molecular regulatory mechanisms primarily governed by the Bcl-2 family proteins. However, since autophagy acts as the double-edge sword in cancer, whether autophagy should be inhibited or activated in cancer treatment remains the subject of debate. Here we report a) a novel autophagy-targeted strategy that targeting the adaptor SQSTM1/p62 induces “inefficient” autophagy due to cargo-loading failure and converts cytoprotective autophagic response to apoptosis via the BH3-only protein NBK/Bik (Part 1); and b) a new mechanism for acquired drug-resistance in which the BH3-only protein Bim acts as a dual-agent regulating both autophagy and apoptosis (Part 2).

14-3-3 proteins are among a family of phospho-binding proteins that are known to regulate many essential cellular mechanisms. By binding to sites of phosphorylation, 14-3-3s are integrated into multiple signaling pathways that govern critical processes, such as apoptosis, cell cycle progression, autophagy, glucose metabolism, and cell motility. These processes are crucial for tumorigenesis and 14-3-3 proteins are known to play a central role in facilitating cancer progression. In this study, my colleagues and I discover two novel 14-3-3 interacting proteins, ATG9A and PTOV1, that are both vital to essential cellular functions and describe various mechanisms that these two proteins regulate. ATG9A is a multi-pass transmembrane lipid scramblase that is found primarily as a homotrimer in the ER or small ATG9A vesicles. It is essential in the cellular recycling process called autophagy and is believed to act at the earliest stages of autophagy by providing the seed for the growth of the double membrane vesicle called an autophagosome. Previous work in our lab demonstrated that upon hypoxic stress, AMPK, the master nutrient-sensing kinase, phosphorylates S761 on the C-terminus of ATG9A. This triggers the binding of 14-3-3ζ to contribute to ATG9A function in hypoxia induced autophagy. Despite this revelation, the exact function of ATG9A is still poorly understood, especially in unstimulated conditions where autophagy functions at a basal level and AMPK is inactive. In this study, we sought to understand ATG9A function more broadly by identifying novel interactors of ATG9A and the role ATG9A plays in basal autophagy. To do this, we employed BioID mass spectrometry and various biochemical approaches to identify LRBA as a bona fide ATG9A interactor and autophagy regulator. Furthermore, using deuterium labeling and quantitative whole proteome mass spectrometry, and various other biochemical techniques, we show that ATG9A regulates the basal degradation of p62 and is recruited to sites of basal autophagy by active poly-ubiquitination to initiate basal autophagy. PTOV1 is an oncogenic protein that is poorly understood. Our current understanding of PTOV1 is limited to a few studies, which demonstrate that PTOV1 is highly expressed in primary prostate tumor samples and is correlated with metastasis, drug resistance, and poor clinical outcomes. In this study, we identify a mechanism by which SGK2, a poorly understood kinase, phosphorylates PTOV1 at S36 to trigger 14-3-3 binding at that site to increase PTOV1 stability in the cytosol and increase c-Jun expression. Upon SGK2 inhibition, 14-3-3 releases PTOV1 and PTOV1 is shuttled into the nucleus where HUWE1, an E3 ubiquitin ligase, ubiquitinates PTOV1 and initiates PTOV1 degradation by the proteasome. This is the first detailed mechanism of regulation identified for the poorly understood oncogene, PTOV1, and sheds light on potential therapeutic targets for cancer treatments.

Malgré les avancées récentes, le cancer reste la première cause de mortalité en France avec ~ 150000 décès recensés par an (INCa 2012). Notamment, le cancer broncho-pulmonaire est l'un des plus agressifs, avec 29100 décès en 2011. La survie à cinq ans de seulement 10% des patients atteints des cancers du poumon non à petites cellules (NSCLC, 80% des cancers bronchiques), pose un problème d'ordre scientifique, médical et de santé publique. Il est communément admis qu'une sous-population de la tumeur acquiert en raison d'une instabilité génétique de nouvelles propriétés agressives nécessaires à sa dissémination, sa prolifération et une plus grande résistance aux chimiothérapies. Une meilleure compréhension de ces propriétés tumorales constitue un enjeu majeur pour prévenir à terme cette progression maligne. Nous avons axé notre recherche sur la macro-autophagie, un processus catabolique lysosomal essentiel à l'homéostasie cellulaire et sur un de ses substrats SQSTM1 (séquestosome ou p62). Au moment où j'ai initié ma thèse, une relation étroite entre l'autophagie, SQSTM1 et la progression tumorale venait d'être mise en lumière. Plusieurs membres de la machinerie autophagique sont fréquemment mutés/déletés dans les cancers. De façon paradoxale, l'autophagie peut également permettre la survie des cellules tumorales face à l'hypoxie, la carence nutritionnelle ou les chimiothérapies.

Introduction: le Virus Respiratoire Syncytial humain (RSV) induit un taux élevé de morbidité et de mortalité chez les enfants, les personnes immunodéprimées et les personnes âgées. Il existe un besoin urgent d'un nouveau traitement antiviral et d'un vaccin efficaces. Les cellules épithéliales des voies aériennes (AEC) sont la cible principale de RSV et constituent la première ligne de défense grâce à des mécanismes distincts, qui incluent une réponse antivirale autonome cellulaire. La protéine p62/SQSTM1 a de multiples fonctions cellulaires, y compris la séquestration spécifique de la cargaison ubiquitinée (c'est-à-dire, les protéines/organelles et les bactéries intracellulaires) pour leur clairance par autophagie. Des données publiées ont mis en évidence un rôle important de p62 dans la régulation de plusieurs virus (par exemple, le virus de la grippe et la dengue), favorisant ou restreignant sa réplication en fonction du virus. L'objectif de notre étude est de déterminer le rôle de p62 dans la régulation du cycle infectieux de RSV. Méthodes et résultats: L'analyse de l'expression de p62 dans les cellules A549 a montré que p62 est induit et phosphorylé au début de l'infection par RSV. Il est ensuite dégradé plus tardivement durant l’infection. La déplétion des niveaux de p62 a diminué l'accumulation intracellulaire des protéines virales, tandis que la relâche des virions infectieux a été augmentée. De plus, nous avons observé que la réplication de recRSV-GFP est diminuée dans des cellules exprimant de façon stable la protéine associée aux microtubules 1A/1B, chaîne légère 3 (LC3). LC3 recrute p62 et ses cargaisons à l'autophagosome pour qu'ils soient dégradés par autophagie. Des études sont actuellement en cours pour déterminer les mécanismes moléculaires, dépendant de p62, impliqués dans la régulation de la réplication de RSV. Conclusion: nos résultats mettent en évidence un rôle clé de p62 dans la réplication et la propagation de RSV. Ces études aideront à définir si p62 pourrait représenter une cible thérapeutique potentielle pour lutter contre l'infection à RSV. / Introduction: Human respiratory syncytial virus (RSV) causes a high rate of morbidity and mortality worldwide in children, immunocompromised and elderly people. There is an urgent need for effective antiviral treatments and vaccines for RSV. Airway epithelial cells (AECs) are the primary target of RSV and constitute the first line of defense through distinct mechanisms, including intrinsic antiviral responses. The p62/SQSTM1 protein has multiple cellular functions including cell signaling and sequestration of specific ubiquitinated cargo (i.e. proteins/organelles and intracellular bacteria) for autophagic degradation. The replication of several viruses has been shown to be sensitive to p62 levels. The goal of our study is to investigate the role of p62 in the regulation of RSV replication. Methods and Results: Analysis of p62 expression in A549 cells showed that p62 is induced and phosphorylated during early stages of RSV infection, followed by degradation at later times. P62 silencing diminished the intracellular accumulation of viral proteins, while causing increased release of infectious virions. Additionally, we observed that the stable expression of Microtubule-associated protein 1A/1B-light chain 3 (LC3), which recruits p62 and its cargos to the autophagosome for autophagy degradation, reduces recRSV-GFP replication. Studies are currently undertaken to determine the molecular mechanisms involved in p62-dependent regulation of RSV replication. Conclusion: Our results highlight a key role of p62 in the replication of RSV. These studies will help to define whether p62 might represent a potential therapeutic target to fight RSV infection.