Immune exhaustion can be a major clinical problem for patients who have cancer or chronic inflammation. Persistent antigen stimulation drives T cells to express multiple surface markers called immune checkpoints. When these markers bind to their corresponding ligands that are expressed by antigen (e.g. tumor cells), T cells become metabolically impaired and lose several important functions; some cell signaling pathways are inhibited, while other intracellular mediators are re-modulated. Eventually, both CD4+ and CD8+ T cells behave dysfunctionally in ways that may facilitate cancer progression. Immune checkpoints are a major hallmark of immune exhaustion. In addition, natural killer (NK) cells, a critical immune cell subset in the peripheral immune system, also express immune checkpoint molecules, and are responsible for detecting and destroying circulating tumor cells. Yet, little research has investigated immune checkpoints on NK cells. Here, we explored the role of Bromodomain and ExtraTerminal domain (BET) proteins (BRD2, BRD3, BRD4), which are important transcriptional co-regulators, and critical for proliferation and metastasis in many cancer types, in the regulation of immune checkpoint molecules in several immune cell subsets, including CD4+ and CD8+ T cells, and NK cells. Through binding to acetylated histone tails of nucleosomal chromatin, BET proteins assist in transcription of multiple genes. Deregulated expression of BET proteins promotes cancer development or tumor cell metastasis, and new data show the BET proteins contribute to immune exhaustion. Furthermore, Type 2 diabetes mellitus (T2DM) is another worrisome problem related to cancer. T2DM patients show increased risk of developing cancer. Patients with both T2DM and any type of cancer show higher risks for metastasis. Significantly, T2DM patients also show immune exhaustion, suggesting a hypothesis that BET proteins may couple immune system dysfunction, abnormal metabolism and cancer incidence or progression. Specifically, T2DM has been defined to be a metabolic and a chronic inflammatory disease. The 5' Adenosine Monophosphate-activated Protein Kinase (AMPK) signaling pathway is a key pivot of cell metabolism and as well a significant target of drugs that normalize blood glucose, such as metformin. Based on published data, we considered that it is important to explore the mechanism of how immune checkpoints are regulated through metabolic pathways, focusing on immune exhaustion in T2DM patients. Moreover, considering that the expression of BET proteins promotes cancer development and progression, and metastasis and immune exhaustion are characteristic of many cancers as well, we suspected a potential relationship among BET proteins, the AMPK metabolic signaling pathway and immune exhaustion is worth exploring. Here, we measure expression of the immune checkpoint molecules TIM-3, TIGIT, PD-1, and CTLA-4 on normal T cells and NK cells by flow cytometry. We demonstrate different degrees of regulation of immune checkpoints by BET proteins on stimulated T cells and NK cells. Comparing stimulated-only cells with stimulated-plus AMPK inhibitor cells, we found that inhibition of the AMPK signaling pathway causes divergent expression patterns for TIM-3 and TIGIT, PD-1 and CTLA-4. Simultaneous inhibition of both BET proteins and the AMPK signaling pathway, shows that BET proteins regulate TIM-3 and TIGIT through an AMPK-independent metabolic pathway and regulate PD-1 and CTLA-4 through an AMPK-dependent pathway. Overall, we show TIM-3 and TIGIT, PD-1 and CTLA-4 display different expression patterns under regulation of the AMPK signaling pathway, and we show that BET proteins regulate TIM-3, TIGIT, PD-1 and CTLA-4 through both AMPK-dependent and -independent pathways. These findings are important because they reveal novel mechanisms of immune checkpoint regulation, which may be valuable for targeting in cancer patients who are being treated with checkpoint inhibitors.

L'incidence des candidoses invasives a considérablement augmenté au cours des dernières décennies, parallèlement à l'augmentation du nombre de patients à risque. Ces pathologies sont associées à une morbi-mortalité élevée. Ce pronostic peut toutefois être amélioré par l'instauration d'un traitement antifongique précoce. Quatre classes de molécules antifongiques sont aujourd’hui disponibles pour la prévention et le traitement des candidoses invasives : échinocandines, antifongiques azolés, polyènes et pyrimidines. Du fait d’une efficacité importante et d’une meilleure tolérance, les échinocandines et les antifongiques azolés sont les plus largement utilisés. L'utilisation massive de ces molécules a conduit à une modification de l'épidémiologie des candidoses invasives, avec l'émergence d'espèces non-albicans intrinsèquement moins sensibles à ces antifongiques, à l'exemple de C. glabrata ou C. parapsilosis. De plus, des souches résistantes voire multi-résistantes aux échinocandines et aux antifongiques azolés sont de plus en plus fréquemment isolées, et associées à des échecs thérapeutiques. L'activité de ces antifongiques est également limitée par le phénomène de tolérance, résultant de la capacité d'adaptation des levures aux stress membranaires et pariétaux induits par ces molécules.Pour toutes ces raisons, l'identification et le développement de nouvelles stratégies antifongiques sont nécessaires. Ce travail de thèse s'inscrit dans cette optique, avec pour objectifs l'étude de la tolérance et de la résistance aux antifongiques chez Candida spp. et la caractérisation de nouvelles cibles thérapeutiques impliquées dans ces processus.La première partie de ce travail a permis de démontrer l'intérêt du séquençage nouvelle génération et d'une approche multigénique pour l'étude des mécanismes impliqués dans la résistance aux antifongiques azolés et aux échinocandines chez Candida spp. Par ce biais, il a été possible d'appréhender de nouveaux mécanismes potentiellement impliqués dans la résistance aux antifongiques, qui nécessitent toutefois d'être confirmés.Dans un deuxième temps, ce travail a permis de mettre en évidence que l'ensemble des protéines de la voie de signalisation du pH ou voie Rim chez les levures est impliqué dans la tolérance aux antifongiques chez C. albicans, l'espèce la plus fréquemment isolée en pathologie humaine. De plus, de nouveaux gènes Rim-dépendants ont été identifiés par RNA-sequencing, à l'exemple de HSP90, codant pour une protéine chaperone responsable de la régulation de multiples processus biologiques, et IPT1, responsable de la biosynthèse du principal sphingolipide membranaire. Ces deux gènes ont précédemment été impliqués dans la tolérance aux antifongiques azolés et aux échinocandines chez C. albicans et pourraient participer à la tolérance aux antifongiques médiée par la voie Rim. Ces résultats ouvrent des perspectives intéressantes : en effet, en ciblant la voie Rim, il pourrait être possible de potentialiser l'activité des molécules antifongiques actuellement commercialisées et de cibler indirectement Hsp90 tout en s'affranchissant des problèmes de toxicité car cette voie de signalisation est spécifique du règne fongique.Enfin, une dernière partie de ce travail réalisée dans le cadre du partenariat de l'ANR FungiBET a permis de montrer que la protéine BET (Bromodomain and Extra-Terminal) Bdf1 de C. glabrata, impliquée dans la régulation épigénétique de la transcription, est essentielle à la croissance in vitro. Plus précisément, l'intégrité des deux bromodomaines BD1 et BD2 de Bdf1, responsables de la liaison de cette protéine aux histones, est essentielle chez cette espèce, qui est la deuxième la plus fréquemment isolée dans les candidoses invasives en Europe et aux Etats-Unis. Ce résultat confirme les premières données obtenues chez C. albicans, et l'intérêt de l'inhibition des protéines BET fongiques comme nouvelle stratégie antifongique / The incidence of invasive candidiasis (IC) has dramatically increased over the past decades, partly due to the increasing number of at-risk patients. IC is associated with high mortality rates: however, its prognosis can be improved by early treatment. Four antifungal classes are available today for the prevention and treatment of IC: echinocandins, azoles, polyenes and pyrimidines. Due to their high efficacy and interesting safety profile, echinocandins and azoles are more commonly used. Massive use of these compounds has led to epidemiological changes in IC, with the emergence of non-albicans species which are intrinsically less susceptible to these antifungals, such as C. glabrata or C. parapsilosis. In addition, Candida spp. strains resistant, or even multiresistant, to azoles and echinocandins are increasingly isolated and associated with therapeutic failures. Antifungal activity is also limited by tolerance, a reversible phenomenon resulting from the yeast's adaptation to membrane and cell wall stresses caused by these molecules.For these reasons, identification and development of new antifungal strategies are needed. This work aimed at studying antifungal resistance and tolerance in Candida spp. and characterizing new therapeutic targets involved in these process.First, this work showed the interest of next-generation sequencing and multigene approaches to study mechanisms involved in resistance to echinocandins and azoles antifungals in Candida spp. It notably allowed to identify new mechanisms involved in antifungal resistance, which still need to be confirmed.In a second part, this work allowed to show that all the proteins of the pH-signaling pathway, known as the Rim pathway in yeasts, are involved in antifungal tolerance in C. albicans, the most frequent species responsible for IC in humans. In addition, new Rim-dependent genes were identified through RNA-sequencing, such as HSP90, coding for a major chaperone involved in the regulation of multiple cellular process, as well as IPT1, responsible for the synthesis of the main membrane sphingolipid.Both genes were previously shown to be involved in azoles and echinocandins tolerance in C. albicans, and could therefore play a role in antifungal tolerance mediated by the Rim pathway. These results offer great perspectives. Indeed, targeting the Rim pathway would allow to enhance the activity of commercially available antifungals and to indirectly target Hsp90, with no or limited toxicity as this signaling pathway is fungal-specific.Thirdly, a last part of this work performed in the context of the ANR FungiBET consortium, allowed to show that the BET protein Bdf1 in C. glabrata, which is involved in epigenetic regulation of transcription, is required for in vitro growth. More precisely, the integrity of both BD1 and BD2 Bdf1 bromodomains are essential in this species, which ranks second among causes of IC. This result confirms the first data obtained in C. albicans and the interest of inhibiting fungal BET proteins as a new antifungal strategy

<p>In eukaryotic systems, the genetic material of the cell –DNA– is packed into a protein-dense structure called chromatin. Chromatin structure is critical for preservation of the genetic material as well as coordination of vital processes such as DNA replication, transcription and DNA damage repair. The fundamental repeating unit of chromatin is nucleosome which is composed of an octamer of small alkaline proteins called histones and the DNA wrapped around this octamer. The nucleosomes are then packed into higher-order structures leading to formation of 3D chromatin architecture. The chromatin is a dynamic structure; the spacing between nucleosomes, or the folding of the larger chromatin segments is subjected to alterations during embryonic development, tissue specifications or <i>simply during any event that require gene expression changes</i>. Failure in proper regulation of chromatin structure has been associated with embryonic defects and disease such as cancer. </p> <p>This work has focused on a class of ATP-dependent chromatin remodeling complexes known as switch/sucrose-non-fermentable (SWI/SNF) or BRG-associated factors (BAF) complex. This family of complexes act on chromatin and alter its physical structure by mobilizing histones or nucleosome particles through the activity of its ATPase –BRG1 or BRM, enabling more accessible DNA for the other factors such as transcription factors to localize and recruit transcription machinery. In particular, we discovered and biochemically defined a novel version of this family of chromatin complexes that we named as GLTSCR1/1L-BAF (GBAF). GLTSCR1 and GLTSCR1L are two uncharacterized paralogous proteins that have been identified as BRG1-interacting proteins. Biochemically surveying the essence of this interaction, we realized that these proteins incorporates into a previously unknown SWI/SNF family complex that lacks well-characterized SWI/SNF subunits such as ARID1/2, BAF170, BAF47; instead, uniquely comprise GLTSCR1/1L and bromodomain-containing protein BRD9. Focusing on the GLTSCR1 subunit, we observed that its absence is well-tolerated by many different cell types except slight growth retardation by prostate cancer cells. Expanding the cohort of prostate cancer cells, we realized that not the paralogous subunits GLTSCR1 or GLTSCR1L but unique and non-redundant subunit BRD9 is the major GBAF-dependence in prostate cancer cells. We observed that especially the androgen-receptor positive cell lines have severe growth defects upon <i>BRD9 </i>knockdown or inhibition. <i>In vivo, </i>we showed that xenografts with <i>BRD9 </i>knockdown prostate cancer cells (LNCaP) have smaller tumor size. We demonstrated that BRD9 inhibition can block the expression of androgen-receptor targets. Similarly, <i>BRD9 </i>knockdown and treatment with antiandrogen drug (enzalutamide) has overlapping transcriptional effects. Mechanistically, we showed that BRD9 interacts with AR and it colocalizes with AR in subset of AR -binding sites. Surprisingly, we realized that BRD9 depletion has similar transcriptional and phenotypic effects as BET protein inhibitors. BET protein family contains 4 bromodomain containing proteins (BRD2, BRD3, BRD4, BRDT). These proteins were previously shown to be critical for AR-dependent gene expression. We detected interaction between BRD9 and BRD2/4. We demonstrated that BRD4 and BRD9 had shared binding sites on genome, a fraction of which are co-bound by AR. At particular target sites we showed that BRD9 localization is dependent on BET proteins, but not the other way around. Taking together, we provided some evidences that GBAF targeting through BRD9 can be a novel therapeutic approach for prostate cancer. Growing body of reports suggested that current therapy options targeting the androgen receptor is failing due to acquired resistance. Therefore, targeting the AR pathways via its coregulators such as BET proteins or SWI/SNF complexes can serve as potent alternative approaches. Further research is needed to elucidate the roles of GBAF and BET proteins in androgen receptor independent prostate cancer cells, which are still responsive to GBAF or BET manipulations although to a lesser extent.</p>

Cells can release lipid bilayer vesicles of endosomal and plasma membrane origin, which are known as exosomes or extracellular vesicles (EVs). EVs contain diverse shuttling lipids, RNA and transmembrane proteins, and play an important role in communicating between neighboring or distant cells. Breast cancer is the most commonly diagnosed malignancy, with over 2 million new cases in 2018, and is the leading cause of cancer mortality in women all over the world. Some observational studies have suggested that breast cancer is more likely to develop among women who have type 2 diabetes; the association is clear in postmenopausal women. Moreover, women with type 2 diabetes diagnosed before, at the same time, or after breast cancer diagnosis, have decreased overall survival compared to women without diabetes. The most recent medical studies provide more clues as to why breast cancer is more common and has poorer prognosis in type 2 diabetes patients, by pointing out the role of insulin-resistant adipocytes in the etiopathology. Here, we demonstrate how insulin-resistant adipocytes engage crosstalk with breast cancer cells through EVs in the microenvironment and drive the tumor cells to be more metastatic and aggressive. These progression mechanisms and the effects of insulin-resistant adipocytes on breast cancer cells require Bromodomain and ExtraTerminal (BET) proteins – an important epigenetic pathway. Targeting this pathway may help reduce morbidity and mortality of women with breast cancer and type 2 diabetes.

The inevitable progression of advanced prostate cancer to castration resistance, and ultimately to lethal metastatic disease, depends on primary or acquired resistance to conventional androgen-deprivation therapy (ADT) and accumulated resistance mechanisms to evade androgen receptor (AR) suppression. Whereas the canonical androgen/AR signaling axis maintains prostate cell growth, differentiation and survival, in prostate cancer cells, AR adaptations that arise in response to ADT are not singular, but diverse, and include gene amplification, mutation and even complete loss of receptor expression. Collectively, each of these AR adaptations contributes to a complex, heterogenous, ADT-resistant tumor that culminates in prostate tumor cells transitioning from epithelial to mesenchymal states (EMT) and the development of metastatic castration-resistant prostate cancer (mCRPC). Here, we examined prostate cancer cell lines that model common CRPC subtypes, each with different AR composition, and focused on novel regulators of tumor progression, the Bromodomain and ExtraTerminal (BET – BRD2, BRD3 and BRD4) family of proteins, to test the hypothesis that each BET family member regulates EMT and underlying characteristics such as cell motility and invasiveness. We systematically manipulated the BET proteins and found that BRD4 regulates cell migration and invasion across all models of CRPC, regardless of aggressiveness and AR status, whereas BRD2 and BRD3 only regulate cell migration and invasion in less aggressive models that retain AR expression or signaling. We determined that BRD4’s contribution to this process occurs through the transcriptional regulation of AHNAK, SNAI1 and SNAI2, which are EMT genes linked to promotion of metastasis in a diverse set of cancers. Furthermore, treatment of CRPC cell lines with low doses of MZ1, a small-molecule, BRD4-selective degrader, inhibits EMT and metastatic potential. Overall, these results reveal a novel, BRD4-regulated EMT gene signature that may be targetable to treat metastatic castration-resistant prostate cancer.

No description available.

Periodontal disease affects 47% of Americans over 30 and is a growing global concern. Current treatments for periodontal disease focus on the mechanical elimination of periodontal biofilms. Very few treatments are available that target the rampant, unregulated host immune response that is ultimately responsible for tissue degradation. BET proteins have been shown to play critical roles in inflammatory gene regulation and are therefore potentially ideal therapeutic targets for treating periodontal disease. RVX-208 is a selective BET-inhibitor with a high affinity for Bromodomain 2 (BD2) as compared to BD1 in BET proteins. Our previous studies have shown that RVX-208 inhibits inflammatory cytokine production and suppresses osteoclast differentiation. Cell culture assays have provided proof of concept for RVX-208 and its feasibility as a treatment for periodontal disease. As such, our long term goal is to develop RVX-208 as a front-line treatment for periodontitis. The objectives of this study were to determine the ability of RVX-208 to reduce bone loss in a ligature-induced periodontitis model, and to further investigate the mechanisms through which RVX-208 mediates its anti-inflammatory and osteoclastogenesis-suppressive effects. The specific aims of this study were: 1) To further validate the in vivo effects of RVX-208 on a ligature-induced periodontitis model in rats, and 2) To determine the molecular mechanisms of RVX-208 on preventing alveolar bone loss in periodontal disease. To investigate, a ligature-induced periodontitis model was created in rodents. Those rodents were treated with increasing dosages of RVX-208 (0-2.5 mM) by subgingival injection every other day. After 2 weeks, the maxillae were harvested and analyzed via a micro-CT protocol that had been created and validated through statistical analyses. To study the ability of RVX-208 to suppress osteoclastogenesis, RAW264.7 cells were induced into osteoclasts by RANKL and then treated with RVX-208. To ensure RVX-208 was not species specific, THP-1 cells were challenged with either E. coli-LPS or P. gingivalis bacteria and then treated with RVX-208. Linear and volumetric micro-CT analysis showed that RVX-208 could significantly ameliorate bone loss in a ligature-induced periodontitis model. RVX-208 was shown to prevent osteoclast differentiation by suppressing the expression of genes closely associated with osteoclast differentiation and maturation. RVX-208 was found to not be species specific, as it was able to mediate its effects on a human cell line, and had consistent anti-inflammatory effects regardless of whole pathogen or LPS-induced inflammatory response. Therefore, RVX-208 is a promising therapeutic for treatment of periodontal diseases.