Dissertations / Theses on the topic 'EZH2i'

Il mesotelioma pleurico maligno (MPM) è un tumore molto aggressivo e rapidamente progressivo che si sviluppa a livello del mesotelio che compone la pleura; questa neoplasia può assumere diversi sottotipi istologici (epitelioide, bifasico e sarcomatoide), i quali sono strettamente correlati alla prognosi. Le modificazioni epigenetiche che avvengono nelle fasi di iniziazione e progressione del MPM possono svolgere un ruolo fondamentale nel regolare negativamente il crosstalk tra tumore e sistema immunitario, contribuendo a mantenere un microambiente tumorale immunosoppressivo. Conoscere più dettagliatamente il panorama epigenetico del MPM può contribuire a definire il razionale per nuove terapie antitumorali e porre le basi per studi di combinazione che prevedano l’utilizzo di farmaci epigenetici con farmaci immunoterapeutici. Con il presente studio abbiamo voluto valutare, in un primo momento, le modificazioni nel profilo di espressione genica di 10 linee di MPM, di diverso istotipo, trattate con la guadecitabina, un agente demetilante il DNA di seconda generazione, tramite la piattaforma nCounter di Nanostring. I risultati ottenuti tramite Ingenuity Pathway Analysis (IPA) hanno mostrato che la guadecitabina era in grado di indurre l’attivazione dei geni coinvolti nel crosstalk tra cellule dendritiche e natural killer nel 50% delle linee cellulari di MPM indagate, accompagnata dall’attivazione di altre componenti coinvolte nella risposta immunitaria a infezioni e infiammazioni. I fattori trascrizionali “upstream” più frequentemente attivati appartenevano al pathway di segnalazione dell’interferon (IFN)-γ. Inoltre, è stata riscontrata l’up-regolazione (fold change medio (mFC) ≥ 1.5) di molecole immuno-relate, come NY-ESO-1 (mFC=13.16), MAGE-B2 (mFC=13.09), CD70 (mFC=5.27) e CTLA-4 (mFC=4.81). Abbiamo inoltre effettuato analisi istotipo-specifiche per esplorare le modificazioni molecolari indotte dalla guadecitabina nei 3 sottotipi di MPM. La guadecitabina ha indotto l’up-regolazione dell’espressione di marcatori del fenotipo epiteliale (es. CDH1, EPCAM e PECAM1), osservata ad alti livelli nelle linee cellulari sarcomatoidi; ciò è stato associato alla down-regolazione di molecole di origine mesenchimale (es. CDH2 e NCAM) e induttori della cascata metastatica (es. CDH11). Successivamente abbiamo comparato gli effetti immunomodulatori della guadecitabina con quelli di altri farmaci epigenetici (gli inibitori delle iston acetiltransferasi (HDAC) VPA e SAHA o l’inibitore di EZH2 EPZ-6438) da soli o in combinazione con la guadecitabina in 5 linee cellulari di MPM (2 sarcomatoidi, 1 bifasica e 2 epitelioidi). Analisi citofluorimetriche e molecolari hanno rivelato che la guadecitabina up-regolava l’espressione delle molecole immuno-relate, quali HLA di classe I (mFC=1.59), ICAM-1 (mFC=3.27), PD-L1 (mFC=2.13), e NKG2DL (MICA mFC=1.88, MICB mFC=2.42, ULBP2 mFC=3.16), inducendo/up-regolando l’espressione dei Cancer Testit Antigens (CTA) NY-ESO-1, MAGE-A1 e MAGE-A3; il VPA up-regolava l’espressione degli antigeni di HLA di classe I (mFC=1.50), PD-L1 (mFC=2.76), NKG2DL (MICA mFC=1.69, MICB mFC=2.67, ULBP2 mFC=3.26) e quella dei CTA MAGE-A1 e MAGE-A3, rispettivamente in 2/5 e 3/5 linee cellulari di MPM; il SAHA up-regolava l’espressione di MICA (mFC=1.57), MICB (mFC=4.05) e MAGE-A1 e MAGE-A3, rispettivamente in 2/5 e 4/5 linee cellulari; per contro, l’EPZ-6438 ha mostrato minime capacità immunomodulanti, inducendo solamente NY-ESO-1 e up-regolando l’espressione di PD-L1, MICB e ULBP2 in 1 linea cellulare ciascuno. Contrariamente ai risultati eterogenei ottenuti dai singoli farmaci, l’associazione di VPA, SAHA o EPZ-6438 alla guadecitabine ha rafforzato le capacità immunomodulanti di quest’ultima, influenzando l’espressione di tutte le molecole indagate. Specificatamente, le combinazioni di guadecitabine con VPA, SAHA o EPZ-6438 up-regolavano l’espressione degli antigeni HLA di classe I (mFC=2.21, 2.03, o 2.29 rispettivamente), di ICAM-1 (mFC=4.09, 4.63, o 5.33), di PD-L1 (mFC=6.95, 2.42, o 2.50), di MIC-A (mFC=3.48, 2.00, o 2.23), di MIC-B (mFC=6.80, 2.48, o 2.81) e di ULBP2 (mFC=13.45, 3.40, o 4.11). Infine, livelli di up- regolazione/induzione maggiori sono stati osservati per i CTA a seguito di tutti e 3 i trattamenti combinati rispetto alla guadecitabina in singolo. La modulazione delle caderine è stata influenzata dal sottotipo istologico di MPM: l’espressione di CDH1 è stata indotta dalla guadecitabina in singolo e dalla sua combinazione con VPA, SAHA e EPZ-6438 nelle 2 linee cellulari sarcomatoidi, costitutivamente negative per l’espressione del gene; l’espressione di CDH2 è stata up-regolata dal VPA e dal SAHA singoli in 1/5 linee cellulari e dalle combinazioni di guadecitabina con VPA o SAHA, rispettivamente in 3/5 o 1/5 linee cellulari di MPM; ciononostante, non è stata osservata alcuna up-regolazione del gene nelle 2 linee cellulari epiteliodi, costitutivamente negative per l’espressione di CDH2. In conclusione, dalle analisi approfondite del pannello di espressione genica abbiamo confermato che la guadecitabina è in grado di up-regolare/indurre l’espressione di molecole immunitarie e immuno- relate cruciali per il crosstalk tra il tumore e il sistema immunitario; inoltre, abbiamo dimostrato che essa induce l’attivazione di geni correlati all’IFN, soprattutto nel fenotipo sarcomatoide, supportando l’ipotesi che i demetilanti possano aumentare la risposta immunitaria contro il MPM, potenzialmente anche del tipo istologico più aggressivo; la modulazione delle molecole di adesione tendente verso il fenotipo epitelioide suggerisce la possibilità di revertire la transizione epitelio-mesenchima, cruciale nel processo di metastatizzazione. Infine, combinando la guadecitabina con farmaci inibitori delle HDAC/EZH2 ha rafforzato la sua attività immunomodulante, fornendo il razionale per studi di associazione di farmaci epigenetici e agenti immunoterapici in modo da aumentare l’efficacia di questi ultimi nel trattamento del mesotelioma.
Malignant pleural mesothelioma (MPM) is a highly aggressive and rapidly progressive tumor that affect the mesothelium componing the pleura; it can acquire different histological subtypes (mainly epithelioid, biphasic, and sarcomatoid MPM), which are of prognostic significance. Epigenetic modifications occurring during MPM initiation and progression may play a relevant role in negatively regulating the crosstalk between the tumor and the immune system, as well as contributing to the highly immunosuppressive microenvironment. A better understanding of MPM epigenetics will contribute to refine antitumor strategies, laying the ground for epigenetic-based immunotherapy. The present study evaluated, in the first instance, changes in the gene expression fingerprint of 10 MPM cell lines of different phenotype treated with the second-generation DNA hypomethylating agent (DHA) guadecitabine, through the Nanostring Oncology panel with nCounter readout. Ingenuity pathway analysis results revealed that guadecitabine induced the activation of natural killer and dendritic cells signaling pathways in 50% of MPM cell lines, followed by the activation of other components involved in the immune system response to infections and inflammation. Besides, the most frequently activated upstream regulators belonging to the interferon (IFN)-γ signaling pathway. Also, the up- regulation (mean fold change (mFC) ≥ 1.5) of key immune-related molecules, such as the NY-ESO-1 (mFC=13.16), MAGE-B2 (mFC=13.09), CD70 (mFC=5.27), and CTLA-4 (mFC=4.81) was reported. We also performed histological type-specific investigations to explore molecular changes induced by guadecitabine among the 3 histotypes. Guadecitabine induced the up-regulation of the expression of epithelial markers (e.g., CDH1, EPCAM, PECAM1), observed at higher levels in sarcomatoid cell lines; this was accompanied by the down-regulation of mesenchymal origin molecules (e.g., CDH2, NCAM), and inductor of metastatic signals (e.g., CDH11). Secondly, the immunomodulatory effects of guadecitabine were compared to those of different epigenetic drugs (the histone deacetylase (HDAC) inhibitors VPA and SAHA, or the EZH2 EPZ- 6438), alone or in combination with guadecitabine, in 5 MPM cell lines (two sarcomatoid, one biphasic, and two epithelioid). We performed cytofluorimetric and molecular qRT-PCR analyses and, in this regard, results showed that guadecitabine up-regulated the expression of immune-related molecules, such as HLA class I antigens (mFC=1.59), ICAM-1 (mFC=3.27), PD-L1 (mFC=2.13), and NKG2DLs (MIC-A mFC=1.88, MIC-B mFC=2.42, and ULBP2 mFC=3.16), and up-regulated/induced Cancer Testis Antigens (CTA: NY-ESO-1, MAGE-A1, and MAGE-A3) expression; VPA up-regulated the expression of HLA class I antigens (mFC=1.50), PD-L1 (mFC=2.76), NKG2DLs (MIC-A mFC=1.69, MIC-B mFC=2.67, and ULBP2 mFC=3.26), and the expression of CTA MAGE-A1 and MAGE-A3 in 2/5 and 3/5 MPM cell lines, respectively; SAHA up- regulated the expression of MICA (mFC=1.57), MICB (mFC=4.05), MAGE-A1 and MAGE-A3 in 2/5and 4/5 MPM cell lines, respectively; conversely, EPZ-6438 induced minimal immunomodulatory effects, inducing only NY-ESO-1 and up-regulating PD-L1, MIC-B, and ULBP2 expression in 1 MPM cell line each. Despite the heterogeneous activities of single epigenetic drugs, the addition of both VPA, SAHA, and EPZ-6438 to guadecitabine strengthened the immunomodulatory effects of the latter, by affecting the expression of all investigated molecules. Specifically, guadecitabine plus VPA, SAHA, or EPZ-6438 upregulated the expression of HLA class I antigens mFC=2.21, 2.03, or 2.29; ICAM-1 mFC=4.09, 4.63, or 5.33; PD-L1 mFC=6.95, 2.42, or 2.50; MIC-A mFC=3.48, 2.00, or 2.23; MIC-B mFC=6.80, 2.48, or 2.81; ULBP2 mFC=13.45, 3.40, or 4.11, respectively. Lastly, higher levels of upregulated/induced CTA expression were observed after all 3 combination treatments versus guadecitabine alone. Cadherins modulation was MPM histotype-related: CDH1 expression was induced in the 2 constitutive-negative sarcomatoid MPM cell lines by guadecitabine alone or combined with VPA, SAHA, or EPZ-6438; CDH2 expression was upregulated by VPA or SAHA in 1/5 cell lines, and by guadecitabine plus VPA or SAHA in 3/5 or in 1/5 MPM cell lines, respectively; however, no induction of CDH2 have been reported in the constitutive negative epithelioid cell lines. Overall, from comprehensive gene expression panel analyses, we confirmed that guadecitabine induced/up-regulated the expression of immune and immune-related molecules, pivotal in the tumor- immune system crosstalk; also, we highlighted that guadecitabine-induced activation of IFN-related genes, especially in the sarcomatoid phenotype, supporting the hypothesis that DHA could increase the immune response against MPM, potentially also with sarcomatoid features; moreover, the modulation of adhesion molecules towards the epithelial type suggests the possibility to revert the epithelial-to- mesenchymal transition (EMT) event, crucial in the invasion-metastasis cascade. Also, combining guadecitabine with HDACi/EZH2i strengthened its immunomodulatory capabilities, laying the rationale for epigenetic drugs-based immunotherapies, to enhance efficacy of these strategy in the MPM clinic.

Occurrence of mutations in the Polycomb (PcG) gene; EZH2 (Enhancer of Zeste Homologue 2) represent a new class of molecular lesions associated with instability in the epigenome of patients with Myelodysplastic Syndrome (MDS). Detection of microdeletion at 7q36.1 or 7q Copy Neutral Loss of Heterozygosity [CN (LOH)] led to the identification of EZH2 mutations. EZH2 is the catalytic component of Polycomb Repressive Complex 2 (PRC2) that trimethylates lysine 27 of histone 3 (H3K27) resulting in gene silencing and recruitment of the sister complex i.e. Polycomb Repressive Complex 1 (PRC1) to target genes. Discovery of EZH2 mutations have shed light on the involvement of other PcG and PcG interacting proteins i.e. Jumonji (jmj) family of demethylases and DNA methyltrasferase 3A (DNMT3A) in MDS. Investigation of Single Nucleotide Polymorphism (SNP) array abnormalities and mutational analysis of these genes have not been ascertained and therefore I examined cytogenetic aberrations affecting twelve Polycomb (PRC1) and seventeen Jumonji genes using high density SNP 6 arrays. SNP6 data analyzed in this study was generated by our group for previous research projects. I visualised this data using CHromosome Analysis Suite (Chas) from Affymetrix and identified five PRC1 genes (BMI1, PHC1, PHC2, RING1A and RING1B) in 17/91 (19 %) patients with either Copy Number Variations (CNVs) like deletions or amplifications or CN (LOH). Interestingly, the frequency of SNP6 aberrations was high (two times) in Jumonji genes as compared to PRC1. 29/91 patients (31 %) showed either CNVs or CN (LOH) in fifteen (JMJD3, JMJD4, JMJD1B, JMJD2A, JARID2, JMJD1C, JARID1B, JMJD2C, UTX, JARID1C, JARID1A, JMJD2D, JHJD1A, JARID1D and JHJD1B) Jumonji genes. Mutational analysis of patients with SNP6 aberrations was carried out using Sanger or 454 sequencing but no mutations were detected in either the PRC1 or Jumonji genes. To elucidate changes in gene expression as a result of amplification or deletion of genomic material, qPCR was performed on 22/29 patients for thirteen Jumonji genes. Gene expression of JARID1A, JARID1C and UTX were modulated concomitant to the CNVs. Deletion of JARID1A locus was associated with reduced gene expression (p value < 0.0001) in two patients while trisomy of JARID1C (n=1) and UTX (n=2) were associated with increased expression (p value < 0.0001) of both the genes. Mutational analysis of PRC2 core components (SUZ12, EED, EZH1) and DNMT3A was carried out in a cohort of 61 MDS patients previously sequenced by our group for EZH2 mutations to examine their mutational overlap. 10/61 patients had heterozygous DNMT3A mutations (clone size 20-44 %) with two patients showing mutations at the R882 site. Interestingly, these mutations were seen predominantly (n= 6) in patients with monosomy 7/del 7q however only one patient had both DNMT3A (R882H) and EZH2 (V626M) mutations suggesting that there is no specific association between mutations of the two genes. In contrast, PRC2 genes were not mutated in this cohort emphasizing the importance of EZH2 mutations alone in MDS pathogenesis. Therefore I examined the functional consequences of the commonly occurring EZH2 (R690C/R690H) and DNMT3A (R882H) mutations in myeloid cell lines. To achieve this, numerous attempts were made to clone DNMT3A R882H mutation into p3XFLAG-myc-CMV-26 to allow transfection and in vitro assessment of the mutant in myeloid cells but all attempts to ligate the plasmid failed and therefore work on DNMT3A was discontinued. EZH2 (R690C/R69H) and Flag tagged wild type EZH2 were constructed in p3XFLAGmyc- CMV-26 vector using a PCR based cloning strategy and transfected into K562 cells. Western blot analysis at 72 hr post transfection, showed elevated levels of both R690C/R690H mutants and Flag tagged wild type EZH2 but no alterations in its target H3K27me3 levels. Affymetrix Human Transcriptome 2.0 gene expression profiling was used to identify modulation of gene signature as result of elevated EZH2 levels and MLLT10 gene was found to be up regulated in cells transfected with Flag-tagged wild type EZH2 (2.3 fold) as well as R690C/ R690H (3.6 – 4.6 fold) mutants. In contrast, PML (promyelocytic leukaemia) (2.16 fold) and FANCL (Fanconi Anaemia, Complementation Group L) (2.18 fold) genes were up regulated exclusively in cells over expressing the Flag tagged wild type EZH2. To compare this gene signature to gene expression changes as a result of EZH2 knock out (KO), shRNA mediated inhibition of EZH2 was carried out in myeloid cells and 95 % KO of both EZH2 and H3K27me3 levels were observed at Day 7 post transduction. Microarray gene expression profiling identified BCL2 (-2.14 fold), FLT1 (-4.03 fold), HOXA10 (-2.2 fold), CD44 (-8.2 fold), CD83 (-2.1 fold), TLSP (-3.24 fold), IFI16 (-3.11 fold) and PAG1 (-3.37 fold) inhibition in cells transduced with shRNA against EZH2 compared to the scrambled and wild type K562 cells. There were no overlapping genes in K562 cells with EZH2 KO and EZH2 mutants R690C/R690H. The differences in expression profiling could be due to the difference in H3K27me3 levels modulated by EZH2. Comparison of gene signature obtained by EZH2 KO on patient samples carrying the R690H mutation, showed contrasting results i.e. up regulation of HOXA10, FLT1, PAG1B, EZH1 and TLSP compared to patients with wild type EZH2 suggesting that EZH2 R690C/R690H mutants do not mimic the transcriptional changes seen in EZH2 KO. This strongly suggests the presence of other mechanisms to compensate for the loss of EZH2 in myeloid cells. However the results obtained here should be examined in additional other myeloid cell lines to validate the findings obtained in K562 cells.

Extensive efforts have shed light on the identity and biology of cancer stem cells, required and sufficient for the propagation of hematological malignancies and solid tumours. Much less is understood about the closely related issue as to the identity and properties of the normal stem and progenitor cells targeted by oncogenic lesions, and how the nature of the targeted cell might impact on the biology and clinical picture of the resulting cancer. To address this, we developed a mouse model allowing targeted inactivation of Ezh2 and Runx1 to different haematopoietic compartments. Inactivating mutations of EZH2 and RUNX1 frequently co-occur in haematological malignancies with markedly different phenotypes including myelodysplastic syndrome (MDS) and early thymic progenitor (ETP) leukaemia. Inactivation of Ezh2 and Runx1 in adult haematopoietic stem cells (HSCs) resulted in perturbed haematopoiesis leading to development of an MDS-like disease. Unexpectedly, this MDS phenotype could be fully reproduced when Ezh2 and Runx1 inactivation was targeted to multipotent progenitors (MPPs) using Flt3-Cre. Furthermore, the disease was transplantable by MPPs, but not more committed progenitor populations, demonstrating that MDS tumour propagating potential is not exclusive to intrinsically self-renewing HSCs. Targeting Ezh2 and Runx1 inactivation to early lympho-myeloid progenitors did not result in an MDS phenotype. These mice showed a marked expansion of ETPs within the thymus, combined with a block in thymocyte differentiation. These expanded ETPs displayed transcriptional features characteristic of ETP leukaemia, a treatment-resistant acute leukaemia subtype hypothesised to originate from ETPs. Combination of inactivation of Ezh2 and Runx1 in ETPs with the constitutively activating Flt3-ITD signalling mutation resulted in an aggressive lympho-myeloid acute leukaemia, which could be propagated by the expanded ETP population. These findings demonstrate the potential of lympho-myeloid progenitors such as ETPs to become leukaemia stem cells which propagate a disease retaining lympho-myeloid features. We used this novel ETP leukaemia model to explore therapeutic targeting of Ezh2-inactivated ETP leukaemias using inhibitors of the bromodomain and extra terminal (BET) proteins. Aberrant transcription resulting from epigenetic changes induced by Ezh2 loss could be reversed by BET inhibitors, and these compounds showed therapeutic efficacy against both mouse and human ETP leukaemias in vitro and in vivo.

Les cortico-surrénalomes (CCS) sont considérés comme des tumeurs malignes endocriniennes rares, associées à un pronostic sombre. Les trois mécanismes moléculaires les plus fréquemment altérés dans les CCS comprennent les mutations inactivatrices du gène suppresseur de tumeur TP53,la surexpression de IGF-II et l'activation constitutive de la voie de signalisation Wnt/β-caténine. En utilisant des modèles de souris transgéniques, nous avons montré que ces altérations, même combinées, ne sont pas suffisantes pour permettre la progression maligne.Nous avons précédemment identifié l'histone méthyltransférase EZH2 comme le modificateur d'histone le plus dérégulé dans les CCS. Nous avons également montré que sa surexpression est associée à une progression tumorale et à un mauvais pronostic. Cependant, les mécanismes sous-jacents de cette agressivité sont largement inconnus. Dans cette étude, nous avons cherché à identifier les gènes cibles de EZH2 dans les CCS, qui sont soient activés, soient réprimés. Ainsi, nous avons effectué une analyse bio-informatique des données du transcriptome de trois cohortes de patients porteurs de CCS. L’analyse montre une forte corrélation entre la surexpression de EZH2 et les gènes régulés positivement, suggérant un rôle majeur d’inducteur transcriptionnel de EZH2 dans les CCS. Nous avons montré que cette activité positive repose sur une interaction entre EZH2 et E2F1, qui entraîne la surexpression de gènes impliqués dans la régulation du cycle cellulaire et la mitose tels que RRM2,PTTG1 et PRC1/ASE1. Nous avons montré que l'inhibition de RRM2 par ARN interférent ou traitement pharmacologique avec le GW8510 inhibe la croissance cellulaire, la capacité à combler les blessures, la croissance clonogénique, la migration et induit l'apoptose des cellules H295R en culture. En revanche, l'expression du facteur pro-apoptotique NOV/CCN3 est diminuée dans les CCS, ce qui est corrélé au développement de tumeurs agressives. Nos analyses moléculaires montrent que l'inhibition de EZH2 augmente l'expression de NOV/CCN3, suggérant que la surexpression de EZH2 pourrait favoriser la progression maligne des CCS en inhibant les stimulateurs de l'apoptose. Le facteur NOV a déjà été identifié comme cible négative du récepteur nucléaire SF1 dans les cellules du CCS, bien que les mécanismes moléculaires à l'origine de cette inhibition n'aient pas été identifiés. De manière intéressante, dans le cancer de la prostate, l'expression de NOV est inhibée par le récepteur des androgènes AR, grâce au recrutement de EZH2 qui pose la marque répressive H3K27me3. Nous avons pu identifier une coopération similaire entre SF1 et EZH2 pour réprimer l'expression de NOV et bloquer ainsi l'apoptose dans les CCS.Au total, ces résultats identifient SF1 et E2F1 comme deux partenaires indépendants de EZH2, induisant la répression de facteurs pro-apoptotiques et l'activation des gènes du cycle cellulaire respectivement, conduisant ainsi à l'agressivité des CCS
Adrenocortical carcinomas (ACC) are regarded as rare endocrinemalignancies associated with dismal prognosis. The three common molecularmechanisms predominantly altered in ACC include inactivating mutations of theTP53 tumor suppressor gene, overexpression of IGF-II and constitutive activationof the Wnt/β-catenin signaling pathway. Using transgenic mouse models, wehave shown that these alterations, even when combined together, were notsufficient to induce malignant progression.We previously identified the histone methyltransferase EZH2 as the mostderegulated histone modifier in ACC. We have also shown that its overexpressionis associated with tumor progression and poor prognosis. Yet, the mechanismsunderlying this aggressiveness are largely unknown. Here, we aimed to identifyEZH2 target genes in ACC, which are either activated or repressed.Thus, we conducted a bio-informatics analysis of transcriptome data fromthree cohorts of ACC patients. The analysis showed a strong correlation betweenhighly expressed EZH2 and positively regulated genes suggesting a major role of‘transcriptional inducer‘ for EZH2 in ACC. We have shown that this positiveactivity relies on an interaction between EZH2 and E2F1 that results in theupregulation of genes implicated in cell cycle regulation and mitosis such asRRM2, PTTG1 and PRC1/ASE1. We showed that Inhibition of RRM2 by RNAinterference or pharmacological treatment with GW8510 inhibits cellular growth,wound healing, clonogenic growth, migration and induces apoptosis of H295Rcells in culture.In contrast, expression of the pro-apoptotic factor NOV/CCN3 is decreasedin ACC, which is correlated with development of aggressive tumours. Ourmolecular analyses show that EZH2 inhibition increases expression ofNOV/CCN3, suggesting that EZH2 overexpression may also favour malignantprogression in ACC by inhibition of apoptosis stimulators. NOV has previouslybeen identified as a negative target of the nuclear receptor SF1 in ACC cells,although the molecular mechanisms underlying this inhibition were unidentified.Interestingly, in prostate cancer, NOV expression is inhibited by the androgenreceptor, through recruitment of EZH2 and deposition of the H3K27me3 mark.We have been able to identify a similar cooperation between SF1 and EZH2 tosuppress NOV expression and block apoptosis in ACC.Altogether, these findings identifiy SF1 and E2F1 as two independentpartners of EZH2, inducing repression of proapoptotic factors, and activation ofcell cycle genes respectively, thus leading to aggressiveness of ACC

INTRODUÇÃO: O tratamento de escolha do carcinoma de mama localmente avançado é a quimioterapia neoadjuvante, porém, em virtude da heterogeneidade tumoral, sabe-se que nem todos os tumores responderão a esse tratamento. Neste contexto, avaliamos a proteína EZH2 (Enhancer of Zest Homolog 2), uma histona-metiltransferase catalisadora da trimetilação da lisina 27 da histona H3, com objetivos de avaliar sua expressão na predição da resposta do carcinoma de mama localmente avançado à quimioterapia neoadjuvante e de correlacionar sua expressão com a análise imunoistoquímica dos marcadores prognósticos usuais (proteínas HER2, Ki- 67, receptores hormonais de estrogênio e progesterona) e do p53. MÉTODOS: Foram obtidos fragmentos de tumor de 37 pacientes com carcinoma invasivo de mama nos estádios IIb e IIIa, submetidas à quimioterapia neoadjuvante com agentes antracíclicos. As pacientes pertenciam a 2 grupos. O Grupo 1 era composto de 19 pacientes que apresentaram resposta objetiva ao tratamento quimioterápico. No Grupo 2, as 18 pacientes não apresentaram essa resposta. Através da construção de arranjo em matriz de amostras teciduais, realizamos análise imunoistoquímica das expressões das proteínas HER2, Ki-67, p53, dos receptores de estrogênio e progesterona e da EZH2. RESULTADOS: O grupo de pacientes que não responde à quimioterapia tem, em média, idade significativamente superior (56,5 anos) ao das pacientes com resposta à quimioterapia (46,5 anos), porém os grupos não diferiram em relação ao número de ciclos de quimioterapia e em relação aos valores dos receptores hormonais e de HER2, Ki-67 e EZH2. A comparação entre a faixa etária, o número de ciclos de quimioterapia e os marcadores biológicos tumorais não demonstrou diferença significativa entre os grupos. A relação linear dos valores da proteína EZH2 com a idade, o número de ciclos de quimioterapia e os valores dos receptores hormonais foi negativa; e com as proteínas HER2 e Ki-67 a relação foi positiva. Para o grupo de pacientes que respondem ou não à quimioterapia neoadjuvante, não houve associação com as taxas de proteína EZH2. CONCLUSÕES: A proteína EZH2 correlaciona-se negativamente com os receptores hormonais de estrogênio e de progesterona e, positivamente com as proteínas HER2 e Ki-67. A expressão dessa proteína não se correlacionou com a resposta clínica do carcinoma de mama localmente avançado à quimioterapia neoadjuvante à base de antracíclicos.
INTRODUCTION: Neoadjuvant chemotherapy is the treatment of choice for patients with locally advanced breast cancer, however, because of tumor heterogeneity, not all tumors will respond to this treatment. In this context, we evaluated the EZH2 protein (Enhancer of Zest Homolog 2), a histone methyltransferase. EZH2 catalyses the trimethylation of lysine 27 of histone H3. The purposes of this study were to evaluate the expression of EZH2 for predicting tumor response to neoadjuvant chemotherapy in locally advanced breast cancer and its relation to usual prognostic markers (HER2, Ki-67, hormonal receptors of estrogen and progesterone - ER and PR) and p53. METHODS: Thirty-seven paraffin-embedded tumor blocks from different patients with stages IIb and IIIa invasive breast cancer. All of them have received neoadjuvant anthracycline-containing chemotherapy. The patients belonged to two different groups. Group 1 comprised 19 patients with objective response to chemotherapy, and Group 2, comprised 18 patients with no response to treatment. A TMA-based (tissue microarray) immunohistochemical analysis of HER2, Ki-67, p53, estrogen and progesterone receptors and EZH2 was performed. RESULTS: The group of patients who did not achieve a response had higher age (56.5 years) than the patients with response to chemotherapy (46.5 years), but the groups did not differ from the number of cycles of chemotherapy, and from the values of hormone receptors and HER2, Ki-67 and EZH2. The analysis of age, number of cycles of chemotherapy and biological tumor markers did not show a significant difference between the two groups. There was a negative linear relationship between EZH2 values and age, number of cycles of chemotherapy and hormone receptors. There was a positive linear relationship between EZH2 and HER2 and Ki-67. There was no association between EZH2 expression and response to chemotherapy. CONCLUSIONS: EZH2 protein is negatively correlated with hormonal receptors (ER and PR), and positively correlated with HER2 and Ki-67. There was no correlation between EZH2 expression and response to neoadjuvant anthracyclinecontaining chemotherapy.

Polycomb group (PcG) proteins are epigenetic modifiers that act as transcriptional repressors modulating chromatin accessibility of target genes. Methylation on lysine-27 of histone H3 (H3K27me3) catalyzed by the PcG protein Ezh2 is a bona fide epigenetic mark linked to lineage specification and identity. Analyses on PcG mutant mice have revealed an essential role of PcG proteins in haematopoiesis. In the B cell compartment, Ezh2 is expressed in progenitor B cells and is strongly induced upon recruitment of naïve B cells into the germinal center (GC) reaction, during a T cell dependent immune response. Using a conditional gene targeting approach in vivo, we addressed the effects of Ezh2 inactivation on GC B cell responses, terminal differentiation, memory B cell generation and function. Conditional inactivation of Ezh2 in GC B cells caused a significant reduction in numbers and frequency of GC B cells. Loss of Ezh2 promoted a significant increase in the fraction of GC B cells undergoing apoptosis. As a result of the impaired GC reaction, serum titers of antigen-specific, class-switched antibodies were significantly decreased and formation of memory B cells significantly impaired in mutant mice. Instead, mutant GC B cells showed premature onset of terminal differentiation, as assessed by the induction of genes coding for the master regulators of plasma cell differentiation Prdm1, Irf4 and Xbp1. In this work we provide evidence that sets the ground for a model where Ezh2 together with GC master regulator Bcl6 supports GC B cell identity and function.

En France comme dans la majorité des pays développés, le cancer de la prostate est le plus fréquent chez l’homme. Il est clairement établi que les altérations génétiques et épigénétiques sont des événements communs dans les cancers de la prostate, se traduisant par l’expression aberrante de gènes critiques. La méthylation des histones participe à la régulation de l’expression des gènes dans la cellule. La marque épigénétique H3K27me3 est associée à la répression génique et se trouve dérégulée dans les cancers de la prostate. Ses niveaux sont déterminés par l’équilibre entre les activités de la méthyltransférase d’histone EZH2 et de la déméthylase d’histone JMJD3. Afin de comprendre le mécanisme de dépôt de H3K27me3 dans la tumorigenèse prostatique, le travail de cette thèse s’est orienté sur l’évaluation simultanée de l’impact de JMJD3 et de EZH2. Dans un premier temps, les niveaux d’expression de JMJD3 et de EZH2 ont été montrés augmentés simultanément dans le cancer de la prostate. Cette augmentation est corrélée à un enrichissement de ces deux protéines sur le promoteur des gènes RARβ2, ERα, RGMA, AR et PGR. Dans un deuxième temps, une analyse transcriptomique a permis d’identifier une signature génique corrélée avec le niveau d’agressivité de la tumeur. L’utilisation des « épidrogues » GSK-J4 et DZNeP ciblant JMJD3 et EZH2 permettent de moduler l’expression de ces gènes. L’ensemble de ces résultats caractérise JMJD3 et EZH2 comme des facteurs clés dans le processus de tumorigenèse prostatique. Le panel de gènes identifié devrait permettre de développer de potentiels marqueurs de diagnostic mais également de pronostic dans le cancer de la prostate et sa modulation par les « épidrogues » permettra de développer de nouvelles stratégies thérapeutiques
In France like in majority of developed countries, prostate cancer is the most common cancer in men. It has been clearly established that genetic and epigenetic alterations are common events in prostate cancer resulting in aberrant gene expression. Histone methylation are involved in gene expression of cells. The H3K27me3 epigenetic mark is a repressive mark and it is deregulated in prostate cancer. H3K27me3 levels are determined by the balance between histone methyltransferase EZH2 and histone demethylase JMJD3 activities. In order to understand the mechanism of H3K27me3 deposition in prostatic tumorigenesis, this thesis focused on the simultaneous assessment of the impact of JMJD3 and EZH2.Firstly, expression levels of JMJD3 and EZH2 were shown to be simultaneously increased in prostate cancer. The increase is correlated to both protein enrichments on RARβ2, ERα, RGMA, AR and PGR gene promotors. Secondly, transcriptomic analysis identified gene signature correlated with tumor aggressiveness. The utilization of GSK-J4 and DZNeP epidrugs targeting JMJD3 and EZH2 allowed us to modulate gene expressionOur results characterized JMJD3 and EZH2 as key factors in prostatic tumorigenesis process. The identified gene panel would be able to develop potential diagnostic and prognostic markers in prostate cancer and their modulation by epidrugs would make new therapeutic strategies

Epigenetic regulators are commonly mutated in cancer. Activating mutations and overexpression of EZH2 occur in lymphoma and other malignancies, while loss-of-function mutations are found in myeloid malignancies. This thesis is a study of this apparent contradiction, examining the importance of cellular context for Ezh2 loss during the evolution of a single malignancy, Acute Myeloid Leukaemia (AML). This work demonstrates diametrically opposite functions for Ezh2 at early and late stages during the evolution of leukemias generated by disparate AML-associated fusion-oncogenes. Ezh2 functions as an oncogene that may be therapeutically targeted during disease maintenance. In contrast, Ezh2 behaves as a tumour suppressor gene during AML induction. Integrated genomic analysis demonstrates that largely different expression programmes are de-repressed during disease induction and maintenance following Ezh2 loss. Studying disease induction, Ezh2 represses a subset of bivalent promoters that resolve towards gene activation upon Ezh2 loss, inducing a feto-oncogene programme including genes like Plag1, whose overexpression phenocopies Ezh2 loss to accelerate AML induction in mouse models. This data highlights the importance of cellular context and phase of disease evolution for Ezh2 function. Moreover, the work herein identifies EZH2 as a potential target in AML, whilst providing reassurance of the safety of this therapeutic strategy.

Accumulating evidence suggests that glioma stem cells (GSCs) are important therapeutic targets in glioblastoma (GBM). In this study, we identified NIMA-related kinase 2 (NEK2) as a functional binding protein of enhancer of zeste homolog 2 (EZH2) that plays a critical role in the posttranslational regulation of EZH2 protein in GSCs. NEK2 was among the most differentially expressed kinase-encoding genes in GSC-containing cultures (glioma spheres), and it was required for in vitro clonogenicity, in vivo tumor propagation, and radioresistance. Mechanistically, the formation of a protein complex comprising NEK2 and EZH2 in glioma spheres phosphorylated and then protected EZH2 from ubiquitination-dependent protein degradation in a NEK2 kinase activity-dependent manner. Clinically, NEK2 expression in patients with glioma was closely associated with EZH2 expression and correlated with a poor prognosis. NEK2 expression was also substantially elevated in recurrent tumors after therapeutic failure compared with primary untreated tumors in matched GBM patients. We designed a NEK2 kinase inhibitor, compound 3a (CMP3a), which efficiently attenuated GBM growth in a mouse model and exhibited a synergistic effect with radiotherapy. These data demonstrate a key role for NEK2 in maintaining GSCs in GBM by stabilizing the EZH2 protein and introduce the small-molecule inhibitor CMP3a as a potential therapeutic agent for GBM.

Les surrénales sont des glandes endocrines permettant la réponse au stress de l’organisme. Alors que la medulla produit des catécholamines, la corticosurrénale sécrète des minéralocorticoïdes au niveau de la zone glomérulée, et des glucocorticoïdes grâce aux cellules de la zone fasciculée. Ces hormones sont notamment impliquées dans l’homéostasie hydrominérale, la réponse immunitaire et la maturation pulmonaire au cours de la vie fœtale. Les insuffisances surrénaliennes peuvent donc être très délétère en absence de traitement. Pour maintenir l’intégrité tissulaire au cours de la vie et pour mieux répondre aux variations des besoins de l’organisme, le cortex surrénalien est en renouvellement cellulaire constant. Des expériences de lignage ont mis en évidence que ce renouvellement repose sur le recrutement de cellules progénitrices capsulaires et situées dans la partie externe du cortex. Lorsqu’ils sont mobilisés, ces progéniteurs se différencient en cellules de la zone glomérulée, qui vont alors migrer de façon centripète le long du cortex et se différencier en cellules de la zone fasciculée après une conversion de lignage, au cours de leur migration. Cette conversion de lignage est orchestrée via un équilibre entre l’activation des voies Wnt/β-caténine, imposant une identité glomérulée, et PKA, permettant une différenciation fasciculée. Les facteurs épigénétiques jouent de nombreux rôles essentiels, du développement embryonnaire jusqu’à la tumorigenèse, en passant par l’homéostasie des tissus. Nous avons montré que la méthyltransférase EZH2 était le facteur épigénétique le plus surexprimé dans les carcinomes corticosurrénaliens et que cette surexpression était associée à l’agressivité de ces cancers. EZH2 est la sous-unité catalytique du complexe multi-protéique PRC2 qui permet, entre autres, la répression de la transcription de ses gènes cibles en posant la marque H3K27me3. L’objectif de ma thèse a été d’identifier les potentiels rôles physiologiques de EZH2 dans la surrénale, qui n’avaient jusque là, jamais été recherchés.En développant un modèle murin d’invalidation génétique de Ezh2 dans le cortex surrénalien, dès l’émergence de l’ébauche surrénalienne au cours du développement embryonnaire, nous avons pu mettre en évidence une hypoplasie corticosurrénalienne, résultant d’une forte atrophie de la zone fasciculée, et associé à une insuffisance primaire en glucocorticoïdes. Nos analyses nous ont permis de démontrer le rôle original et inattendu de Ezh2 dans le contrôle de la voie de signalisation PKA, en réprimant l’expression d’inhibiteurs de cette voie comme les phosphodiestérases (PDE) et la sous-unité régulatrice Prkar1b. EZH2 régule ainsi la zonation fonctionnelle du cortex surrénalien via son activité histone méthyltransférase. A l’inverse, on n’observe pas d’altération marquée de la voie Wnt/β-caténine, suggérant que Ezh2 n’est pas essentiel au contrôle de cette voie dans la surrénale. Nous avons également pu mettre en évidence une dédifférenciation de cellules corticales qui retrouvent, suite à la perte de Ezh2, une identité progénitrice en exprimant des marqueurs adréno-gonadique tels que Gata4 et Wt1. Cette dédifférenciation est un phénomène naturel que l’on retrouve avec le vieillissement et qui pourrait être associée avec la diminution progressive de l’expression de Ezh2 dans les cellules stéroïdogènes. L’ensemble de ces résultats, met en évidence une nouvelle fonction de Ezh2 dans le contrôle de la voie de signalisation PKA et de l’homéostasie de la glande surrénale
Adrenals are endocrine glands allowing the stress response of the organism. While the medulla produces catecholamines, the adrenal cortex secretes mineralocorticoids in the glomerular zone, and glucocorticoids through cells in the fasciculated zone. These hormones are notably involved in hydromineral homeostasis, the immune response and pulmonary maturation during fetal life. Adrenal insufficiency can therefore be very deleterious in the absence of treatment. To maintain tissue integrity over the course of life and to better respond to the changing needs of the body, the adrenal cortex is in constant cell renewal. Lineage experiments have shown that this renewal is based on the recruitment of capsular progenitor cells and progenitors located in the outer part of the cortex. When mobilized, these progenitors differentiate into cells of the glomerular zone, which then migrate centripetally along the cortex and differentiate into cells of the fasciculated zone after lineage conversion, during their migration. This lineage conversion is orchestrated via a balance between the activation of the Wnt/β-catenin pathway, imposing a glomerular identity, and PKA pathway, allowing fasciculated differentiation. Epigenetic factors play many important roles, from embryonic development to tumorigenesis, passing by tissue homeostasis. We have shown that methyltransferase EZH2 is the most overexpressed epigenetic factor in adrenocortical carcinomas and this overexpression is associated with cancer agressivity. EZH2 is the catalytic subunit of the multiprotein complex PRC2 that allow, among others things, the repression of the transcription of its target genes by posing the mark H3K27me3. The aim of my thesis was to indentify the putative physiological roles of EZH2 in the adrenal, never investigated yet.By developing a murine model of genetic invalidation of Ezh2 in the adrenal cortex, from the emergence of the adrenal anlagen during embryonic development, we have been able to demonstrate adrenocortical hypoplasia, resulting from a strong atrophy of the zona fasciculata, and associated with primary glucocorticoid insufficiency. Our analyses allowed us to demonstate the original and unexpected role of EZH2 in the controle of the PKA pathway, by repressing expression of this pathway inhibitors such as phosphodiesterases (PDE) and regulatory subunit Prkar1b. EZH2 thus regulate functionel zonation of adrenal cortex via its histone methyltransferase activity. On the contrary, we don’t observe marked alteration of the Wnt/β-catenin pathway, suggesting EZH2 is not essential for the control of this pathway in the adrenal. We could also show a dedifferenciation of cortical cells which, after the loss of Ezh2, exhibit progenitors identity by expressing adreno-gonadal marks as Gata4 and Wt1. This dedifferenciation is a natural phenomenon that appear with ageing and could be associated with processive decrease of Ezh2 expression in steroidogenic cells. All of these results, highlights a new function of Ezh2 in the control of the PKA signaling pathway and in the homeostasis of the adrenal gland

L’acronimo NAFLD (“non-alcoholic fatty liver disease”) comprende sia la condizione di steatosi epatica semplice sia la steatoepatite non-alcoolica (NASH: “non-alcoholic steatohepatitis”) caratterizzata da necroinfiammazione e dalla possibile presenza di fibrosi epatica. È stato dimostrato che la NASH può talvolta progredire fino a determinare l’insorgenza di cirrosi e/o di epatocarcinoma cellulare (HCC). Negli ultimi anni, numerosi studi hanno evidenziato un sostanziale aumento nel numero di casi con HCC correlato alla NAFLD, sia in presenza che in assenza di cirrosi epatica. Indipendentemente dall’eziologia, durante il processo di epatocarcinogenesi, numerose vie di trasduzione del segnale e di controllo di espressione genica risultano essere alterate. Tuttavia, i meccanismi molecolari alla base della progressione della NAFLD verso un suo fenotipo pro-carcinogenico non risultano ancora del tutto chiariti. Tra le numerose proteine coinvolte nell’insorgenza e nella progressione dell’HCC, la proteina FAK (Focal Adhesion Kinase) è spesso trovata overespressa o iper-fosforilata in pazienti affetti da tumore, suggerendo un ruolo chiave per questa proteina nell’induzione del fenotipo neoplastico. Recentemente, anche la metil-transferasi EZH2 è stata associata al processo di epatocarcinogenesi e, in particolare, un recente studio ha evidenziato una correlazione positiva tra l’over-espressione di FAK ed EZH2 e l’aggressività del tumore in pazienti affetti da neoplasia endometriale, suggerendo un potenziale collegamento tra le due proteine. In questo studio, pertanto, abbiamo volto l’attenzione al ruolo di FAK ed EZH2 in modelli in vitro ed in vivo di NAFLD o di HCC. Gli esperimenti condotti sia sul modello in vitro che sul modello in vivo di NAFLD, hanno mostrato un aumento significativo della proteina FAK, della sua fosforilazione in tirosina 397 e della sua proteina target Paxillina. Tuttavia, il silenziamento di FAK in una linea cellulare di epatoblastoma umano è risultato essere associato con l’attivazione del processo di de novo lipogenesis e con un conseguente aumento dell’accumulo lipidico intra-cellulare. D’altro canto, gli esperimenti condotti su un modello murino di progressione della NAFLD verso l’HCC hanno evidenziato che livelli di FAK e della sua fosforilazione in tirosina 397 aumentavano parallelamente alla progressione del danno epatico fino all’insorgenza dei noduli tumorali. Abbiamo inoltre dimostrato, per la prima volta, il coinvolgimento di EZH2 nella NAFLD, riportando una correlazione inversa tra l’espressione di EZH2 e il grado di severità della malattia. Infine, un modello murino di epatocarcinoma è stato utilizzato per caratterizzare il ruolo di FAK nell’epatocarcinogenesi. In particolare, i nostri dati dimostrano che il silenziamento di FAK riduce drasticamente la crescita del tumore che esprime anche livelli più bassi di EZH2. Allo stesso modo, studi in vitro hanno evidenziato l’azione anti-proliferativa/pro-apoptotica del silenziamento di FAK in cellule di HCC. Entrando nel merito del potenziale meccanismo molecolare che potrebbe connetter le due proteine di nostro interesse, nelle cellule silenziate per FAK abbiamo riscontrato una riduzione del trascritto, della localizzazione nucleare e dell’attività di trimetilazione di EZH2. Sulla base di ulteriori risultati abbiamo dimostrato che p53 e E2F2/3 sono coinvolti nella regolazione della trascrizione di EZH2 mediata da FAK. Pertanto, in conclusione, in questo studio abbiamo dimostrato il ruolo chiave di FAK nella NAFLD e nell’epatocarcinoma, e abbiamo fornito evidenze dell’esistenza di una inter-connessione tra le proteine FAK ed EZH2, suggerendo p53 e E2F2/3 come possibili mediatori.
NAFLD is one of the most common liver disease worldwide and it encompasses a wide range of liver injuries, ranging from simple steatosis (non-alcoholic fatty liver “NAFL”) to non-alcoholic steatohepatitis (“NASH”). NASH can be sometimes associated with hepatic fibrosis and may potentially progress to irreversible cirrhosis and in some cases to hepatocellular carcinoma (HCC). The number of HCC new cases with a NAFD-dependent aetiology has strongly increased during the last decade but the molecular mechanisms regulating NAFLD-related hepatocarcinogenesis remain to be explored yet. Among all the factors involved in HCC onset and progression different epigenetic mechanisms and signalling pathways, affecting cell homeostasis (e.g. cell proliferation, apoptosis, migration and invasion) may play a major role, but their action in NAFLD is still obscure and their connection with hepatocarcinogenesis is unknown. Interestingly, the focal adhesion tyrosine kinase (FAK) is often found overexpressed or hyper-phosphorylated in HCC patients suggesting a key role of this protein in the control of cancer cells behaviour. Similarly, the methyltransferase EZH2 has been recently associated with the process of hepato-carcinogenesis. Further, a recent study reported a positive correlation between FAK and EZH2 expression and their association with tumour aggressiveness in endometrial cancer. Therefore, a potential direct/indirect interplay between these proteins might affect the development of different tumours, including HCC. In this study, we point to investigate the role of FAK and EZH2 in in vitro and in vivo models of diet-induced NAFLD and of HCC. Our results demonstrated an increased expression of Tyr-397 phosphorylated FAK and of its target paxillin in vivo and in vitro NAFLD. Moreover, the silencing of FAK also promoted increased lipid accumulation via the activation of the de novo lipogenesis pathway in HepG2 cells. Interestingly, in a model of NAFLD-induced HCC, both total and pTyr397 FAK correlated with disease severity. We reported the first evidence of EZH2 connection to NAFLD observing a down-regulation of EZH2 in our in vitro and in vivo models. Furthermore, the pharmacological inhibition of EZH2 worsened liver steatosis and inflammation. Results obtained from human HCC xenografts on NOD/SCID micrevealed a crucial role of FAK in HCC development and progression. Accordingly, we found that silencing of FAK reduced cell proliferation and invasion, and induced apoptosis in HCC cells. Additionally, we demonstrated that the silencing of FAK critically affected EZH2 transcription, nuclear localization and H3K27 tri-methylation activity. Importantly, we found that p53 and E2F2/3 are key mediators of FAK-dependent effects on EZH2 expression/activity. In conclusion, we demonstrated a master role of FAK in NAFLD and HCC and provided strong evidence of its connection with EZH2, introducing a new protein network active in the control of cancer cells’ proliferation, in which p53 and E2F may act as mediators.

A esquistossomose é um grave problema de saúde pública, com alta mortalidade e morbidade em países endêmicos, causada pelo verme trematódeo do gênero Schistosoma. O praziquantel é a única droga disponível para tratamento da doença, é usada em larga escala para tratamento de populações de áreas endêmicas, porém não previne a reinfecção e tem efeito somente em vermes adultos. Drogas estudadas em câncer como inibidores de histona deacetilases (iHDACs) modificam o padrão epigenético da célula desencadeando a morte celular, e em Schistosoma mansoni já foi mostrado que a inibição de HDACs além de aumentar a acetilação de histonas alterou o fenótipo de miracídios e provocou morte em esquistossômulos e vermes adultos. O presente estudo investigou o efeito do iHDAC Trichostatin A (TSA) na regulação da transcrição gênica em esquistossômulos, detectando por meio de ensaios de microarray centenas de genes diferencialmente expressos, relacionados a replicação de DNA, metabolismo e complexos modificadores de histonas. A inibição de HDAC em vermes adultos levou a um aumento da acetilação nas marcas de histonas H3K9ac, H3K14ac e H4K5ac relacionadas à indução de transcrição. Com imunoprecipitação de cromatina seguida de PCR (ChIP-qPCR) detectou-se o aumento de deposição de H3K9ac e H3K14ac na região promotora de genes com expressão aumentada ou diminuída, porém a marca de repressão H3K27me3 não sofreu alteração na região promotora de nenhum gene analisado. Análises adicionais indicaram um conjunto de genes diferencialmente expressos que codificam proteínas histone readers, que fazem parte de complexos modificadores de histonas, como EED capaz de identificar a marca de repressão H3K27me3 e regular a atividade de EZH2, apontando um novo alvo terapêutico. O efeito sinérgico entre iHDAC e um iEZH2 foi testado e detectou-se o aumento da mortalidade de esquistossômulos. A estrutura de SmEZH2 foi modelada por homologia e usada para análises computacionais que sugeriram uma alta afinidade de ligação de SmEZH2 com o iEZH2, abrindo uma perspectiva de desenvolvimento de novas drogas específicas para tratamento da esquistossomose.
Schistosomiasis is a serious public health problem, with high mortality and morbidity in endemic countries, caused by trematode worms of the genus Schistosoma. Praziquantel is the only available drug for treatment of the disease; it is used extensively to treat populations in endemic areas, but does not prevent reinfection and is effective only in adult worms. Drugs studied in cancer as histone deacetylase inhibitors (iHDACs) modify the epigenetic status of the cell, triggering cell death, and it has been shown in Schistosoma mansoni that inhibition of HDACs increase histone acetylation, alter the phenotype of miracidia and cause death in schistosomules and adult worms. The present study investigated the effect of iHDAC Trichostatin A (TSA) on the regulation of gene transcription in schistosomules, detecting by means of microarray assays hundreds of differentially expressed genes related to DNA replication, metabolism and histone remodeling complexes. Inhibition of HDAC in adult worms led to an increase in histone acetylation marks H3K9ac, and H3K14ac H4K5ac related to transcriptional induction. With chromatin immunoprecipitation followed PCR (ChIP-qPCR) we detected an increased deposition of H3K9ac and H3K14ac at the promoter region of genes with increased or decreased expression, but the repressive mark H3K27me3 was not changed at all analyzed gene promoter regions. Additional analysis indicated a set of differentially expressed genes that encode histone reader proteins that are part of histone modifier complexes such as EED, which is able to identify the repression mark H3K27me3 and to regulate EZH2 activity, pointing to a new therapeutic target. The synergistic effect between iHDAC and one iEZH2 has been tested and found to cause an increase in schistosomules mortality. The SmEZH2 structure was modeled by homology and used for computational analyses, which suggested a high affinity binding of SmEZH2 with iEZH2, opening the opportunity for development of new specific drugs for treatment of schistosomiasis.

EGR1, one of the immediate-early response genes, plays an important role as a mediator for transmitting extracellular stimuli. EGR1 is down regulated in many cancers. Many studies show that it functions as a tumor suppressor gene in a variety of cancers. EGR1 also acts as an oncogene in number of cancers. We found that in rhabdomyosarcoma (RMS), which is a muscle derived pediatric cancer, EGR1 was expressed in both RMS subtypes, embryonal and alveolar, but with a much higher expression profile in embryonal RMS. This suggests different mechanisms of down regulation of EGR1 in these two subtypes. Molecular and cellular approaches were used to characterize the functional role of EGR1 in RMS. We found that over expression of EGR1 in alveolar RMS significantly decreased cell proliferation, mobility, and anchorage-independent growth. We showed that exogenous EGR1 up regulated the cell cycle regulator, p21, which is normally repressed in RMS. EGR1 also promoted differentiation in RMS cells by up regulating several genes involved in muscle differentiation including myosin heavy chain (MyHC), MyoD and myogenin. We found that EGR1 interacts with the oncogene TBX2 in RMS cells and that TBX2 inhibits EGR1 function. To understand how TBX2 inhibits EGR1, we depleted TBX2 in RMS and we found an up regulation of the EGR1 targeted tumor suppressor gene, PTEN, and the cysteine protease inhibitor gene, CST6. Also, we performed luciferase assays and found that TBX2 decreased the expression of luciferase constructs fused with the PTEN promoter when TBX2 was co-transfected with EGR1. Our novel findings on the EGR1 function in RMS highlights the significant role of EGR1 in muscle development and tumor growth. Significantly, our work also suggests the EGR1 could promote tumor regression in RMS through inducing programmed cell death, or apoptosis. We found that EGR1 induced apoptosis through triggering the intrinsic apoptosis pathway and activating caspase cascades involving caspase 3 and caspase 9, which are essential mitochondrial apoptotic factors. Also, we observed the activation of two pro-apoptotic factors, BAX and dephosphorylated BAD, which are both located upstream of the caspase cascades in the intrinsic pathway. Also, we found in our study that EGR1 is repressed by the catalytic subunit of PRC2 complex, EZH2, which mediates gene silencing through methylation of lysine 27 on histone 3 (H3K27me3). EGR1 also sensitized RMS cells to chemotherapeutic agents, which could be a future direction for improved therapeutic targeting. Therefore, this work provides a novel and powerful molecular therapeutic target for RMS cancer.

TBX2 and TBX3, which function as repressors, are members of the T-Box transcription factor family which are conserved throughout the metazoan lineage. TBX2 is highly expressed in rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in children, and many other cancers. Previously, our lab dissected the oncogenic properties of TBX2 and its regulation of p14, p21 and PTEN. TBX3 is also expressed in some cancer types, however, its expression profile in RMS is severely down-regulated. TBX3 is shown to repress TBX2 in chondrocytes, but the characterization and regulation of TBX3 is poorly understood in the muscle lineage. Polycomb Repressive Complex 2 (PRC2), a gene silencing complex, acts to methylate histone H3 lysine 27 (H3K27me) of target gene promoters. The catalytic subunit of PRC2, EZH2, is up-regulated in RMS and data from our lab has shown that depletion of EZH2 up-regulated TBX3 and down-regulated TBX2 in C2C12 cells, an immortalized murine cell line. The hypothesis of this project was that there would be a PRC2-TBX3-TBX2 axis in RMS cells. To examine if TBX3 represses TBX2, TBX3 was transiently expressed in RMS cells representing both subtypes of RMS and we found that TBX2 was downregulated in each cell line. In a stable RH30 cell line with ectopic TBX3, TBX2 was down-regulated and PTEN expression was up-regulated. To determine if TBX2 repression by TBX3 was direct, a TBX3 ChIP assay was performed on the TBX2 promoter as well as the PTEN promoter. We found a strong enrichment of TBX3 on the TBX2 promoter but not on the PTEN promoter. Accordingly, we also observed that TBX3 over-expression impaired tumorigenesis through reduced cell proliferation, migration, and anchorage dependent growth. Also, we found that a stable RD cell line with ectopic TBX3 could promote differentiation, strongly suggesting that these results could have therapeutic value. Next, a shEZH2 plasmid was transfected into RMS cell lines ask if TBX3 was regulated by the PRC2 complex as we had observed in C2C12 cells. Just as we hypothesized, TBX3 was up-regulated and TBX2 was down-regulated. Similar to the previous TBX3 overexpression experiments, the EZH2 depleted RMS cell lines also showed decreased cell proliferation and migration rate. Also, an EZH2 knock down treatment induced differentiation in RMS cell lines. Therefore, understanding this potent regulation axis could provide an excellent opportunity for treatment of RMS cancer in the future.

Our lab has recently found that E2F3, an essential cell cycle regulator, regulates the self-renewal capacity of neural precursor cells (NPCs) in the developing mouse brain. Chromatin immunoprecipitation (ChIP) and immunoblotting techniques revealed several E2F3 target genes, including the polycomb group (PcG) protein, EZH2. Further ChIP and immunoblotting techniques identified the neural stem cell self-renewal regulators p16INK4a and Sox2 as shared gene targets of E2F3 and PcG proteins, indicating that E2F3 and PcG proteins may co-regulate these target genes. E2f3-/- NPCs demonstrated dysregulated expression of EZH2, p16INK4a, and SOX2 and decreased enrichment of PcG proteins at target genes. Restoring EZH2 expression to E2f3+/+ levels restores p16INK4a and SOX2 expression levels to near E2f3+/+ levels, and also partially rescues NPC self-renewal capacity toward E2f3+/+ levels. Taken together, these results suggest that E2F3 controls NPC self-renewal by modulating expression of p16INK4a and SOX2 via regulation of PcG expression, and potentially PcG recruitment.

Le processus qui aboutit à la formation de plaquettes est appelé mégacaryopoïèse. Les mégacaryocytes (MK) sont de grandes cellules de la moelle osseuse qui par fragmentation dans la circulation sanguine produisent des plaquettes. La régulation extrinsèque ou intrinsèque de ce processus a été largement étudiée. Cependant la régulation épigénétique reste mal connue bien que de nombreuses mutations dans des gènes de régulateurs épigénétiques soient retrouvées dans les hémopathies malignes de la lignée MK. En particulier des mutations du gène de la méthyltransférase EZH2, composant catalytique du Polycomb Repressive Complex 2 (PRC2) ont été détectées dans plusieurs types d’hémopathies. Ces mutations sont soit gain soit perte de fonction suggérant qu’EZH2 peut être à la fois un oncogène ou un gène suppresseur de tumeur. Dans les TE (Thrombocythémie Essentielle) et les MFP (Myélofibrose Primaire), deux néoplasmes myéloprolifératifs (NMPs), qui affectent principalement la lignée MK, des mutations d’EZH2 perte de fonction ont été retrouvées ainsi que dans les DS-AMKL (Down syndrome acute megakaryoblastic leukemia). Cela suggère qu’EZH2 joue un rôle important dans la mégacaryopoïèse normale. La caractérisation de cette fonction pourrait être utile pour mieux appréhender le rôle des mutations d’EZH2 dans les pathologies malignes mégacaryocytaires. Cette thèse peut être divisée en deux parties : 1) Caractérisation du rôle joué par EZH2 dans la mégacaryopoïèse normale et pathologique 2) Développement d‘un outil permettant d’étudier la coopération entre mutations dans les DS-AMKL.1) Lors des temps précoces de la différenciation in vitro des cellules CD34+ de sang de cordon vers la lignée mégacaryocytaire l’inhibition d’EZH2 entraîne l’acquisition plus rapide des marqueurs MK de surface (CD41 et CD42) pour un nombre de mitoses égal. Ceci suggère qu’EZH2 régule la spécification MK des progéniteurs hématopoïétiques. Plus tard dans la différenciation, l'inhibition constante d’EZH2 via des inhibiteurs ou des shRNA, arrête la prolifération et diminue le niveau de ploïdie des MKs en arrêtant la réplication de l’ADN. Ceci est du à la surexpression de plusieurs CDKi (Cyclin dependent kinase inhibiteurs), dont CDKN2D. L'analyse par Chip-Seq a montré que la transcription de CDKN2D est régulée par H3K27me3 au niveau de son promoteur et donc que CDKN2D est une nouvelle cible de PRC2. Dans les MKs les plus matures, l’inhibition d’EZH2 diminue la formation des proplaquettes, ceci est corrélé à des modifications d’expression de gènes régulant le cytosquelette d’actine. L’ensemble de ces résultats a été confirmé sur des MKs de patients porteurs de la mutation JAK2V617F.2) Par la technique CRISPR-Cas9, nous avons introduit dans des iPSC (induced pluripotent stem cells) disomiques et trisomiques pour le chromosome 21, la mutation GATA1s présente chez tous les patients avec une DS-AMKL. Nous avons montré que ces mutations modifiaient le cadre de lecture dans l’exon 2 et entrainaient l’expression de la forme courte de GATA1 (GATA1s). Nous sommes en train d'effectuer des études fonctionnelles ainsi que d’introduire d’autres mutations, y compris celles d’EZH2 pour modéliser la maladie.Au cours de cette thèse nous avons montré que l’inhibition d’EZH2 régule les temps initiaux de la mégacaryopoïèse en accélérant la spécification cellulaire au niveau des progéniteurs et ensuite la maturation terminale en inhibant profondément la polyploïdisation par surexpression de plusieurs CDKi dont CDKN2D et en inhibant la formation des plaquettes par un effet sur le cytosquelette d’actine. Ces résultats pourront être utiles pour mieux comprendre le rôle de la perte de fonction d’EZH2 dans les hémopathies malignes de la lignée mégacaryocytaire
The process that leads to platelet production is called megakaryopoiesis. Megakaryocytes (MK) are the large bone marrow cells that produce platelets by fragmentation in the blood flow. The extrinsic and intrinsic regulation of megakaryopoiesis has been largely studied. However, the epigenetic regulation remains poorly known although numerous mutations in genes of epigenetic regulators have been found in patients with MK hematological malignancies. The methyltransferase EZH2, the catalytic component of Polycomb Repressive Complex 2 (PRC2) is among the most studied epigenetic regulators. EZH2 is also mutated in many malignant hematological disorders where it can be an oncogene or a tumor suppressor gene. Particularly in ET (Essential Thrombocythemia) and PMF (Primary Myelofibrosis), two myeloproliferative neoplasms (MPNs) that affect mainly the MK lineage, loss of function EZH2 mutations have been found as well as in DS-AMKL (Down syndrome acute megakaryoblastic leukemia)Altogether these observations suggest that EZH2 controls normal megakaryopoiesis and characterization of this function could be helpful to understand the role of EZH2 in MK malignant diseases.This thesis can be divided in two parts:1) Characterization of the role of EZH2 in normal and pathological megakaryopoiesis 2) Establishment of a cellular tool to study the cooperation between the different mutations of DS-AMKL. RESULTS1) Using CD34+ cells isolated from cord blood, we showed that at early stages of differentiation, EZH2 inhibition accelerates the acquisition of MK surface markers (CD41a and CD42a) without increasing proliferation suggesting that EZH2 regulates the specification towards the MK lineage. Later in differentiation the constant inhibition of EZH2 via inhibitors or shRNAs, produced a proliferation arrest and a decrease in ploidy level that was related to an arrest in DNA replication due to an upregulation of several CDKi (Cyclin dependent kinase inhibitors), more particularly CDKN2D. Chip-Seq analysis demonstrated that CDKN2D is effectively regulated by H3K27me3 and is a new target of PRC2. This inhibition of ploidization by EZH2 inhibition was confirmed in MK from JAK2V617F patients. Furthermore in the more mature MKs (normal or JAK2V617F) we observed a defect in proplatelet formation, which was associated with an abnormal expression of genes regulating the actin filament. 2) By CRISPR-Cas 9, in iPSCs either disomic or chromosome 21 trisomic, we introduced, the GATA1s mutation present in all DS-AMKL patients. We confirmed at the gene and protein level that this genome editing has been correctly performed and that it induces as previously observed a blockage in erythroid differentiation. We are now carrying out the complete functional characterization together with the introduction of other mutations of DS-AMKL including EZH2.CONCLUSIONThis study describes EZH2 as a regulator of megakaryopoiesis via an initial control of cell specification and then of MK maturation. These results will be useful to better understand the role that EZH2 plays in diseases affecting the MK lineage such as MPNs and DS-AMKL

Le lymphome folliculaire (LF) représente le 2ème lymphome par ordre de fréquence et reste considéré à l’heure actuelle comme incurable. De nombreuses questions sur le processus de lymphomagénèse sont encore non résolues et il n’existe aucun marqueur génomique ou moléculaire unanimement reconnu permettant de prédire l’évolution des patients. Nos travaux de recherche s’inscrivent dans l’objectif de mieux comprendre l’impact des altérations moléculaires identifiées dans ces tumeurs, grâce à une approche intégrative visant à combiner des données génomiques, transcriptomiques et mutationnelles. Ce travail a permis de construire un score, basé sur l’expression d’un panel de gènes, prédictif du risque de progression de la maladie. Ce score a été confirmé sur une seconde cohorte de patients, validant son utilité potentielle en pratique clinique. Par ailleurs, nos résultats suggèrent que les cellules tumorales peuvent acquérir des propriétés évocatrices d’un profil de cellules souches et associées à un pronostic particulièrement défavorable. Une 2ème partie de notre travail a porté sur les altérations touchant le gène EZH2, muté chez 25% des patients. Nous avons démontré qu’un gain génomique au niveau du locus EZH2 pouvait également avoir des conséquences sur le profil transcriptomique et un impact pronostique, soulignant l’importance de prendre en compte l’ensemble des anomalies touchant ce gène. Enfin, nous rapportons qu’un polymorphisme constitutionnel situé dans ce gène est associé au risque de progression des patients traités par un anticorps anti-CD20. L’ensemble de ces résultats apporte un éclairage nouveau sur la biologie du LF et peut contribuer à améliorer la prise en charge des patients
Follicular Lymphoma (FL) is the 2nd most frequent lymphoma subtype and is usually considered incurable with current strategies. Several questions regarding the lymphomagenesis process are still pending, and no molecular or genomic marker has been unanimously recognized yet to predict outcome. We performed an integrative analysis combining genomic, transcriptomic and mutational data in the view to bringing new highlights in the molecular alterations acting in FL. Based on gene-expression profiling data we developed a model able to predict progression-free survival in FL patients. We confirmed its predictive value in another cohort of patients, thereby allowing its potential use in clinical practice. Furthermore, our results highlight that some tumors show a stem-cell-like gene-expression profile that was associated with highly unfavorable outcome. In the second part of our work, we focused on alterations of the gene EZH2. Although mutations have been reported in 25% of FL patients, we questioned whether genomic gains at EZH2 locus could also contribute to lymphomagenesis. We showed that such gain may impact the transcriptional profile and have a prognostic significance, thus highlighting the crucial interest of determining both kinds of alterations. Finally, we report that a germ-line polyporphism in the EZH2 gene was significantly associated with progression-free survival in patients treated by anti-CD20 therapy. Taken together, these results bring new highlights on FL biology and may help to improve the clinical management of FL patients

Les protéines de la famille TET catalysent l'oxydation des 5-méthylcytosines (5mC) en 5-hydroxyméthylcytosines (5hmC) et jouent ainsi un rôle dans la régulation épigénétique de la transcription et dans le processus de déméthylation de l'ADN. Les interactions entre la méthylation de l'ADN et les autres marques épigénétiques sont encore mal connues.Des mutations inactivatrices du gène TET2 ont été décrites dans les hémopathies myéloïdes et lymphoïdes et l'inactivation conditionnelle (cKO) de ce gène évaluée chez la souris a permis d'identifier de multiples anomalies de l'hématopoïèse, ainsi que le développement tardif d'hémopathies myéloïdes. Cette latence importante suggère la nécessité d'évènements oncogéniques coopératifs pour la transformation hématopoïétique. Chez l'Homme, les mutations de TET2 sont observées en association avec de nombreuses autres mutations, et en particulier avec des mutations du gène DNMT3A impliqué dans la méthylation de novo des cytosines de l'ADN, et avec des mutations du gène EZH2 responsable de methylation de la lysine 27 de l'histone H3. Nous avons testé fonctionnellement ces associations en utilisant des modèles murins.L'utilisation d'un modèle de transplantation de moelle osseuse nous a permis d'identifier une coopération de l'inactivation de Tet2 et du mutant DNMT3AR882H dans la transformation des lignées myéloïdes et lymphoïde T, correspondant aux hémopathies humaines porteuses de ces mutations. Dans la transformation lymphoïde, nos données indiquent que la dérégulation de la méthylation entraine une surexpression du gène NOTCH1 et de l'activité de la voie de signalisation correspondante.L'analyse de souris invalidées de manière conditionnelle pour Tet2 et Ezh2 a montré que les souris correspondantes meurent d'aplasie médullaire, dont l'origine est imputée à la disparition de cellules souches hématopoïétiques capables de reconstituer l'hématopoïèse à long terme (LT-HSC). Ezh2 et Tet2 ont donc des rôles primordiaux dans le maintien de l'autorenouvellement des cellules souches hématopoïétiques, dont les mécanismes moléculaires, génétiques et épigénétiques restent à définir
TET family proteins catalyzing the conversion of 5-methylcytosines (5mC) into 5-hydroxymethylcytosines (5hmC) are crucial for epigenetic regulation of transcription and for DNA demethylation. Interactions between DNA methylation and other epigenetic marks are not fully understood.TET2 inactivating mutations have been identified in both myeloid and lymphoid malignancies. The conditional inactivation (cKO) of this gene in mice highlights pleiotropic hematopoietic abnormalities as well as myeloid transformation at late stages. This latency suggest cooperativity between Tet2 and other oncogenic events during transformation. Human TET2 mutations are frequently found associated with other mutations, and more particularly with mutations in DNMT3A, involved in de novo methylation of cytosines and with mutations in EZH2, responsible for lysine 27 of histone H3 methylation. We decided to functionally assess these mutation associations in mice.Bone marrow transplantation of Tet2 inactivated and DNMT3AR882H mutated cells allowed us to identify myeloid and T-cell transformations, corresponding to human hematological disorders harboring these mutations. Our results on T-cell transformations clearly demonstrate that the deregulation of methylation leads to NOTCH1 overexpression and activation of the corresponding signaling pathway.Analyses of Tet2 and Ezh2 inactivated mice show that these Ezh2 Tet2 mice succumb to bone marrow exhaustion, attributed to long term hematopoietic stem cells (LT-HSC) disappearance. Ezh2 and Tet2 show major roles in LT-HSC maintenance whose molecular, genetic and epigenetic mechanism remains to be investigated

Au cours de ces dernières décennies, l'augmentation de la consommation de glucides, d'acides gras et de cholestérol liée aux changements des habitudes alimentaires dans la plupart des pays industrialisés est à l'origine de nombreuses pathologies telles que l'obésité, les troubles cardiovasculaires, le développement du diabète de type II et la survenue de cancers. Plusieurs arguments bibliographiques suggèrent notamment que le cholestérol puisse être un élément à risque dans la survenue du cancer de la prostate. D'une part, l'hypercholestérolémie est associée à une augmentation des cas de cancer de la prostate et, d'autre part, les cellules cancéreuses présentent des dérèglements du métabolisme des lipides associés à l'accumulation de cholestérol dans les tumeurs solides. Les objectifs de ces travaux ont été d'analyser le rôle du cholestérol dans le développement du cancer de la prostate et d'étudier le rôle des récepteurs nucléaires LXRs (liver X receptors), régulateurs fondamentaux de l'homéostasie du cholestérol, dans les mécanismes associés à l'initiation et à la progression tumorale. Nos résultats montrent qu'une accumulation de cholestérol, induite par un régime chez les souris déficientes en LXRs, peut initier les premières étapes du développement tumoral par des mécanismes épigénétiques mettant en jeu l'action répressive de l'histone méthyltransférase EZH2 sur des gènes suppresseurs de tumeur. En parallèle, l'activation pharmacologique des LXRs dans des cellules cancéreuses humaines réduit la croissance tumorale en augmentant la mort des cellules par des mécanismes faisant intervenir les rafts lipidiques. Au total, nos travaux révèlent l'existence d'une relation entre la consommation excessive de cholestérol et la modification d'empreintes épigénétiques, mécanisme de plus en plus associé aux processus carcinogéniques. Nos données indiquent également que les LXRs, en s'opposant à l'accumulation de cholestérol intracellulaire, ralentissent l'initiation et la progression du cancer de la prostate. Ainsi, toute stratégie thérapeutique visant à diminuer le cholestérol intra-tumoral, telle que l'activation pharmacologique des LXRs, peut être considérée comme une piste thérapeutique dans le cadre du cancer de la prostate.

The Polycomb group protein Ezh2 catalyzes the Histone H3 lysine-27 trimethylation (H3K27me3) within the Polycomb Repressive Complex 2 (PRC2). PRC2 exerts a critical control over the expression of a large set of target genes controlling important biological functions, including cell proliferation, differentiation and stem cell self-renewal. Aberrant Ezh2 function is commonly observed in several cancer types and is due to deregulated enzymatic activity and/or expression of the Polycomb protein. Studies in preclinical models have started to reveal the importance of Ezh2 in B cell lymphomagenesis. In contrast, little is known about the effects of Ezh2 deregulated function/constitutive expression in B cell tumor maintenance and progression. The present study addresses this issue taking advantage of a MYC-driven mouse lymphoma model, featuring high Ezh2 expression as a result of malignant B cell transformation. Conditional, genetic inactivation of Ezh2 methyltransferase activity in aggressive primary Burkitt-like mouse B cell lymphomas led to the identification of two classes of tumors, differentially responding to the loss of Polycomb function. In type-1 lymphomas, Ezh2 inactivation impaired clonal tumor growth starting from single lymphoma cells. Instead, type-2 lymphomas were largely resistant to the loss of Ezh2 catalytic function, giving rise to a substantial number of Ezh2 mutant clones. Transcriptome analyses allowed the identification of a molecular signature discriminating type-1 from type-2 lymphomas, including genes controlling cell cycle progression, DNA replication and cell survival, which were more expressed in type-2 tumors. These results correlated with a more aggressive behavior of type-2 lymphomas when transplantated into immunoproficient hosts. The growth of rare Ezh2 mutant subclones, established from type-1 lymphomas, was impaired by the treatment with an Ezh1/2 small molecule inhibitor, identifying the Ezh2 paralog, Ezh1, as a determinant of resistance of tumor cells to Ezh2 inactivation. Ezh2 inhibition led to genome wide loss of H3K27me3, which was comparable between lymphoma types. However, while the loss of H3K27me3 at target genes in type-1 lymphomas failed to alter their expression, in type-2 lymphomas Ezh2 targets were in most cases deregulated following the loss of the histone mark. Based on these results, we propose that Ezh2 mutant subclones from type-1 lymphomas select an H3K27me3-independent mechanism to ensure correct regulation of Ezh2 target genes, which is needed for tumor growth. We also find that residual H3K27me3 is deposited at the promoter of new genes by a non-canonical PRC2/Ezh1, in Ezh2 mutant subclones from type-1 lymphomas. This activity alters the expression of target genes contributing to tumor growth. We finally report the isolation of clonal variants from type-1 lymphomas that acquire secondary resistance to pharmacological Ezh1/2 inhibition. The latter tumors (together with type-2 lymphomas) will be instrumental to unravel the genetic bases of resistance of MYC-driven lymphomas to PRC2 inhibition. Anti-Ezh2 inhibitors are currently being tested in phase-1 and -2 clinical trials for the treatment of both solid and blood cancers including B cell lymphomas. Our studies highlight the importance of understanding in more detail the mechanisms of action of Ezh2/PRC2 in tumors, in order to identify those that may benefit from anti-Ezh2 therapies. Our results also provide evidence for mechanisms of lymphoma resistance to Ezh2 inhibition and suggest strategies to circumvent such resistance.

The developing brain is highly sensitive to environmental influences. Unfavorable nutrition is one kind of stress that can cause acute metabolic disorders during the neonatal period [1,2,3] and severe diseases in later life [4,5]. These early life experiences occurring during heightened periods of brain plasticity help determine the lifelong structural and functional aspects of brain and behavior. In humans, for example, weight gain during the first week of life increased the propensity for developing obesity several decades later [5]. This susceptibility is, if not all, related to the dynamic reversible epigenetic imprints left on the histones [6,7,8], especially during the prenatal and postpartum period [9]. Histones are highly dynamic and responsive towards environmental stress [10,11]. Through covalent modification of the histone tail, histones are able to direct DNA scaffolding and regulate gene expression [10,12]. Thus far, various types of post translational modifications have been identified on various histones tails [12]. Among them, the methylation and acetylation on lysine residue (K) 27 on histone 3 (H3) has been tightly linked to gene repression [13,14] and activation [15], respectively. EZh2 (enhancer of zeste 2) in the polycomb repressive complex 2 (PRC2) is the only methyltransferase that has been linked to catalyze this methylation reaction. In addition, SUZ (suppressor of zeste) and EED (embryonic ectoderm development) are two other key proteins in PRC2 function core that help EZH2. As previous reported, increased H3K27 methylation was monitored after fasting stress during neonatal period in chicks' paraventricular nucleus (PVN). In this study, we investigated the detailed mechanism behind changes in H3K27 methylation following fasting stress. After 24 hours fasting on 3 days-of-age (D3), chicks exhibited elevated mRNA levels of PRC2 key components, including EZH2, SUZ and EED, in the PVN on D4. Western blots confirmed this finding by showing increased global methylation status at the H3K27 site in the PVN on D4. In addition, until 38 days post fasting, SUZ and EZH2 remained inhibited. A newly identified anorexigenic factor, Brain-derived neurotrophic factor (BDNF), was used as an example of multiple hormones expressed in PVN to verify this finding. Both BDNF protein and mRNA exhibited compatible changes to global changes of tri- (me3) and di-methylated (me2) H327. Furthermore, by using chromatin immunoprecipitation assays (ChIP), we were able to monitor the changes of H3K27me2/me3 deposition along the Bdnf gene. Fasting significantly increased H3K27me2/me3 as well as EZH2 at the Bdnf's promoter, transcription start site and 3'-untranslated region. These data show that fasting stress during the early life period could leave epigenetic imprinting in PVN for a long time. Next, we tried to understand the function of this epigenetic imprinting in the chicks' PVN. Thus, we compared naive chicks (never fasted) to chicks that received either a single 24 hour fast on D3 or two 24 hour fast on both D3 and 10 days-of-age (D10). We found that the D3 fasted group significantly increased the level of PRC2 key components and its product H3K27me2/me3 compared to the naive group. However, D3 fasting and D10 fasting together decreased the surges of H3K27me2/me3, SUZ and EED (not EZH2) compared to the naive group. We called this phenomenon "epigenetic memory". The Western blot, qPCR and CHIP assay results from BDNF all confirmed the existence of "epigenetic memory" for PRC2. These data suggested that fasting stress during the early period of brain development could leave long term epigenetic modifications in neurons. These changes could be beneficial to the body, which keeps homeostasis of inner environment and prevent massive response to future same stress. The EZH2 protein was knocked down and the H3K27 methylation status changes were monitored after applying the same treatment. We first confirmed that EZH2 antisense oligonucleotides (5.5 ug), but not EZH2 siRNA and artificial cerebrospinal fluid (ACSF), inhibit EZH2 protein by 86 % in the PVN. Then, on D3, chicks were subjected to a 24 hour fasting stress (D3-fasting) post either EZH2 antisense or ACSF injection. The EZH2 antisense blocked the surge of both EZH2 mRNA and H3K27 methylation after D3-fasting. At the same time, BDNF exhibited elevated expression levels and less methylated H3K27 deposition along the Bdnf gene. In addition, we were also interested in the changes of "epigenetic memory" post EZH2 antisense injection. We found that after EZH2 antisense injection, chicks' PVN no longer exhibited any "epigenetic memory" to repetitive fasting stress. While EZH2 mRNA was constantly inhibited, SUZ, EED and H3K27me2/3 levels were unpredictable. These findings suggested that neurons in the PVN utilized PRC2 as a major H3K27 methylation tool. Knockdown of EZH2 in the PRC2 impaired the proper response in PVN to fasting stress and PVN's ability to acclimate to repetitive fasting stresses. Thus, EZH2 is an important H3K27 methyltransferase inside chicken hypothalamus to maintain homeostasis. In conclusion, fasting stress during the early life period could leave epigenetic markers on chromosomes of neurons in the feeding regulation center. These epigenetic markers will be left on chromosomes for a long period of time and have a beneficial role in keeping homeostasis when individuals face future fasting stress again. H3K27 methylation is one of these epigenetic markers and inhibits expression of various genes inside neurons. EZH2 is so far the only detected methyltransferases for H3K27 that form the PRC2. Thus EZH2 plays a key function in the body's response to fasting.
Ph. D.

Environmental factors in within tumours are known to affect cancer progression and behaviour. In solid tumours, hypoxia (a low-oxygenated state) is a common condition associated with a more aggressive disease and with an increased risk of metastasis. This project focused on evaluating the potential of blocking an epigenetic enzyme, G9a, in breast cancer. The presented work demonstrates that G9a is crucial for the hypoxic response in cancer and that its inhibition is effective in reducing cancer cell survival and blocking metastatic formation. Overall, the described work represents a proof-of-concept demonstrating that G9a is a promising therapeutic target in breast cancer.

Les complexes PRC1 et PRC2 contrôlent l’expression génique via l’organisation de la structure de la chromatine. Ce contrôle se fait par l’ajout de la marque H2AK119ub1 par le PRC1 et l’ajout de la marque H3K27me3 par le PRC2. Cette étude s’attache à étudier le rôle de la protéine Pcgf1 (membre du complexe PRC1) et de la protéine Ezh2 (membre du complexe PRC2) lors du développement du poisson-zèbre. Les gènes sont inactivés par TALEN. Le complexe PRC1 est formé par différentes protéines dont les Pcgf. Il existe de nombreux homologues Pcgf qui ont des fonctions distinctes. Cette étude s’intéresse au rôle de la protéine Pcgf1 lors du développement du poisson-zèbre. Les individus pcgf1-/- sont viables et fertiles. Cependant, leur développement précoce est retardé et les adultes montrent des signes de vieillissement accéléré. Ce mutant est le premier modèle de vertébré qui met en évidence le rôle de Pcgf1 dans la prolifération cellulaire lors du développement et son association au vieillissement. La protéine Ezh2 est impliquée dans le devenir cellulaire et la différenciation. Les embryons se développent normalement puis les larves meurent à 12 jours post-fécondation. De façon intéressante, les embryons de poisson-zèbre peuvent gastruler en l’absence d’Ezh2, contrairement au modèle murin. Les organes sont correctement mis en place à 5 jours post-fécondation. Les larves présentent un défaut de maintien de la paroi du bulbe intestinal. La protéine Ezh2 est importante pour le maintien du pancréas exocrine. L’absence d’Ezh2 cause une augmentation importante du nombre de cellules apoptotiques. Ezh2 est essentiel lors de la régénération de la nageoire caudale
PCR1 and PRC2 are complexes that control gene expression via chromatin structure reorganization. This expression regulation is maintained by adding epigentics marks H2AK119ub1 by the PRC1 and adding of H3K27me3 by the PRC2. The study devotes to study the role of the protein Pcgf1 (part of the PRC1 complex) and of the Ezh2 protein (part of the PRC2 complex) during the zebrafish development. The PRC1 complex is formed by different proteins including Pcgf proteins. There are several Pcgf homologs that have different functions. The study reveals that some Pcgf proteins have a different expression during caudal fin regeneration and development. We are interested in Pcgf1 protein during the zebrafish development. The pcgf1 gene was inactivated by using TALEN. The fish pcgf1-/- are viable and fertile. However, the early development is delayed and adults show signs of accelerated aging. This mutant is the first vertebrate model showing the role of Pcgf1 in cells proliferation during development and aging. Ezh2 protein is involved in cell-fate decisions and differenciation. Inactivation of ezh2 gene by TALEN reveals the essential role of Ezh2 during development. Indeed, at the beginning embryos develop normally then larvae die at 12 days post-fertilization. Interestingly, zebrafish embryo can gastrulate without Ezh2. This contradicts with observations in mouse model. The organs are properly formed at 5 days postfertilization. Larvae show defects in the intestinal bulb wall. Ezh2 is important for exocrine pancreas maintenance. The absence of Ezh2 causes an increase in apoptic cells. Ezh2 is essential during caudale fin regeneration

Although prostate cancer causes morbidity and mortality many men have indolent disease with few health consequences. There is an urgent need to identify which patients with prostate cancer require clinical intervention, thereby preventing both under- and overtreatment. The Polyeomb Group (PeG) gene EZH2 is up-regulated in hormone-refractory metastatic prostate cancer, suggesting that EZH2 promotes progression to an advanced tumour phenotype. EZH2 is a transcriptional repressor during normal embryonic development by virtue of its histone methyltransferase activity. At the start of this work the role ofEZH2 during tumour progression was unclear. This thesis identifies EZH2 as a dual-function promoter of prostate cancer progression. EZH2 function promotes proliferation of both androgen-responsive and androgen-independent prostate cancer cells, suggesting that aberrant EZH2 function may occur earlier than originally thought during prostate cancer progression. It was discovered that EZH2 function promotes prostate cancer cellular invasiveness, demonstrating a mechanism which can account for the association between increased EZH2 expression and advanced disease. It was observed that androgen-induced expression of the TMPRSS2:ERG gene fusion in prostate cancer cells was associated with a trend towards increased expression of HDAC1 and the PeG genes EZH2 and SUZ12. It was also observed that EZH2 regulates actin polymerisation in prostate cancer cells, which may account for the abrogation of cellular invasiveness observed following EZH2 knock-down. Given that EZH2 is a transcriptional repressor, a search for candidate EZH2 target genes was performed. EZH2 may promote transcriptional repression ofp21 and MMP7. . Effects of overexpression of EZH2 and a dominant gain-of-function allele, EZH2R732K, were investigated in prostate cancer cells. No discernible phenotype was observed, suggesting additional factors are required to promote the progression to an aggressive prostate cancer phenotype. The results presented in this thesis suggest mechanisms that can account for the association between increased EZH2 expression and advanced prostate cancer.

La sénescence induite par chimiothérapie permet l’arrêt pérenne de la division des cellules tumorales. Néanmoins, ce mécanisme de suppression tumorale peut être neutralisé par certaines cellules traitées, ce qui se traduit généralement par une rechute des patients. Récemment, nous avons décrit dans des cellules colorectales un mécanisme d’échappement à la sénescence induite par le SN-38 dépendant de la protéine de survie MCL1. Cette étude montre que les cellules sénescentes (PLS) favorisent la prolifération des cellules non-sénescentes (PLD) par l’intermédiaire de signaux mitogéniques activant la kinase Cdk4 et par conséquent la reprise de la division. Nous démontrons que Cdk4 inhibe Rb par phosphorylation de la sérine 780, permettant l’activation des fonctions transcriptionnelles des facteurs E2F sur les gènes du cycle cellulaire. La perte d’activité de Cdk4 par ARN interférence ou par le Palbociclib réduit l’émergence de clones proliférants. La méthyltransférase EZH2 est une cible de E2F exclusivement exprimée par les PLD et son expression dépend de l’activité de Cdk4. Par ailleurs, l’utilisation d’ARN interférence dirigé contre EZH2 ou des inhibiteurs chimiques DNZepA et GSK343 réduit également l’émergence de clones proliférants. Enfin, son inhibition potentialise à la fois l’arrêt de la division et la sénescence en réponse au Palbociclib dans les cellules ayant échappé au SN-38. Ainsi, ces travaux ont permis de mettre en évidence un rôle important de EZH2 en tant qu’effecteur de Cdk4 dans le mécanisme d’échappement au SN-38, une voie susceptible d’apporter des nouvelles cibles thérapeutiques dans le traitement du cancer
Chemotherapy-induced senescence enables to trigger a durable division arrest of tumor cells. However, this tumor suppressor mechanism is neutralized in some treated cells leading mostly to cancer relapse in patients. Recently, we have described a MCL1-dependent mechanism of escape in SN-38-induced senescence from colorectal cell lines. In this study, we showed that senescent cells (PLS cells) promoted the non senescent cells (PLD cells) proliferation through mitogenic signals stimulating Cdk4 kinase activity and subsequently the cell cycle. We demonstrated that Cdk4 phosphorylated Rb on the serine 780 to inhibit its activity, allowing E2F- family transcriptional functions activation on cell cycle targets. Loss of Cdk4 expression or activity induced by RNA interference or Palbociclib reduced the emergence of proliferating clones. TheEZH2-methyltransferase, a E2F transcriptional target, is only expressed by PLD cells and this expression depends on Cdk4 activity. Moreover, loss of EZH2 expression or activity, by RNA interference or by DZNepA and GSK343 inhibitors, reduced the emergence of proliferating cells. Finally, EZH2 inhibition promotes both cell division arrest and senescence in response to Palbociclibin the SN-38-escaped cells. To conclude, this study enabled to highlight a major role of EZH2 as effector of Cdk4 in the escape mechanism induced by SN-38 a signaling pathway offering newtargeted cancer therapies

Ca2+/Calmodulin-dependent protein kinase type II (CaMK-II) is a Serine/Threonine protein kinase that is activated by Ca2+ and Calmodulin to phosphorylate substrates involved in myriad developmental processes. This project implicates CaMK-II in specification of HSCs, and zebrafish provide an ideal embryonic model to study hematopoiesis. Zebrafish genetic manipulation was achieved through: incubation in chemical inhibitors; injection of notochord-targed WT and DN CaMK-II constructs with Transposase; and injection of camk2g1 translation-blocking morpholino antisense oligonucleotide (MO). Whole-mount in situ hybridization (WISH) and immunolocalization on zebrafish embryos allowed visualization of key HSC markers and pathway components that implicated CaMK-II in the specification of HSCs. CaMK-II is a negative regulator of shh expression during HSC specification, but CaMK-II does not influence Shh during its well-documented role in vasculogenesis. CaMK-II appears to affect the spatial distribution of Shh protein, which accumulates near the notochord source and differentially affects expression of Shh target genes based on their distance from the notochord. This project also identifies the specific timing requirement for CaMK-II during HSC specification, as inhibition of CaMK-II consistently reduces HSC specification, but only if administered before 18hpf. CaMK-II also downregulates ezh2 in the DA during the time of HSC specification, and the Ezh2 inhibition rescues the loss of HSCs, suggesting that CaMK-II regulates the secretion of Shh from the notochord to epigenetically regulate expression of key HSC specification genes in the DA through EZH2 methyltransferase.

Gliomas represent 80% of the central nervous system tumors. Historically, they have been classified by the World Health Organization according to their supposed cell of origin and to their malignancy grade. Among gliomas, astrocytomas are the most common and are classified histologically with a four grade system where grade IV corresponds to the most malignant form, also known as glioblastoma multiforme (GBM). Furthermore, in recent years a molecular characterization has been defined for GBM. Different subtypes have been identified, each of them over expressing a specific group of genes. For decades, it has been assumed that cancer was caused only by genetic alterations. Now the view is changed and cancer is considered both a genetic and epigenetic disease. It was demonstrated indeed that epigenetic silencing of genes through DNA or histone methylation at their promoters can be an alternative way to achieve their loss of function; in addition, DNA demethylation of constitutive heterochromatin can promote genome instability. In this context, we refer to epigenetics as the sum of heritable changes in phenotype and/or gene expression without altering the primary DNA sequence. Methylation of lysine 27 on histone H3 is a post-translational modification that is mediated by the histone methyltransferase complex known as Polycomb Repressive Complex 2 (PRC2) through its active subunit, Enhancer of Zeste Homolog 2 (EZH2). This facultative heterochromatin mark promotes the recruitment of Polycomb Group (PcG) proteins to achieve gene silencing. PcG proteins have been shown to play a major role in embryonic development and adult somatic cell differentiation. Initial studies on embryonic stem cells (ESCs) showed that Polycomb Complexes are required to maintain stem cell identity. However further investigation showed that this process is much more elaborated and the current model proposes that PcG proteins function dynamically during development and differentiation to lock off the expression of alternative cell fate regulators in any particular lineage. Moreover EZH2 and other Polycomb members have been found to be dysregulated in a variety of cancer types. EZH2 is overexpressed in tumors like prostate, breast and bladder, and BMI1, a member of Polycomb Repressive Complex 1 (PRC1), is over expressed in GBM, causing aberrant expression of neural stem cell (NSC) markers and preventing apoptosis. It was also demonstrated that genes that are directly regulated by Polycomb in ESCs are up to 12-fold more likely to have cancer specific DNA hypermethylation at their promoters than other genes. In my project I have pursued two main questions. First, I wanted to assess whether EZH2 is required for glioma initiation. Moreover, since tumor-initiating cells have been found also in GBM, I wanted to check if EZH2 is required to maintain this stem cell pool. In order to address those questions, I chose a well established animal model of GBM which relies on the loss of Ink4a/Arf together with the over expression of EGFRvIII. A conditional knockout allele for Ezh2 was introduced in order to remove this protein at different stages of the disease. With this work I could demonstrate that while EZH2 is required for the establishment of the tumor, it can be dispensable for its maintenance. The Polycomb axis acts early on during tumor formation, causing the relocation of H3K27me3 in an instructive manner and this process seems to be fundamental in order to achieve the full transformation of the cells. I was able to show that EZH2 is dispensable for glioma maintenance, pointing to a unique window of Polycomb sensitivity that characterizes the primary phase of gliomagenesis prior to the establishment of the glioma propagating cell (GPC) compartment that is able to reconstitute tumors.

Les infections virales sont responsables de 15 à 20 % des cancers humains. L étude des mécanismes moléculaires avec lesquels les virus oncogènes induisent la transformation cellulaire est essentielle pour la compréhension des cancers qui en résultent. Cela permettra également la découverte de nouveaux mécanismes pouvant être impliqués dans le développement de cancers, qui peuvent être ciblés par des approches thérapeutiques. Les virus du papillome humain (HPV) sont des petit virus à ADN qui futs isolés de la peau de patients souffrants de Epidermodysplasia Verruciformis (EV) qui cause un risque élevé d'infection par les HPV et le développement de cancer de la peau non mélanique (NMSC). Certains HPV cutanés, tels que HPV5, 8 et 38, sont suspectés de jouer un rôle dans de développement du cancer de la peau. Cependant, le lien direct entre les HPV cutanés et l'étiologie du cancer n'est pas encore clairement établi. Des études de notre laboratoire ont montré que les oncoprotéines HPV38 E6 et E7 sont capables d'immortaliser des kératinocytes primaires humains in vitro et in vivo. Pour immortaliser des cellules, d'importantes voies de signalisations, telles que les voies de p53 et celle de NF-KB, doivent être affectées. Dans cette étude, nous avons cherché à mettre en évidence les mécanismes moléculaires menant à la dérégulation de p53 et de NF-KB par E6 et E7 de HPV38, dans des kératinocytes humains. Nous avons montré que HPV38 E6 et E7 induisent la formation d'un complexe protéique incluant IKKβ, ΔNp73α, EZH2 et DNMT1. La formation de ce groupement protéique corrèle avec l'inhibition de la transcription de certains gènes cibles de p53, tel que PIG3. Nous avons également mis en évidence l'activation de la voie NF-KB par les oncoprotéins E6 et E7 de HPV38. Cette activation est importante par le rôle joué par NF-KB dans la protection des cellules de l apoptose induite par TNF-α et par l'exposition aux rayonnements UVB. De plus nous avons observé que E7 est la principale oncoprotéine de HPV38 responsable de la dérégulation des voies p53 et NF-KB. Nos études mettent en évidence de nouveaux mécanismes moléculaires qui peuvent être essentiels dans le processus de transformation cellulaire par HPV38
Viral infections contribute to 15–20% of all human cancers. Studying the mechanisms employed by the oncogenic viruses to induce cellular transformation is essential for a better understanding of the resulting cancers and the discovery of new mechanisms involved in cancer development which can be targeted in therapeutic approaches. Human papillomaviruses (HPVs) are small dsDNA viruses which have been clearly associated with certain cancers. They were first isolated from the skin of patients suffering from Epidermodysplasia Verruciformis (EV) having an increased susceptibility to infection by specific HPV types and to the development of non-melanoma skin cancer (NMSC). Certain cutaneous HPV types, such as 5, 8, and 38, are suspected to play a role in skin cancer development. However the direct role of cutaneous HPV in the etiology of cancer is still under debate. Previous studies from our laboratory have reported that HPV38 E6 and E7 proteins are able to immortalize human primary keratinocytes in vitro and in vivo. Cellular immortalization can be achieved through the deregulation of important signaling pathways including p53 and NF-KB. In the present work, we have investigated the molecular mechanisms of p53 and NF-KB pathways deregulation by E6 and E7 oncoproteins from HPV38 in human keratinocytes. We show here that HPV38 E6E7 induce the formation of a transcription repressor complex including IKKβ, ΔNp73α, and polycomb group members EZH2 and DNMT1. The formation of this protein complex correlates with the inhibition of several p53-target genes, such as PIG3. We also report in these studies that HPV38 E6E7 activate NF KB pathway, which plays an important role in the survival of HPV38 E6E7-immortalized human keratinocytes upon TNF-α– and UVB-mediated apoptosis. In addition our data highlight E7 being the main HPV38 protein mediating p53 and NF-KB deregulation. Our studies shed light on novel molecular mechanisms that could be important for HPV38-mediated cellular transformation

Si de nombreuses études soutiennent l’existence d’une relation étroite entre les désordres nutritionnels, les modifications épigénétiques et l’étiologie du cancer colorectal (CCR), les mécanismes sous-jacents restent à éclaircir. Les gènes suppresseurs de tumeurs de la famille UNC5H (UNC5A, B, C et D) qui codent des récepteurs membranaires contrôlant la balance survie/apoptose font partie des gènes fréquemment réprimés au cours de la carcinogenèse colique par des mécanismes épigénétiques encore peu compris. Dans le modèle murin de carcinogenèse colique AOM/DSS, nous avons montré que l’expression d’UNC5A, UNC5B et UNC5C était diminuée dans les tumeurs mais exclusivement chez les souris soumises à un régime riche en sucres (HCD) durant toute la durée de l’expérience, reliant ainsi la nutrition à leur perte d’expression dans le CCR. La O-GlcNAcylation est une modification post-traductionnelle ciblant des milliers de protéines nucléocytoplasmiques et mitochondriales intervenant dans divers processus cellulaires fondamentaux parmi lesquels la régulation épigénétique de l’expression génique et dont les niveaux sont augmentés au cours de la carcinogenèse colique. Les niveaux de O-GlcNAcylation dépendent étroitement du nucléotide sucre donneur de la réaction, l’UDP-GlcNAc, qui lui-même est au carrefour de plusieurs métabolismes définissant cette glycosylation comme un senseur nutritionnel. Dans ce contexte, nous avons émis l’hypothèse selon laquelle la O-GlcNAcylation puisse représenter un des relais moléculaires entre la nutrition et la répression des gènes de la famille UNC5H au cours de la carcinogenèse colique. Dans des cellules cancéreuses coliques humaines, par une combinaison d'approches incluant inhibitions pharmacologiques et interférence à l’ARN couplées à des analyses en RT-qPCR et à des tests d’activité promotrice, nous avons montré l’action conjointe de la O-GlcNAcylation et d’EZH2 (la sous-unité catalytique du complexe PRC2 responsable du dépôt de la marque épigénétique répressive H3K27Me3) dans la régulation de l’expression d’UNC5A. Plus précisément, des expériences de CUT&RUN nous ont permis de prouver que la O-GlcNAcylation d’EZH2 permet son recrutement sur le promoteur d’UNC5A afin de réprimer sa transcription. L’ensemble de nos résultats soutiennent donc l'hypothèse selon laquelle la O-GlcNAcylation pourrait représenter une nouvelle connexion entre la nutrition et la régulation épigénétique de gènes suppresseurs de tumeurs clés régissant la cancérisation de la muqueuse colique
Although many studies support a close relationship between nutritional disorders, epigenetic changes and the etiology of colorectal cancer (CRC), the underlying mechanisms remain to be elucidated. The UNC5H tumor suppressor genes (UNC5A, B, C and D) that code for membrane receptors controlling the survival/apoptosis balance are among the genes frequently repressed during colonic carcinogenesis by epigenetic mechanisms that are still poorly understood. In the AOM/DSS mouse model of colonic carcinogenesis, we showed that UNC5A, UNC5B and UNC5C expression was decreased in tumors but exclusively in mice subjected to a High Carbohydrate Diet (HCD) during all the time course of the experiment, thus linking nutrition to their repression in CRC. O-GlcNAcylation is a post-translational modification targeting thousands of nucleocytoplasmic and mitochondrial proteins involved in various fundamental cellular processes including epigenetic regulation of gene expression and whose levels are increased during colonic carcinogenesis. O-GlcNAcylation levels depend of UDP-GlcNAc, the sugar nucleotide donor of the reaction, which itself is at the crossroad of several metabolisms, thus defining this glycosylation as a nutritional sensor. In this context, we hypothesized that O-GlcNAcylation could be one of the molecular relays between nutrition and UNC5H genes repression during colonic carcinogenesis. In human colon cancer cells, by using a combination of pharmacological inhibitions and siRNA approaches coupled to RT-qPCR analyses and promoter activities studies, we showed that O-GlcNAcylation and EZH2 (the catalytic subunit of the PRC2 complex responsible for the deposition of the epigenetic repressive mark H3K27Me3) act jointly to repress UNC5A expression. More precisely, by CUT&RUN experiments, we demonstrated that O-GlcNAcylation of EZH2 allows its recruitment onto the UNC5A promoter to repress its transcription. To conclude, all these results confirm the hypothesis that O-GlcNAcylation could be a new connection between nutrition and epigenetic regulation of tumor suppressor genes governing the cancerization of the colonic mucosa

EZH2 is a SET domain-containing methyltransferase and the catalytic component of the multimeric Polycomb- group (PcG) protein complex, PRC2. When in complex with other PRC2 members (EED, SUZ12, AEBP2, and RBBP4), EZH2 catalyzes methylation of H3K27, a histone modification associated with transcriptional repression and developmental regulation. As several PRC2 components are upregulated or mutated in a variety of human cancers, efforts to discover small-molecule modulators of PRC2 and understand its regulation may yield therapeutic insights. Identification of small-molecule probes with distinct chemotypes, MOAs, and selectivity profiles are not only of great value, but necessary in establishing comprehensive probe sets capable of illuminating the various roles of EZH2 in oncogenesis. Here we describe efforts to identify and characterize small-molecule modulators of PRC2 and further understand its regulation. Chapter II outlines the expression and purification of 5-component PRC2 (EZH2-EED-SUZ12-AEBP2-RBBP4) and the establishment of biochemical and cellular HTS assays. These assays were used to screen a diverse set of small molecules (>120,000), identifying biochemical PRC2 inhibitors and activators (described in Chapter III). One biochemical PRC2 inhibitor, BRD1835, appeared to inhibit PRC2 activity through a novel artifactual mechanism involving interaction with peptide substrate, leading to apparent peptide-competitive behavior and putative cellular activity (described in Chapter IV). The characterization of novel biochemical PRC2 activators, BRD3934 and BRD8284, is discussed in Chapter V. Chapter VI describes the use of an HCS assay to identify known bioactive compounds that alter intracellular levels of H3K27me3 through modulating H3K27me3-connected regulatory nodes or by targeting PRC2 directly. These efforts led to the discovery that an antifungal agent, miconazole, is capable of activating PRC2 activity in vitro, while a mucolytic agent, bromhexine, selectively ablates cellular H3K27me3 levels through targeting an activity distinct from PRC2. Finally, Chapter VII discusses novel PRC2-connected crosstalk mechanisms identified through screening libraries of uniquely modified histone peptides for their ability to bind or support methylation by PRC2. These studies enhance our understanding of PRC2 regulation by revealing the effects of H3R26 and H3K23me1 modifications on enzymatic activity, implicating their respective methyltransferases in PRC2 regulation.

Les tumeurs cortico-surrénaliennes bénignes et malignes sont associées à une morbidité élevée résultant de l'hypersécrétion des hormones cortico-surrénaliennes, retrouvée chez près de 60% des patients. Au delà des perturbations endocrines, les carcinomes cortico-surrénaliens (CCS) sont des tumeurs de mauvais pronostic avec 16 à 38% de survie à 5 ans. Cette agressivité résulte à la fois de la présence de métastases chez de nombreux patients, au moment du diagnostic (30 à 40% des cas) et de l'absence d'approches thérapeutiques, au delà de la résection chirurgicale de la tumeur primaire. Au début de ma thèse, les mécanismes moléculaires impliqués dans le développement des tumeurs bénignes et malignes de la cortico-surrénale, étaient largement méconnus. L'activation anormale de la voie de signalisation Wnt/ßcaténine dans 48% des tumeurs bénignes et 37% des tumeurs malignes, suggérait que cette voie pouvait, comme dans d'autres tissus, participer au développement tumoral dans la cortico-surrénale. Afin de confirmer cette hypothèse, nous avons développé et caractérisé un modèle de souris transgéniques dans lesquelles la ßcaténine est constitutivement activée, spécifiquement dans le cortex surrénalien (souris ∆Cat). Grâce à ces souris, nous avons démontré pour la première fois que la ßcaténine agit comme un oncogène dans la cortico-surrénale, mais que son activation constitutive ne suffit pas à déclencher systématiquement le développement de tumeurs malignes. Chez plus de 90% des patients, la formation des CCS est associée à la surexpression du facteur de croissance IGF2. Grâce à des modèles de souris transgéniques qui surexpriment Igf2, nous avons pu montrer que cette surexpression n'a que peu d'effet sur l'initiation ou la progression tumorale, suggérant que d'autres altérations sont requises pour favoriser la transition maligne. Des résultats préliminaires encourageants suggèrent que la surexpression de l'histone méthyl-transférase EZH2 et les altérations épigénétiques résultantes, pourraient être la clé du développement des CCS. Parallèlement, nous avons montré que l'activation constitutive de la ßcaténine conduit au développement d'un hyperaldostéronisme primaire chez les souris ∆Cat, suggérant que l'activation de la voie Wnt/ßcaténine pourrait participer à la formation d'adénomes surrénaliens producteurs d'aldostérone (APA) chez les patients. Effectivement, nous avons mis en évidence que l'activation constitutive de la ßcaténine est l'altération moléculaire la plus fréquente dans les APA, avec une prévalence de 68%. Des analyses in vitro m'ont permis de montrer que la ßcaténine stimule la production d'aldostérone en contrôlant directement et indirectement l'expression de deux enzymes clés de la synthèse d'aldostérone - CYP21 et CYP11B2 - et du récepteur à l'angiotensine II (le sécrétagogue naturel de l'aldostérone), AT1R. Nous avons par ailleurs montré que la production excessive d'aldostérone chez les souris ∆Cat, pouvait être maîtrisée par un régime enrichi en quercétine, un inhibiteur naturel de l'activité transcriptionnelle de la ßcaténine. L'ensemble de ces résultats démontre l'importance de la voie Wnt/ßcaténine dans la tumorigenèse surrénalienne et dans l'hypersécrétion d'aldostérone ce qui fait d'elle une nouvelle cible thérapeutique potentielle.

Sabe-se que TRAIL é capaz de matar células tumorais de forma seletiva e que TRAIL tem sua expressão reduzida em diversos tumores, porém pouco se sabe sobre os mecanismos responsáveis pela sua inibição. Tendo em vista que a expressão de TRAIL pode ser regulada pelo Ácido Retinóico; que PRAME é capaz de inibir a via do ácido retinóico através da proteína EZH2 e que nós observamos anteriormente que a expressão de TRAIL esta diminuída em pacientes com LMC, nós decidimos investigar a associação entre PRAME, EZH2 e TRAIL na LMC. Nós demonstramos que PRAME, mas não EZH2, tem sua expressão aumentada em células BCR-ABL+ e sua expressão está associada com a progressão da LMC. Alem disto, existe uma correlação positiva entre PRAME e BCR-ABL e negativa entre PRAME e TRAIL nestes pacientes. A inibição da expressão de PRAME ou EZH2 por RNAi induziu um aumento da expressão de TRAIL. Estes dados revelam um novo mecanismo de regulação responsável por diminuir a expressão de TRAIL, e geram novos possíveis alvos para a terapia da LMC e, possivelmente, também para outros tumores.
TRAIL was shown to selectively kill tumor cells. Not surprisingly, TRAIL is down-regulated in a variety of tumor cells, but the mechanism responsible for TRAIL inhibition remains elusive. Because TRAIL can be regulate by retinoic acid; PRAME was shown to inhibit transcription of retinoic acid receptor target genes through the polycomb protein EZH2; and we have found that TRAIL is inversely correlated with BCR-ABL in CML patients, we decided to investigate the association of PRAME, EZH2 and TRAIL in BCR-ABL-positive leukemia. Here, we demonstrate that PRAME, but not EZH2, is up-regulated in BCR-ABL cells and is associated with the progression of disease in CML patients. In addition, PRAME expression is positively correlated with BCR-ABL and negatively with TRAIL in these patients. Importantly, knocking down of PRAME or EZH2 by RNA interference restores TRAIL expression. Our data reveal a novel regulatory mechanism responsible for lowering TRAIL expression and provide the basis of alternative targets for combined therapeutic strategies for CML.

Multiple myeloma (MM) is a hematological malignancy of antibody producing plasmablasts/plasma cells. MM is characterized by extensive genetic and clonal heterogeneity, which have hampered the attempts to identify a common underlying mechanism for disease establishment and development of appropriate treatment regimes. This thesis is focused on understanding the role of epigenetic regulation of gene expression mediated by the polycomb repressive complexes 1 and 2 (PRC1 and 2) in MM and their impact on disease biology and therapy. In paper I the genome-wide distribution of two histone methylation marks; H3K27me3 and H3K4me3 were studied in plasma cells isolated from newly diagnosed MM patients or age-matched normal donors. We were able to define targets of H3K27me3, H3K4me3 and bivalent (carry both marks) which are, when compared to normal individuals, unique to MM patients. The presence of H3K27me3 correlated with silencing of MM unique H3K27me3 targets in MM patients at advanced stages of the disease. Notably, the expression pattern of H3K27me3-marked genes correlated with poor patient survival. We also showed that inhibition of the PRC2 enzymatic subunit EZH2 using highly selective inhibitors (GSK343 and UNC1999) demonstrated anti-myeloma activity using relevant in vitro models of MM. These data suggest an important role for gene repression mediated by PRC2 in MM, and highlights the PRC2 component EZH2 as a potential therapeutic target in MM. In paper II we further explored the therapeutic potential of UNC1999, a highly selective inhibitor of EZH2 in MM. We showed that EZH2 inhibition by UNC1999 downregulated important MM oncogenes; IRF-4, XBP-1, BLIMP-1and c-MYC. These oncogenes have been previously shown to be crucial for disease establishment, growth and progression. We found that EZH2 inhibition reactivated the expression of microRNAs genes previously found to be underexpressed in MM and which possess potential tumor suppressor functions. Among the reactivated microRNAs we identified miR-125a-3p and miR-320c as predicted negative regulators of the MM-associated oncogenes. Notably, we defined miR-125a-3p and miR-320c as targets of EZH2 and H3K27me3 in MM cell lines and patients samples.  These findings described for the first time PRC2/EZH2/H3K27me3 as regulators of microRNA with tumor suppressor functions in MM. This further strengthens the oncogenic features of EZH2 and its potential as a therapeutic target in MM. In paper III we evaluated the therapeutic potential of targeting PRC1 in MM using the recently developed chemical PTC-209; an inhibitor targeting the BMI-1 subunit of PRC1. Using MM cell lines and primary cells isolated from newly diagnosed or relapsed MM patients, we found that PTC-209 has a potent anti-MM activity. We showed, for the first time in MM, that PTC-209 anti-MM effects were mediated by on-target effects i.e. downregulation of BMI-1 protein and the associated repressive histone mark H2AK119ub, but that other subunits of the PRC1 complex were not affected. We showed that PTC-209 reduced MM cell viability via significant induction of apoptosis. More importantly, we demonstrated that PTC-209 shows synergistic anti-MM activity with other epigenetic inhibitors targeting EZH2 (UNC1999) and BET-bromodomains (JQ1). This work highlights the potential use of BMI-1 and PRC1 as potential therapeutic targets in MM alone or in combination with other anti-MM agents including epigenetic inhibitors.

Prostate cancer (PCa) is the most common cancer in American men. Although initial androgen deprivation therapy (ADT) confers a five year survival rate of 99%, the relapse of metastatic and drug resistant PCa (CRPC- Castration-Resistant PCa) continues to account for most deaths. How certain PCa cells develop into CRPC is the key question in the field. In addressing it, attention has focused on epigenetic factors that contribute to CRPC development. Herein we investigated the role and regulation of histone demethylase PHF8 during PCa neuroendocrine differentiation (NED) and progression into CRPC. We utilized bioinformatic analyses and biochemical approaches in PCa/CRPC cell line and mouse models to unravel the following results: First, we discovered that PHF8 post-transcriptionally clusters with cell cycle genes during NED and into CRPC via an AR/MYC/miR-22 regulatory axis. We showed that this axis is dysregulated in CRPC cells to allow enhanced cell proliferation and resistance to the clinical AR antagonist drug Xtandi® (enzalutamide). Second, we revealed that PHF8 is necessary for hypoxia induced NED by demethylating repressive H3K9me2 and H3K27me2, above maintaining active H3K4me3 on select NED genes. Importantly, we unveiled that PHF8 sustains HIF1α expression in CRPC cells via a regulatory role associated with full length AR. Third, we recapitulated the role of PHF8 in vivo by excising its floxed allele in the prostate of TRAMP mice -Transgenic Adenocarcinoma of the Mouse Prostate. We observed that KO of Phf8 lowered tumor burden in part by sustaining Ezh2 expression during NED transition into CRPC. In conclusion, our data implicates PHF8 in multiple oncogenic roles and regulations during PCa NED into CRPC. Our results lay a foundation for understanding the dynamics of histone modifying enzymes during PCa progression and hint at designing small molecule inhibitors against PHF8 as a novel CRPC therapeutic target.

Background: Recent evidence suggests that the prostate cancer (PCa)-specific up-regulation of certain genes such as AMACR, EZH2, PSGR, PSMA and TRPM8 could be associated with an aberrant expression of non-coding microRNAs (miRNA). Methods: In silico analyses were used to search for miRNAs being putative regulators of PCa-associated genes. The expression of nine selected miRNAs (hsa-miR-101, -138, -186, -224, -26a, -26b, -374a, -410, -660) as well as of the aforementioned PCa-associated genes was analyzed by quantitative PCR using 50 malignant (Tu) and matched non-malignant (Tf) tissue samples from prostatectomy specimens as well as 30 samples from patients with benign prostatic hyperplasia (BPH). Then, correlations between paired miRNA and target gene expression levels were analyzed. Furthermore, the effect of exogenously administered miR-26a on selected target genes was determined by quantitative PCR and Western Blot in various PCa cell lines. A luciferase reporter assay was used for target validation. Results: The expression of all selected miRNAs was decreased in PCa tissue samples compared to either control group (Tu vs Tf: -1.35 to -5.61-fold; Tu vs BPH: -1.17 to -5.49-fold). The down-regulation of most miRNAs inversely correlated with an up-regulation of their putative target genes with Spearman correlation coefficients ranging from -0.107 to -0.551. MiR-186 showed a significantly diminished expression in patients with non-organ confined PCa and initial metastases. Furthermore, over-expression of miR-26a reduced the mRNA and protein expression of its potential target gene AMACR in vitro. Using the luciferase reporter assay AMACR was validated as new target for miR-26a. Conclusions: The findings of this study indicate that the expression of specific miRNAs is decreased in PCa and inversely correlates with the up-regulation of their putative target genes. Consequently, miRNAs could contribute to oncogenesis and progression of PCa via an altered miRNA-target gene-interaction.