No / PURPOSE: This phase I dose-escalation study was undertaken to establish the maximum tolerated dose of the sequence-selective minor groove DNA binding agent SJG-136 in patients with advanced solid tumors. The study also investigated antitumor activity and provided pharmacokinetic and pharmacodynamic data. EXPERIMENTAL DESIGN: Sixteen patients were assigned sequentially to escalating doses of SJG-136 (15-240 microg/m(2)) given as a 10-minute i.v. infusion every 21 days. The dose was subsequently reduced in incremental steps to 45 microg/m(2) due to unexpected toxicity. RESULTS: The maximum tolerated dose of SJG-136 was 45 microg/m(2). The main drug-related adverse event was vascular leak syndrome (VLS) characterized by hypoalbuminemia, pleural effusions, ascites, and peripheral edema. Other unexpected adverse events included elevated liver function tests and fatigue. The VLS and liver toxicity had delayed onset and increased in severity with subsequent cycles. Disease stabilization was achieved for >6 weeks in 10 patients; in 2 patients this was maintained for >12 weeks. There was no evidence of DNA interstrand cross-linking in human blood lymphocytes with the use of the comet assay. Evidence of DNA interaction in lymphocytes and tumor cells was shown through a sensitive gamma-H2AX assay. SJG-136 had linear pharmacokinetics across the dose range tested. CONCLUSIONS: SJG-136 was associated with dose-limiting VLS and hepatotoxicity when administered by short injection every 21 days. DNA damage was noted, at all dose levels studied, in circulating lymphocytes. The etiology of the observed toxicities is unclear and is the subject of further preclinical research. Alternative clinical dosing strategies are being evaluated.

L'épilepsie du lobe temporal (ELT) est la forme la plus fréquente de l'épilepsie chez l'adulte. Elle se traduit par des crises spontanées et récurrentes, qui sont résistantes à tout traitement dans 90% des cas. Une agression initiale du cerveau (traumatisme crânien, méningite, convulsions fébriles etc.), est souvent à l'origine de la transformation d'un cerveau « sain » en cerveau épileptique. L'ensemble des processus responsables de cette transition s'appelle l'épileptogenèse. Pouvoir bloquer et/ou retarder l'épileptogenèse chez les patients à risque est une question de santé majeure. En plus des crises, l'ELT soulève d'autres questions. Elle est souvent associée à des déficits cognitifs, qui sont la conséquence de la réorganisation des circuits neuronaux. Ces déficits pourraient être traités de façon indépendante de l'épilepsie elle-même. Le projet de recherche de cette thèse s'inscrit dans ce cadre général. / Temporal Lobe Epilepsy (TLE) is the most common form of epilepsy in adults. It translates into spontaneous and recurrent seizures, which are resistant to any treatment in 90% of cases. An initial brain insult (head injury, meningitis, febrile seizures etc.), is often the cause of the transformation of a "healthy" brain into an epileptic one. The process responsible for this transition is called epileptogenesis. Blocking and/or delaying epileptogenesis in at-risk patients is a key issue for public health. In addition to the seizures, TLE raises other problems. It is often associated with cognitive deficits, which are the result of the reorganization of neuronal circuits. These deficits may be treated independently of epilepsy itself. The work presented here fits into this general framework.

Alterações epigenéticas, como metilação do DNA e modificações pós traducionais em histonas, tem importante papel na carcinogênese mamária. A modulação de eventos epigenéticos constitui relevante alvo terapêutico, devido ao seu caráter reversível. Experimentalmente, o ácido docosahexaenoico (DHA), um membro da família dos ácidos graxos ômega-3, é capaz de diminuir proliferação, induzir morte celular e diminuir o potencial invasivo de células tumorais de mama. No entanto, os mecanismos antitumorais do DHA e sua capacidade de modular eventos epigenéticos ainda não estão totalmente elucidados. Nosso objetivo foi verificar, in vitro, a ação do DHA em eventos epigenéticos em diferentes linhagens de carcinoma mamário humano. Três linhagens celulares de câncer de mama (MDA-MB-231, SKBR-3 e MCF-7) foram tratadas durante 72 horas com 100 ?M de DHA ou etanol (controle). As modificações pós traducionais em histonas, acetilação no resíduo de lisina 9 da histona 3 (H3K9ac) e no resíduo 16 da histona 4 (H4K16ac), bem como trimetilação no resíduo de lisina 9 da histona 3 (H3K9me3) e no resíduo de lisina 27 da histona 3 (H3K27me3) foram avaliadas pela técnica de western blot. A análise da expressão do genes RASSF1A, DNMT1, DNMT3A e DNMT3B foi feita pela técnica da reação em cadeia da polimerase quantitativa via transcriptase reversa (RT-qPCR). A avaliação do padrão de metilação de região promotora do gene RASSF1A foi realizada pela técnica de reação em cadeia da polimerase metilação específica (MS-PCR). Foram também analisadas as fases do ciclo celular por citometria de fluxo. Comparado ao controle, o DHA induziu a acetilação no resíduo 16 da histona 4 (H4K16ac) nas linhagens MCF7 (p = 0,04) e MDA-MB-231 (p = 0,03). Observamos que a H3K9me3 foi parcialmente inibida nas linhagens MDA-MB-231 e SKBR-3, após o tratamento com DHA, mas sem alcançar valor estatisticamente significante. Encontramos também diminuição dos níveis de H3K27me3 após o tratamento com DHA nas três linhagens estudadas, porém não foi estatisticamente significativo. O DHA aumentou a expressão do gene RASSF1A na linhagem MCF-7 (1,98 vezes, p = 0,03), mas não nas linhagens MDA-MB-231 e SKBR-3. Não houve mudanças estatisticamente significativas na expressão dos genes DNMT1, DNMT3A e DNMT3B. As análises qualitativas da metilação demonstraram que a região promotora analisada do gene RASSF1A apresentou-se hipermetilada nas três linhagens celulares. Após o tratamento com DHA, houve tendência de desmetilação na região promotora do RASSF1A na linhagem MCF-7 e SKBR3, mas não na linhagem MDA-MB-231. Não houve diferença significativa na porcentagem de morte e distribuição das células MDA-MB-231, SKBR-3 e MCF-7 nas diferentes fases do ciclo celular após tratamento com DHA. Em conclusão, o DHA pode atuar em mecanismos epigenéticos e, ainda, reativar o gene supressor de tumor, como RASSF1A, anteriormente silenciado por hipermetilação, em células MCF-7. Espera-se que esses resultados contribuam para melhor compreensão do potencial papel anticâncer do DHA no câncer de mama / Epigenetic changes, such as DNA methylation and post-translational histone modifications, play an important role in mammary tumorigenesis. Epigenetic events are important as therapeutic targets, because of its reversible nature. Experimentally, docosahexaenoic acid (DHA), a member of the omega-3 fatty acids family, can reduce proliferation, induce apoptosis and decrease the invasive potential of breast tumor cells. However, the antitumor mechanism of DHA and its ability to modulate epigenetic events are not completely understood. Our objective was to verify, in vitro, the action of DHA in epigenetic events related to transcriptional reactivation of tumor suppressor gene, such as RASSF1A, in different human breast cancer cell lines. Three breast cancer cell lines (MCF-7, MDA-MB-231, SKBR-3) were treated with DHA (100 ?M) or vehicle (ethanol) for 72 hours. Western blot was used to analyze histone modification, as histone H3 lysine 9 (H3K9ac) and histone H4 lysine 16 (H4K16ac) acetylation, H3K9 trimethylation (H3K9me3) and H3K27 trimethylation (H3K27me3). Real time quantitative PCR (RT-qPCR) was performed for gene expression quantification of RASSF1A, DNMT1, DNMT3A and DNMT3B. DNA methylation of the promoter region of RASSF1A was evaluated by methylation specific PCR (MS-PCR). Moreover, we evaluated the phases of the cell cycle by flow cytometry. Compared to control cells, DHA induced H4K16ac in MCF-7 (p=0.04) and MDA-MB-231 (p=0.03). We observed that H3K9me3 was partially inhibited in MDA-MB-231 and SKBR-3 cells, after treatment with DHA, but did not reach a statistically significant value. We also found decreased levels of H3K27me3 after treatment with DHA in the three cell lines studied, but not statistically significant. DHA increased RASSF1A expression on MCF-7 (1.98 fold; p=0.03), but not in MDA-MB-231 and in SKBR-3 cells. There were no statistically significant changes in expression of genes DNMT1, DNMT3A and DNMT3B. These three breast cancer cells lines show methylation in specific region of RASSF1A promoter. DHA treatment increased RASSF1A promoter region hypomethylation in MCF-7 and SKBR-3. No significant difference was observed in the percentage of cell death nor cell distribution of MDA-MB-231, SKBR-3 and MCF-7 at different stages of the cell cycle after treatment with DHA. In conclusion, we suggest that DHA may act beneficially in epigenetic mechanisms and reactivation of tumor suppressor gene, RASSF1A as previously silenced by hypermethylation. It is hoped that these results can contribute to better understanding of the anticancer role of DHA in breast cancer

El gen humano 11-HSD2 es un modelo para investigar la contribución de los efectos de los receptores de esteroides en células de cáncer de mama. El análisis del promotor mostró que la región distal está implicada en la mayor parte de la activación dependiente de hormona. En respuesta a hormona, STAT5A se recluta a la región distal y PR a las regiones distal y proximal del promotor. El reclutamiento de PR se debe a dos mecanismos diferentes, la unión directa de PR a la región proximal, y la implicación vía JAK/STAT en el reclutamiento a la región distal. La inducción del gen 11-HSD2 por hormonas disminuye parcialmente por inhibidores de MAPK y PI3K/Akt y totalmente por inhibidores de JAK/STAT. Así, los efectos citoplasmáticos del PR están implicados en la inducción del gen progesterona. La forma activa de la ARN-polimerasa II es reclutada por la inducción con hormonas a la región distal del promotor 11-HSD2 y la región distal tiene respuesta a hormonas por sí misma, indicando que la inducción del gen por hormonas empieza antes del sitio de inicio de transcripción descrito previamente. / The human 11-HSD2 gene is a model to investigate the contribution of steroid hormone receptors effects on a progesterone responsive promoter in breast cancer cells. Deletion analysis of the 11-HSD2 promoter showed that the distal region is involved in most of the hormone-dependent activation. ChIP showed hormone-dependent STAT5A-recruitment to the distal region and PR-recruitment to the distal and proximal promoter regions. Results suggest two different mechanisms of hormone-induced PR-recruitment, since cells stably expressing PR containing a mutated DNA-binding domain have affected hormone-dependent PR-recruitment to proximal promoter, and JAK/STAT pathway inhibition blocks PR-recruitment to distal promoter. Hormone-stimulated 11-HSD2 gene-expression was partially decreased by MAPK and PI3K/AKT pathway inhibitors and totally blocked by JAK/STAT pathways inhibitors, indicating that cytoplasmic PR effects involvement in progestin-induced 11-HSD2 expression. Importantly, upon hormone induction active RNA-polymerase II is recruited from the 11-HSD2 distal promoter region and the distal minimal promoter has hormone-responsiveness by itself, suggesting that progesterone-dependent 11-HSD2 expression starts upstream the previously characterized transcription start site.

Alterações epigenéticas, como metilação do DNA e modificações pós traducionais em histonas, tem importante papel na carcinogênese mamária. A modulação de eventos epigenéticos constitui relevante alvo terapêutico, devido ao seu caráter reversível. Experimentalmente, o ácido docosahexaenoico (DHA), um membro da família dos ácidos graxos ômega-3, é capaz de diminuir proliferação, induzir morte celular e diminuir o potencial invasivo de células tumorais de mama. No entanto, os mecanismos antitumorais do DHA e sua capacidade de modular eventos epigenéticos ainda não estão totalmente elucidados. Nosso objetivo foi verificar, in vitro, a ação do DHA em eventos epigenéticos em diferentes linhagens de carcinoma mamário humano. Três linhagens celulares de câncer de mama (MDA-MB-231, SKBR-3 e MCF-7) foram tratadas durante 72 horas com 100 ?M de DHA ou etanol (controle). As modificações pós traducionais em histonas, acetilação no resíduo de lisina 9 da histona 3 (H3K9ac) e no resíduo 16 da histona 4 (H4K16ac), bem como trimetilação no resíduo de lisina 9 da histona 3 (H3K9me3) e no resíduo de lisina 27 da histona 3 (H3K27me3) foram avaliadas pela técnica de western blot. A análise da expressão do genes RASSF1A, DNMT1, DNMT3A e DNMT3B foi feita pela técnica da reação em cadeia da polimerase quantitativa via transcriptase reversa (RT-qPCR). A avaliação do padrão de metilação de região promotora do gene RASSF1A foi realizada pela técnica de reação em cadeia da polimerase metilação específica (MS-PCR). Foram também analisadas as fases do ciclo celular por citometria de fluxo. Comparado ao controle, o DHA induziu a acetilação no resíduo 16 da histona 4 (H4K16ac) nas linhagens MCF7 (p = 0,04) e MDA-MB-231 (p = 0,03). Observamos que a H3K9me3 foi parcialmente inibida nas linhagens MDA-MB-231 e SKBR-3, após o tratamento com DHA, mas sem alcançar valor estatisticamente significante. Encontramos também diminuição dos níveis de H3K27me3 após o tratamento com DHA nas três linhagens estudadas, porém não foi estatisticamente significativo. O DHA aumentou a expressão do gene RASSF1A na linhagem MCF-7 (1,98 vezes, p = 0,03), mas não nas linhagens MDA-MB-231 e SKBR-3. Não houve mudanças estatisticamente significativas na expressão dos genes DNMT1, DNMT3A e DNMT3B. As análises qualitativas da metilação demonstraram que a região promotora analisada do gene RASSF1A apresentou-se hipermetilada nas três linhagens celulares. Após o tratamento com DHA, houve tendência de desmetilação na região promotora do RASSF1A na linhagem MCF-7 e SKBR3, mas não na linhagem MDA-MB-231. Não houve diferença significativa na porcentagem de morte e distribuição das células MDA-MB-231, SKBR-3 e MCF-7 nas diferentes fases do ciclo celular após tratamento com DHA. Em conclusão, o DHA pode atuar em mecanismos epigenéticos e, ainda, reativar o gene supressor de tumor, como RASSF1A, anteriormente silenciado por hipermetilação, em células MCF-7. Espera-se que esses resultados contribuam para melhor compreensão do potencial papel anticâncer do DHA no câncer de mama / Epigenetic changes, such as DNA methylation and post-translational histone modifications, play an important role in mammary tumorigenesis. Epigenetic events are important as therapeutic targets, because of its reversible nature. Experimentally, docosahexaenoic acid (DHA), a member of the omega-3 fatty acids family, can reduce proliferation, induce apoptosis and decrease the invasive potential of breast tumor cells. However, the antitumor mechanism of DHA and its ability to modulate epigenetic events are not completely understood. Our objective was to verify, in vitro, the action of DHA in epigenetic events related to transcriptional reactivation of tumor suppressor gene, such as RASSF1A, in different human breast cancer cell lines. Three breast cancer cell lines (MCF-7, MDA-MB-231, SKBR-3) were treated with DHA (100 ?M) or vehicle (ethanol) for 72 hours. Western blot was used to analyze histone modification, as histone H3 lysine 9 (H3K9ac) and histone H4 lysine 16 (H4K16ac) acetylation, H3K9 trimethylation (H3K9me3) and H3K27 trimethylation (H3K27me3). Real time quantitative PCR (RT-qPCR) was performed for gene expression quantification of RASSF1A, DNMT1, DNMT3A and DNMT3B. DNA methylation of the promoter region of RASSF1A was evaluated by methylation specific PCR (MS-PCR). Moreover, we evaluated the phases of the cell cycle by flow cytometry. Compared to control cells, DHA induced H4K16ac in MCF-7 (p=0.04) and MDA-MB-231 (p=0.03). We observed that H3K9me3 was partially inhibited in MDA-MB-231 and SKBR-3 cells, after treatment with DHA, but did not reach a statistically significant value. We also found decreased levels of H3K27me3 after treatment with DHA in the three cell lines studied, but not statistically significant. DHA increased RASSF1A expression on MCF-7 (1.98 fold; p=0.03), but not in MDA-MB-231 and in SKBR-3 cells. There were no statistically significant changes in expression of genes DNMT1, DNMT3A and DNMT3B. These three breast cancer cells lines show methylation in specific region of RASSF1A promoter. DHA treatment increased RASSF1A promoter region hypomethylation in MCF-7 and SKBR-3. No significant difference was observed in the percentage of cell death nor cell distribution of MDA-MB-231, SKBR-3 and MCF-7 at different stages of the cell cycle after treatment with DHA. In conclusion, we suggest that DHA may act beneficially in epigenetic mechanisms and reactivation of tumor suppressor gene, RASSF1A as previously silenced by hypermethylation. It is hoped that these results can contribute to better understanding of the anticancer role of DHA in breast cancer

Chez la levure Saccharomyces cerevisiae, l’acétylation de l’histone H3 sur la Lysine 56 (H3K56ac) a lieu sur toutes les histones H3 nouvellement synthétisées qui sont déposées derrière les fourches de réplication. L’acétylation de H3K56 joue un rôle primordial dans l’assemblage de l’ADN lors la réplication et la réparation. L’acétylation de H3K56 joue également un rôle important dans la stabilité génomique et la stabilisation des fourches de réplication bloquée. En effet, les cellules dépourvues de H3K56ac sont sensibles au méthane sulfonate de méthyle (MMS) et à d’autres agents génotoxiques qui causent du stress réplicatif. Notre projet visait à investiguer les liens entre la protéine du réplisome Ctf4 et l’acétyltransférase d’histone Rtt109. Dans un premier lieu, la délétion de CTF4 a partiellement contré la sensibilité des cellules rtt109Δ au MMS. Notre analyse génétique a aussi montré que Ctf4, Rtt109, et le complexe Rtt101-Mms1-Mms22 agissent dans la même voie de réponse face à un stress réplicative. Nos résultats montrent que les cellules ctf4Δ et rtt109Δ présentent des foyers intenses du complexe de liaison à l'ADN simple-brin RPA en réponse au stress réplicatif, suggérant la formation excessive de régions d'ADN simple-brin aux fourches de réplication bloquées, ce qui conduit à une hyper activation des points de contrôle des dommages à l'ADN. Ces mutants présentent des ponts anaphase et des foyers persistants des protéines de recombinaison homologues Rad51 et Rad52 en réponse aux génotoxines, suggérant ainsi que la structure anormale des réplisomes bloqués peut compromettre leur récupération. Nos résultats indiquent également que la délétion des gènes de la RH (RAD51, RAD52, RAD54, RAD55 et MUS81) avec ctf4Δ et rtt109Δ respectivement, engendre une sensibilité synergique au MMS, suggérant que les cellules qui sont déficientes en H3K56 acétylation utilisent la RH pour réparer les dommages causés suite à un stress réplicatif. En conclusion, nos résultats suggèrent que les cellules déficientes en H3K56ac présentent des défauts de RH en réponse aux dommages à l’ADN induits par le MMS durant la phase S. / In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) occurs on all newly synthesized histones H3 that are deposited behind DNA replication forks. H3K56ac plays critical role in chromatin assembly during DNA replication and repair. H3K56ac is also required for genome stability and stabilization of stalled replication fork. Cells lacking H3K56ac are sensitive to methyl methane sulfonate and other drugs that cause replicative stress. In this thesis, we investigated the links between the replisome protein Ctf4 and the H3K56 acetyltransferase Rtt109. Deletion of CTF4 partially rescued the sensitivity of rtt109Δ cells to methyl methane sulfonate. Genetic analyses also showed that Ctf4, Rtt109, and the Rtt101-Mms1-Mms22 complex act in the same pathway to response to replicative stress. ctf4Δ and rtt109Δ cells displayed intense foci of the single-stranded DNA binding complex RPA during replicative stress, suggesting formation of excess single-stranded DNA regions at stalled replication forks, leading to hyper activation of DNA damage checkpoints. These mutants accumulated anaphase bridges and persistent foci of the homologous recombination proteins Rad51 and Rad52 in response to genotoxins, suggesting that abnormal DNA structure formed at stalled replisome may compromise their recovery. Deletion of HR genes (RAD51, RAD52, RAD54, RAD55 and MUS81) together with ctf4Δ and rtt109Δ presents synergistic sensitivity to MMS, suggesting that H3K56ac deficient cells use HR to repair the damages caused by replicative stress. Overall our results demonstrate that H3K56ac deficient cells cannot recover MMS- induced damages because HR is compromised in these mutants.

Indiana University-Purdue University Indianapolis (IUPUI) / Fetal alcohol spectrum disorders (FASD) is the leading neurodevelopment deficit in children born to women who drink alcohol during pregnancy. The hippocampus and cortex are among brain regions vulnerable to alcohol-induced neurotoxicity, and are key regions underlying the cognitive impairment, learning and memory deficits shown in FASD individuals. Hippocampal and cortical neuronal differentiation and maturation are highly influenced by both intrinsic transcriptional signaling and extracellular cues. Epigenetic mechanisms, primarily DNA methylation and histone modifications, are hypothesized to be involved in regulating key neural development events, and are subject to alcohol exposure. Alcohol is shown to modify DNA methylation and histone modifications through altering methyl donor metabolisms. Recent studies in our laboratory have shown that alcohol disrupted genome-wide DNA methylation and delayed early embryonic development. However, how alcohol affects DNA methylation in fetal hippocampal and cortical development remains elusive, therefore, will be the theme of this study. We reported that, in a dietary alcohol-intake model of FASD, prenatal alcohol exposure retarded the development of fetal hippocampus and cortex, accompanied by a delayed cellular DNA methylation program. We identified a programed 5-methylcytosine (5mC) and 5-hydroxylmethylcytosine (5hmC) cellular and chromatic re-organization that was associated with neuronal differentiation and maturation spatiotemporally, and this process was hindered by prenatal alcohol exposure. Furthermore, we showed that alcohol disrupted locus-specific DNA methylation on neural specification genes and reduced neurogenic properties of neural stem cells, which might contribute to the aberration in neurogenesis of FASD individuals. The work of this dissertation suggested an important role of DNA methylation in neural development and elucidated a potential epigenetic mechanism in the alcohol teratogenesis.

Les glycosides cardiotoniques sont des inhibiteurs des pompes sodium / potassium utilisés pour le traitements des insuffisances cardiaques, qui détiennent également des activités anticancéreuses et épigénétiques récemment caractérisées. Toutefois, dans l’objectif de repositionner ces médicaments comme traitement anticancéreux, les mécanismes sousjacents aux activités anticancéreuses et épigénétiques des glycosides cardiotoniques restent à être déterminés. Dans nos travaux, nous révélons que la proscillaridine A est le glycoside cardiotonique qui détient des activités anticancéreuses et épigénétiques les plus puissantes dans des lignées de cancer du côlon, de leucémies et de sarcomes pédiatrique. De plus, nous avons identifié que l’activité anticancéreuse de la proscillaridine A corrèle positivement avec le niveau d’expression protéique du proto-oncogène MYC dans un panel de 14 lignées cellulaires cancéreuses. Dans les lignées cellulaires exprimants un haut niveau de MYC telles que les lignées leucémiques, la proscillaridine A agit comme un inhibiteur de MYC et module sa stabilité protéique ainsi que la régulation transcriptionnelle et translationnelle de ces cibles. Cette inhibition est induite par la baisse significative de l’expression des enzymes épigénétiques les lysines acétyltransférases (KATs), qui contrôlent l’ajout des résidus d’acétylcoenzyme A sur les histones et sur d'autres protéines dont MYC. La baisse d’expression des KATs résultent à une baisse de l’acétylation des résidus de l’histone 3 et à une reprogrammation de l’acétylome des cellules cancéreuses surexprimant MYC. Ces changements au niveau de la chromatine induisent une reprogrammation transcriptionnelle et phénotypique des cellules surexprimant MYC, qui se traduit par une perte de la transcription des programmes oncogéniques et l’induction des programmes associés à la différenciation cellulaire. Pour finir, nous avons évalué le potentiel synergique anticancéreux et épigénétique de la proscillaridine A avec le médicament épigénétique la décitabine dans des lignées cancéreuses exprimants des niveaux différentiels de MYC. Dans une lignée résistante à la proscillaridine A et exprimant de faible niveau de MYC (lignée de cancer de côlon), la décitabine et la proscillaridine A démontrent des activités épigénétiques synergiques tandis que dans une lignée sensible à la proscillaridine A et surexprimant MYC (lignée de sarcome pédiatrique), la décitabine et la proscillaridine A démontrent des activités antiprolifératives synergiques. Dans ces travaux, nous avons donc démontré le potentiel de repositionner la proscillaridine A dans les cancers surexprimant MYC. Également, nous démontrons le potentiel synergique anticancéreux et épigénétique de la proscillaridine A avec la décitabine et nous suggérons d’étudier cette combinaison de médicaments dans les cancers plus résistants à la proscillaridine A. / Cardiac glycosides are sodium/potassium pomps’ inhibitors used for the treatment of heart failure, and whose anticancer and epigenetic activities have been recently characterized. However, in order to repurpose cardiac glycosides as anticancer drugs, mechanistic studies are required to identify the anticancer and epigenetic mechanism of actions. In our experiments, proscillaridin A exhibited the most powerful anticancer and epigenetic activities in colon cancer, leukemia, and sarcoma cell lines. Moreover, we demonstrated that in a panel of 14 cancer cell lines, proscillaridin A anticancer activities positively correlated with MYC protooncogene expression level. In high MYC expressing cell lines such as leukemia, proscillaridin A inhibited MYC expression through protein destabilization and through transcriptomic and translational regulation of MYC targets. Theses inhibitions are induced by the loss of lysine acetylatransferase (KAT) expressions, which are epigenetic enzymes controlling the addition of acetyl-coenzyme A on histones and other proteins such as MYC. KAT inhibitions are responsible for the global loss of histone 3 acetylation and acetylome reprogrammation in high MYC expressing cancer cells. These chromatin changes induced transcriptomic and phenotypic reprogrammation, defined by a loss of the transcription of oncogenic programs and the induction of cell differentiation. To finish, we evaluated the anticancer and epigenetic synergic potential of proscillaridin A in combination with the epigenetic drug the decitabine in cancer cell lines expressing different MYC levels. In a cancer cell line resistant to proscillaridin A treatments and expressing low MYC level (colon cancer cell line), the combination of decitabine and proscillaridin A demonstrated synergistic epigenetic activity although, in a cell line sensitive to proscillaridin A treatments and expressing high MYC level (sarcoma cell line), the combination of decitabine and proscillaridin A exhibited synergistic anti-proliferative activity. To conclude, we highlighted the potential of repurposing proscillaridin A as an anticancer treatment in high MYC expressing cells. Furthermore, we demonstrated the anticancer and epigenetic synergistic potential of proscillaridin A in combination with decitabine and we propose to study the drug combination in cancers that are resistant to proscillaridin A treatment.

Myeloid-derived suppressor cells (MDSCs) prolong sepsis by promoting immunosuppression. We reported that sepsis MDSC development requires long non-coding RNA Hotairm1 interactions with S100A9. Using a mouse model that simulates the immunobiology of sepsis, we find that histone demethylase KDM6A promotes Hotairm1 transcription by demethylating transcription repression H3K27me3 histone mark. We show that chemical targeting of KDM6A by GSK-J4 represses Hotairm1 transcription, which coincides with decreases in transcription activation H3K4me3 histone mark and transcription factor PU.1 binding to the Hotairm1 promoter. We further show that immunosuppressive IL-10 cytokine promotes KDM6A binding at the Hotairm1 promoter. IL-10 knockdown repletes H3K27me3 and reduces Hotairm1 transcription. GSK-J4 treatment also relocalizes nuclear S100A9 protein to the cytosol. To support translation to human sepsis, we demonstrate that inhibiting H3K27me3 demethylation by KDM6A ex vivo in MDSCs from patients with protracted sepsis decreases Hotairm1 transcription. These findings suggest that epigenetic targeting of MDSCs in human sepsis might resolve post-sepsis immunosuppression and improve sepsis survival.

No description available.