The accurate maintenance of genomic integrity in stem cells (SCs) is essential for tissue homeostasis and its deregulation leads to developmental defects, cancer and ageing. We have shown that Brca1, key homologous recombination (HR) gene and critical regulator of the choice of the DNA double strand break (DSB) repair pathway, is specifically required for hair follicle formation and the establishment and maintenance of adult hair follicle SC pool in a conditional knock-out (CKO) mouse model. Brca1 loss leads to DNA damage-induced cell death in the hair follicle (HF), particularly in the matrix transient amplifying progenitors and moderately so in prospective quiescent adult HF SCs. This cell loss causes compensatory hyper-proliferation of the prospective HF SCs and their subsequent depletion. In striking contrast, the interfollicular epidermis (IFE) and its resident SCs remain unaffected by Brca1 deletion. I uncovered two mechanisms underlying the ability of the SCs and progenitors of the IFE to survive the deletion of Brca1. Collectively, this data reveals how distinct SCs and progenitors respond differently to Brca1 loss. Furthermore we show how the IFE can survive Brca1 loss through the use of two particular mechanisms as to sustain tissue homeostasis. The mechanisms uncovered here are likely to be relevant in other tissue-specific SCs and will have important implications in understanding cancer initiation and ageing. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Les dérivés du platine (i.e. cisplatine et oxaliplatine) représentent une des classes pharmacologiques les plus utilisées en oncologie, notamment dans les cancers colorectaux. Cependant, leur efficacité est limitée par l’émergence de résistances acquises. Nous avons alors étudié in vitro dans différentes lignées cancéreuses coliques humaines (HCT116, LoVo, SW480, HT29) les conséquences à long terme des dérivés du platine lorsqu’ils sont utilisés dans des conditions tenant compte de la sensibilité cellulaire. Ils provoquent des cassures double-brin (objectivées par l’expression de -H2AX), dont le taux dépend du système p53/p21, du complexe MRN et du niveau de stabilité microsatellitaire. Ils induisent aux plus fortes concentrations ( IC50), une cytostase qui s’accompagne de la formation de cellules géantes macrocytaires et endopolyploïdes, dont certaines acquièrent un phénotype sénescent. Dans le même temps, l’activation des mécanismes de réparation des CDB varie en fonction du dérivé du Pt et de la lignée considérée. A plus long terme, des cellules « résistantes » se développent : elles ont une ploïdie normale, et se caractérisent par une plus grande résistance aux dérivés du platine et la présence de novo d’anomalies chromosomiques récurrentes leur conférant un avantage sélectif potentiel en terme de prolifération. Ces mécanismes pourraient contribuer à expliquer en clinique la survenue d’une résistance à une chimiothérapie pourtant initialement efficace. Parallèlement, nous avons évalué in vitro et in vivo de nouveaux complexes de platine obtenus par pharmacomodulation, et associant un noyau intercalant dérivé de la phénanthroline ou de l’acridine. Les résultats in vitro montrent globalement une amélioration significative de la cytotoxicité. Toutefois, un des composés les plus cytotoxiques in vitro, le [(5,6-diméthyl-1,10-phénanthroline) (S,S-diaminocyclohexane)platine(II)], n’exerce pas d’effet antitumoral dans un modèle syngénique de cancer colique chez le rat BD-IX, mais montre une néphrotoxicité marquée. Ces données soulignent l’insuffisance du criblage in vitro et la discordance in vitro/in vivo. / Platinum compounds (i.e. cisplatin and oxaliplatin) represent a class of DNA-damaging agents widely used in clinic especially in the treatment of colorectal cancer. However, their effectiveness is restricted because of emergence of acquired resistance. Therefore, long-term effects of platinum compounds, used at conditions reflecting the in vitro cellular sensibility, were assessed in vitro in several human colon cancer cell lines (HCT116, LoVo, SW480, HT29). Their cytotoxicity is related to double-strand break formation (objectived by -H2AX expression), which depends on p53/p21 status, MRN complex and microsatellite stability of the cell line. Furthermore, at the highest concentrations ( IC50), cells stopped their proliferation and exhibited phenotypic alterations resulting from progressive polyploidy and/or senescence. In the same time, DNA repair systems are activated differently according to the platinum derivate and the cell line. At later stages, cells that are more resistant to platinum compounds than their parental counterpart emerged. They have recovered their basal level of ploidy and acquired de novo recurrent chromosomal aberrations. Such mechanisms could contribute to the recurrence of clinical malignancies, even after an effective initial response to chemotherapy. On the other hand, pharmacological evaluation of new platinum compounds with phenanthroline or acridine intercalating ligand was performed in vitro and in vivo. Globally, many compounds exhibited a higher cytotoxic effect than cisplatin or oxaliplatin in all cell lines studied. Unfortunately, in vivo investigations of one of the most cytotoxic compounds ([(5,6-dimethyl-1,10-phenanthroline) (S,S-diaminocyclohexane)platinum(II)]) did not exhibit antitumor effect in BD-IX rats bearing peritoneal carcinomatosis, whatever the route of administration used (systemic or local), but it displayed nephrotoxicity. These results query the in vitro/in vivo correlation and reconsider the place of the in vivo screening.

Le système ubiquitine-protéasome représente une plateforme de signalisation cellulaire chez les eucaryotes et joue un rôle majeur dans la coordination des processus cellulaires. Des progrès récents suggèrent que l’ubiquitination joue un rôle important dans les phénomènes de séparation de phase liquide-liquide (LLPS), un processus permettant la localisation d’une quantité accrue de protéines dans un compartiment subcellulaire, afin de réaliser une fonction biologique. En effet, il a été démontré que l’ubiquitination joue un rôle central dans les mécanismes qui gouvernent la LLPS durant la formation des granules de stress dans le cytoplasme ou les foci de réparation de l’ADN dans le noyau. D’autre part, chez la levure, des travaux ont montré que le protéasome est capable de s’assembler sous forme de granules dans le cytoplasme suite à un stress métabolique. Toutefois, les mécanismes par lesquels le système ubiquitine-protéasome ainsi que ses régulateurs contrôlent les processus de LLPS restent à déterminer. Dans la première étude de cette thèse, nous avons investigué le mécanisme d’action de la déubiquitinase USP16, qui a été suggérée comme un régulateur négatif de la LLPS, empêchant la formation des foci de réparations de dommages à l’ADN. Cependant, nos résultats démontrent que USP16 est majoritairement cytoplasmique et que seulement une entrée forcée de USP16 dans le noyau empêche la formation des foci de réparation des cassures double brin induites par des radiations ionisagntes et ce en favorisant la déubiquitination de l’histone H2A. De plus, aucune translocation nucléaire de USP16 n’a été observée durant le cycle cellulaire ou suite à des dommages à l’ADN. Nos travaux montrent que USP16 est activement exclue du noyau via son signal d’export nucléaire et régulerait indirectement la LLPS menant à la formation des foci de réparation de l’ADN. Dans la deuxième étude, nous décrivons le comportement dynamique des protéines du protéasome lors d’une LLPS induite par un stress métabolique. Nos résultats indiquent que le protéasome forme des foci distincts dans le noyau des cellules humaines en réponse à une privation de nutriments. Nous avons constaté que ces foci sont enrichis en ubiquitine conjuguée et nous avons démontré que le récepteur d’ubiquitine Rad23B ainsi que l’absence des acides aminés non essentiels sont des éléments clés nécessaires à l’assemblage de ces foci du iv protéasome. De plus, des expériences de survie cellulaire montrent que la présence de ces foci est associée à la mort des cellules par apoptose. En conclusion, nos travaux mettent en lumière l’importance du système ubiquitine-protéasome dans la formation et la régulation des foci cellulaires suite à une LLPS. De même, cette étude aidera également à approfondir notre compréhension sur les mécanismes qui gouvernent l’homéostasie des protéines, la survie cellulaire et le développement du cancer. / The ubiquitin-proteasome system represents a major cell-signaling platform in eukaryotes and plays a pivotal role in the coordination of cellular processes. Recent studies provided evidence that ubiquitination plays a role in liquid-liquid phase separation (LLPS), a process that results in the localization of highly increased levels of a protein in a defined subcellular compartment, in order to achieve a biological function. Indeed, ubiquitination has been shown to play a central role in the mechanisms that govern LLPS and subsequent formation of stress granules in the cytoplasm or the DNA repair foci in the nucleus. On the other hand, several studies have shown that the proteasome itself is able to form granules in the cytoplasm following metabolic stress in yeasts. However, the mechanisms by which the ubiquitin-proteasome system and its regulators control LLPS processes remain to be determined. In the first study of this thesis, we investigated the mechanism of action of USP16 deubiquitinase, which has been suggested as a negative regulator of LLPS preventing the formation of DNA damage repair foci. However, our results demonstrate that USP16 is predominantly cytoplasmic and that only enforced nuclear entry of USP16 prevents the formation of repair foci after double strand breaks induced by ionizing radiation, and this by promoting the deubiquitination of histone H2A. In addition, no nuclear translocation of USP16 was observed during cell cycle or following DNA damage. Our study shows that USP16 is actively excluded from the nucleus via its nuclear export signal and would indirectly regulate LLPS that lead to DNA repair foci. In the second study, we describe the dynamic behavior of proteasome proteins during metabolic stress, a process that involves LLPS. Our results indicate that the proteasome forms distinct foci in the nucleus of human cells in response to nutrients deprivation. We found that these foci are enriched with conjugated ubiquitin and demonstrated that the ubiquitin receptor Rad23B as well as the absence of nonessential amino acids are the key elements necessary for the assembly of these proteasome foci. In addition, cell survival experiments show that the presence of these foci is associated with cell death by apoptosis. In conclusion, our work has shed new light on the importance of the ubiquitin-proteasome system in the formation and regulation of cell foci following LLPS. Likewise, this vi study will also help deepen our understanding of the mechanisms leading to protein homeostasis, cell survival and cancer development.

Abstract Hereditary mutations in DNA damage response (DDR) genes often lead to genomic instability and ultimately tumor development. However, the molecular mechanism of how these DDR deficiencies promote genomic instability and malignancy is not well understood. Thus, the specific aim of this thesis is to identify the functional and molecular framework behind the elevated breast cancer risk observed in heterozygous PALB2 and ABRAXAS mutation carriers. The heterozygous germline alteration in PALB2 (c.1592delT) causes a haploinsufficiency phenotype in the mutation carrier cells. Due to PALB2 haploinsufficiency, elevated Cdk activity and consequently aberrant DNA replication/damage response was observed in the PALB2 mutation carrier cells. Excessive origin firing that is indicative of replication stress was also seen in the PALB2 mutation carrier cells. In addition to replication stress, PALB2 mutation carrier cells also experience G2/M checkpoint maintenance defects. The increased malignancy risk in females associated with heterozygosity for the Finnish PALB2 founder mutation is likely to be due to aberrant DNA replication, elevated genomic instability and multiple different cell cycle checkpoint defects. The heterozygous germline alteration in ABRAXAS (c.1082G>A) causes a dominant-negative phenotype in the mutation carrier cells. Decreased BRCA1 protein levels as well as reduced nuclear localization and foci formation of BRCA1 and CtIP was observed in the ABRAXAS mutation carrier cells. This causes disturbances in basal BRCA1-A complex localization, which is reflected by a restraint in error-prone DNA double-strand break (DSB) repair pathway usage, attenuated DNA damage response, deregulated G2/M checkpoint control and apoptosis. Most importantly, mutation carrier cells display a change in their transcriptional profile, which we attribute to the reduced nuclear levels of BRCA1. Thus, the Finnish ABRAXAS founder mutation acts in a dominant-negative manner on BRCA1 to promote genome destabilization in the heterozygous carrier cells. / Tiivistelmä Perinnölliset muutokset DNA-vauriovasteen geeneissä johtavat usein genomin epävakauteen ja lopulta syövän kehittymiseen. Molekyylitason mekanismeja, joilla vauriovasteen vajaatoiminta ajaa genomin epävakautta ja syöpää, ei kuitenkaan ymmärretä kunnolla. Tämän väitöskirjan tavoitteena on tunnistaa solutoiminnan ja molekyylitason vaikuttajat heterotsygoottisten PALB2- ja ABRAXAS-geenimuutosten kantajien kohonneen rintasyöpäriskin taustalla. Heterotsygoottinen ituradan suomalainen perustajamuutos PALB2-geenissä (c.1592delT) aiheuttaa haploinsuffisienssin kantajahenkilöiden soluissa. PALB2:n haploinsuffisienssin seurauksena kantajasoluissa havaittiin kohonnutta Cdk-proteiinin aktiivisuutta ja siitä johtuvaa kiihtynyttä DNA:n kahdentumista. PALB2-mutaatiota kantavissa soluissa nähtiin myös liiallista replikaation aloituskohtien käyttöä, mikä viittaa replikaatiostressiin. Replikaatiostressin lisäksi PALB2-mutaation kantajasoluilla havaittiin vaikeuksia ylläpitää solusyklin G2/M-tarkastuspisteen toimintaa. Näiden solutoiminnan poikkeavuuksien takia heterotsygoottisen PALB2 c.1592delT -mutaation kantajilla todettiin genomin epävakautta ja kohonnut syöpäriski. Heterotsygoottinen ituradan mutaatio ABRAXAS-geenissä (c.1082G>A) aiheuttaa dominantti-negatiivisen fenotyypin mutaation kantajasoluissa. ABRAXAS-mutaatiota kantavissa soluissa havaittiin BRCA1-proteiinitasojen laskua sekä BRCA1- ja CtIP-proteiinien vähentynyttä lokalisaatiota tumaan ja DNA-vauriopaikoille. Tämä aiheuttaa häiriöitä BRCA1-A-kompleksin paikallistumisessa, mikä johtaa häiriöihin virhealttiiden DNA-kaksoisjuoste¬katkoksien korjausmekanismien käytössä, DNA-vauriovasteessa, G2/M-tarkastus-pisteen säätelyssä ja ohjelmoidussa solukuolemassa. Tärkeimpänä löydöksenä havaittiin mutaation kantajasoluissa muuttunut transkriptioprofiili, joka johtunee BRCA1-proteiinitasojen laskusta tumassa. Näin ollen suomalainen ABRAXAS-perustajamutaatio toimii dominantti-negatiivisena BRCA1:n suhteen, aiheuttaen genomin epävakautta heterotsygoottisissa kantajasoluissa.

Prostate cancer is one of the most frequently diagnosed cancers and failure to manage localized disease contributes to the majority of deaths. Radiation therapy (RT) is a common treatment for localized prostate cancer and uses ionizing radiation (IR) to damage DNA. Although RT is potentially curative, tumors often recur and progress to terminal disease. The cellular response to RT is multidimensional. For example, cells respond to a single dose of IR by activating the DNA damage response (DDR) to repair the DNA. Targeting proteins involved in the DDR is an effective clinical strategy to sensitize cancer cells to RT. However, multiple radiation treatments, as in fractionated ionizing radiation (FIR), can promote neuroendocrine differentiation (NED). FIR-induced NED is an emerging resistance mechanism to RT and tumors that undergo NED are highly aggressive and remain incurable.<br><br> Currently, the only clinical approach that improves RT for prostate cancer treatment is androgen deprivation therapy (ADT). ADT blocks androgen receptor (AR) signaling which inhibits the repair of DNA damage. In 2017, my lab reported that targeting Protein arginine methyltransferase 5 (PRMT5) blocks AR protein expression. Therefore, targeting PRMT5 may also sensitize prostate cancer cells to RT via a novel mechanism of action.<br><br> This dissertation focuses on the role of PRMT5 in the cellular response to IR and the goal of my work is to validate PRMT5 as a therapeutic target to enhance RT for prostate cancer treatment. I demonstrate that PRMT5 has several roles in the cellular response to IR. Upon a single dose of IR, PRMT5 cooperates with pICln to function as a master epigenetic activator of DDR genes and efficiently repair IR-induced DNA damage. There is an assumption in the field that the methyltransferase activity and epigenetic function of PRMT5 is dependent on the cofactor MEP50. I demonstrate that PRMT5 can function independently of MEP50 and identify pICln as a novel epigenetic cofactor of PRMT5. During FIR, PRMT5, along with both cofactors MEP50 and pICln, are essential for initiation of NED, maintenance of NED, and cell survival. Targeting PRMT5 also sensitizes prostate cancer xenograft tumors in mice to RT, significantly reduces and delays tumor recurrence, and prolongs overall survival. Incredibly, while 100% of control mice died due to tumor burden, targeting PRMT5 effectively cured ~85% of mice from their xenograft tumor. Overall, this work provides strong evidence for PRMT5 as a therapeutic target and suggests that targeting PRMT5 during RT should be assessed clinically.<br>

Indiana University-Purdue University Indianapolis (IUPUI) / Break-induced replication (BIR) is a mechanism to repair double-strand breaks (DSBs) that possess only a single end that can find homology in the genome. This situation can result from the collapse of replication forks or telomere erosion. BIR frequently produces various genetic instabilities including mutations, loss of heterozygosity, deletions, duplications, and template switching that can result in copy-number variations (CNVs). An important type of genomic rearrangement specifically linked to BIR is half crossovers (HCs), which result from fusions between parts of recombining chromosomes. Because HC formation produces a fused molecule as well as a broken chromosome fragment, these events could be highly destabilizing. Here I demonstrate that HC formation results from the interruption of BIR caused by a defective replisome or premature onset of mitosis. Additionally, I document the existence of half crossover instability cascades (HCC) that resemble cycles of non-reciprocal translocations (NRTs) previously described in human tumors. I postulate that HCs represent a potent source of genetic destabilization with significant consequences that mimic those observed in human diseases, including cancer.