DNMT1 Inhibition Reprograms Pancreatic Cancer Stem Cells via Upregulation of the miR-17-92 Cluster.

Pancreatic ductal adenocarcinoma (PDAC) and other carcinomas are hierarchically organized, with cancer stem cells (CSC) residing at the top of the hierarchy, where they drive tumor progression, metastasis, and chemoresistance. As CSC and non-CSC share an identical genetic background, we hypothesize that differences in epigenetics account for the striking functional differences between these two cell populations. Epigenetic mechanisms, such as DNA methylation, play an important role in maintaining pluripotency and regulating the differentiation of stem cells, but the role of DNA methylation in pancreatic CSC is obscure. In this study, we investigated the genome-wide DNA methylation profile of PDAC CSC, and we determined the importance of DNA methyltransferases for CSC maintenance and tumorigenicity. Using high-throughput methylation analysis, we discovered that sorted CSCs have a higher level of DNA methylation, regardless of the heterogeneity or polyclonality of the CSC populations present in the tumors analyzed. Mechanistically, CSC expressed higher DNMT1 levels than non-CSC. Pharmacologic or genetic targeting of DNMT1 in CSCs reduced their self-renewal and in vivo tumorigenic potential, defining DNMT1 as a candidate CSC therapeutic target. The inhibitory effect we observed was mediated in part through epigenetic reactivation of previously silenced miRNAs, in particular the miR-17-92 cluster. Together, our findings indicate that DNA methylation plays an important role in CSC biology and also provide a rationale to develop epigenetic modulators to target CSC plasticity and improve the poor outcome of PDAC patients. Cancer Res; 76(15); 4546-58. ©2016 AACR

An open-label, randomized, non-inferiority trial of the efficacy and safety of ciprofloxacin versus streptomycin + ciprofloxacin in the treatment of bubonic plague (IMASOY): study protocol for a randomized control trial.

BACKGROUND: Bubonic plague is the primary manifestation of infection with Yersinia pestis, accounting for 90% of all plague cases and with 75% of global cases reported in Madagascar. All drugs in use for treating plague are registered based on experimental data and anecdotal evidence, and no regimen currently recommended is supported by a randomized clinical trial. The IMASOY trial intends to fill this knowledge gap by comparing two 10-day regimens included in the national guidelines in Madagascar. The primary objective of the trial is to test the hypothesis that ciprofloxacin monotherapy is non-inferior to streptomycin followed by ciprofloxacin for the treatment of bubonic plague, thus avoiding the need for injectable, potentially toxic, aminoglycosides. METHODS: A two-arm parallel-group randomized control trial will be conducted across peripheral health centres in Madagascar in five districts. Males and non-pregnant females of all ages with suspected bubonic or pneumonic plague will be recruited over the course of three plague 'seasons'. The primary endpoint of the trial is to assess the proportion of patients with bubonic plague who have a therapeutic response to treatment (defined as alive, resolution of fever, 25% reduction in the size of measurable buboes, has not received an alternative treatment and no clinical decision to continue antibiotics) as assessed on day 11. DISCUSSION: If successful, the trial has the potential to inform the standard of care guidelines not just in Madagascar but in other countries afflicted by plague. The trial is currently ongoing and expected to complete recruitment in 2022. TRIAL REGISTRATION: ClinicalTrials.gov NCT04110340 . Registered on 1 October 2019

Genes that reprogram adult cells to pluripotent stem cells : expression and implications in human plasma cell cancers

Le myélome multiple (MM) est une néoplasie lymphocytaire B qui se caractérise par l'accumulation d'un clone de cellules plasmocytaires tumorales dans la moelle osseuse interagissant de manière étroite avec le microenvironnement. L'étude du profil d'expression génique à l'aide des puces à ADN a permis de préciser l'hétérogénéité de cette pathologie et de découvrir de nouveaux acteurs susceptibles d'avoir un rôle importent dans sa physiopathologie. La surexpression de Oct-3/4, Sox2, c-Myc et KLF4 dans des cellules adultes les reprogramme à l'état de cellules souches. J'ai montré une surexpression significative de 3 de ces 4 gènes – KLF4, MYC, SOX2 - dans le MM. Ces gènes sont également fréquemment surexprimés dans 18 types de cancers sur 40 étudiés. Par ailleurs, leur expression peut y être associé à un mauvais pronostique ou un signe de progression tumorale, dus peut-être à leur capacité à induire des caractéristiques de cellules souches cancéreuses. Nous avons par la suite débuté l'étude de la fonction des protéines codées par ces gènes dans le MM, en commençant par KLF4, facteur de transcription activateur ou répresseur. KLF4 est exprimé dans les plasmocytes (PC) normaux, mais son expression est perdue dans les PC tumoraux (cellules myélomateuses, MMC) de 2 patients sur 3 atteints de MM au diagnostic. Parmi les patients pour lesquels les MMC expriment KLF4, se trouve un groupe de patients à haut risque, le groupe MMSET portant la translocation t(4 ;14). L'utilisation d'un modèle inductible d'expression de KLF4 dans des lignées de MM avec transduction lentivirale, a mis en évidence un arrêt du cycle associé à l'expression de P27/KIP1 pour les lignées avec des mutations inactivatrices de P53, mais également de P21/WAF1 pour des lignées avec P53 sauvage. Cette sortie du cycle due à l'expression de KLF4 pourrait protéger les plasmocytes tumoraux de l'apoptose induite par certaines drogues ciblant le cycle, comme nous l'observons in vitro avec le Melphalan.L'objectif majeur de notre équipe est de comprendre le fonctionnement du PC normal et celui du PC tumoral. Afin d'atteindre cet objectif, il est nécessaire d'obtenir un système efficace permettant d'introduire un gène dans un PC. Nous avons montré que des lentivirus pseudotypés avec des glycoprotéines tronquées (hemagglutinine et fusion) issus du virus de la rougeole, transduisent de façon stable et efficace des PC normaux et tumoraux.Multiple myeloma (MM) is a B-cell neoplasia characterized by the accumulation of a clone of malignant plasma cells in bone marrow closely interacting with its microenvironment.Gene expression profiling using DNA microarrays has clarified the heterogeneity of this disease and has allowed the finding of new actors that may have an important function in MM pathophysiology.Overexpression of Oct-3/4, Sox2, c-Myc and KLF4 in adult cells causes their return to the state of stem cells, commonly called induced pluripotent stem cells (iPS). Our team has shown a significant overexpression of at least one of these four factors in 18 out of 40 cancers studied. Moreover, their expression may be associated with poor prognosis or may be a sign of tumor progression, perhaps due to their ability to induce characteristics of cancer stem cells.We therefore began the study of the function of these genes in MM, starting with KLF4, which can either be an activator or a repressor of transcription, depending on the promoter. KLF4 is expressed in normal plasma cells (PC), but its expression is lost in 2 out of 3 patients with MM at diagnosis. Among patients for whom the PCs express KLF4, is a group of high-risk patients, the MMSET group, bearing the t(4;14) translocation.An inducible model of KLF4's expression in MM cell lines was obtained using lentiviral transduction. Our model revealed a KLF4 induced cycle arrest, associated with the expression of P27/KIP1 when P53 is mutated, but also P21/WAF1 in case of wild type P53. This cell cycle blockade due to the expression of KLF4 could protect malignant plasma cells from the apoptosis induced by certain drugs targeting the cell cycle, as shown by our in vitro observations using melphalan.The main goal of our team is to understand the normal function of PCs and the PC tumor. To achieve this, it is necessary to obtain an effective system for introducing a gene in a given PC. We have shown that lentiviruses pseudotyped with truncated glycoproteins (Hemagglutinin and Fusion) from measles virus, can a stably and efficiently transduce normal and malignant PCs

Gènes reprogrammant des cellules adultes en cellules souches pluripotentes (expression et implication dans les cancers plasmocytaires humains.)

Le myélome multiple (MM) est une néoplasie lymphocytaire B qui se caractérise par l'accumulation d'un clone de cellules plasmocytaires tumorales dans la moelle osseuse interagissant de manière étroite avec le microenvironnement. L'étude du profil d'expression génique à l'aide des puces à ADN a permis de préciser l'hétérogénéité de cette pathologie et de découvrir de nouveaux acteurs susceptibles d'avoir un rôle importent dans sa physiopathologie. La surexpression de Oct-3/4, Sox2, c-Myc et KLF4 dans des cellules adultes les reprogramme à l'état de cellules souches. J'ai montré une surexpression significative de 3 de ces 4 gènes KLF4, MYC, SOX2 - dans le MM. Ces gènes sont également fréquemment surexprimés dans 18 types de cancers sur 40 étudiés. Par ailleurs, leur expression peut y être associé à un mauvais pronostique ou un signe de progression tumorale, dus peut-être à leur capacité à induire des caractéristiques de cellules souches cancéreuses. Nous avons par la suite débuté l'étude de la fonction des protéines codées par ces gènes dans le MM, en commençant par KLF4, facteur de transcription activateur ou répresseur. KLF4 est exprimé dans les plasmocytes (PC) normaux, mais son expression est perdue dans les PC tumoraux (cellules myélomateuses, MMC) de 2 patients sur 3 atteints de MM au diagnostic. Parmi les patients pour lesquels les MMC expriment KLF4, se trouve un groupe de patients à haut risque, le groupe MMSET portant la translocation t(4 ;14). L'utilisation d'un modèle inductible d'expression de KLF4 dans des lignées de MM avec transduction lentivirale, a mis en évidence un arrêt du cycle associé à l'expression de P27/KIP1 pour les lignées avec des mutations inactivatrices de P53, mais également de P21/WAF1 pour des lignées avec P53 sauvage. Cette sortie du cycle due à l'expression de KLF4 pourrait protéger les plasmocytes tumoraux de l'apoptose induite par certaines drogues ciblant le cycle, comme nous l'observons in vitro avec le Melphalan.L'objectif majeur de notre équipe est de comprendre le fonctionnement du PC normal et celui du PC tumoral. Afin d'atteindre cet objectif, il est nécessaire d'obtenir un système efficace permettant d'introduire un gène dans un PC. Nous avons montré que des lentivirus pseudotypés avec des glycoprotéines tronquées (hemagglutinine et fusion) issus du virus de la rougeole, transduisent de façon stable et efficace des PC normaux et tumoraux.Multiple myeloma (MM) is a B-cell neoplasia characterized by the accumulation of a clone of malignant plasma cells in bone marrow closely interacting with its microenvironment.Gene expression profiling using DNA microarrays has clarified the heterogeneity of this disease and has allowed the finding of new actors that may have an important function in MM pathophysiology.Overexpression of Oct-3/4, Sox2, c-Myc and KLF4 in adult cells causes their return to the state of stem cells, commonly called induced pluripotent stem cells (iPS). Our team has shown a significant overexpression of at least one of these four factors in 18 out of 40 cancers studied. Moreover, their expression may be associated with poor prognosis or may be a sign of tumor progression, perhaps due to their ability to induce characteristics of cancer stem cells.We therefore began the study of the function of these genes in MM, starting with KLF4, which can either be an activator or a repressor of transcription, depending on the promoter. KLF4 is expressed in normal plasma cells (PC), but its expression is lost in 2 out of 3 patients with MM at diagnosis. Among patients for whom the PCs express KLF4, is a group of high-risk patients, the MMSET group, bearing the t(4;14) translocation.An inducible model of KLF4's expression in MM cell lines was obtained using lentiviral transduction. Our model revealed a KLF4 induced cycle arrest, associated with the expression of P27/KIP1 when P53 is mutated, but also P21/WAF1 in case of wild type P53. This cell cycle blockade due to the expression of KLF4 could protect malignant plasma cells from the apoptosis induced by certain drugs targeting the cell cycle, as shown by our in vitro observations using melphalan.The main goal of our team is to understand the normal function of PCs and the PC tumor. To achieve this, it is necessary to obtain an effective system for introducing a gene in a given PC. We have shown that lentiviruses pseudotyped with truncated glycoproteins (Hemagglutinin and Fusion) from measles virus, can a stably and efficiently transduce normal and malignant PCs.MONTPELLIER-BU Médecine UPM (341722108) / SudocSudocFranceF

Embryonic stem cell markers expression in cancers.

International audienceThe transcription factors Oct4 and Sox2 are highly expressed in embryonic stem (ES) cells. In conjunction with Klf4 and c-Myc, their over-expression can induce pluripotency in both mouse and human somatic cells, indicating that these factors are key regulators of the signaling network necessary for ES cell pluripotency. Self-renewal is a hallmark of stem cells and cancer and stemness program could play an important role in cancer. Therefore we compared the expression of Oct4, Sox2, Klf4 and c-Myc in 40 human tumor types to that of their normal tissue counterparts using publicly available gene expression data, including the Oncomine Cancer Microarray database. We found significant overexpression of at least 1/4 pluripotency factors Oct4, Sox2, Klf4 or c-Myc in 18 out of the 40 cancer types investigated. Furthermore, within a given tumor category these genes are associated with tumor progression or bad prognosis. A key goal in cancer research is to identify the mechanism by which cancer stem cells arise and self-renew. The overexpression of Oct3/4, Sox2, Klf4 and/or c-Myc could contribute to the pathologic self-renewal characteristics of cancer stem cells

Gene expression profiling and real-time PCR analyses identify novel potential cancer-testis antigens in multiple myeloma.: Novel cancer-testis genes in multiple myeloma

International audienceCancer-testis (CT) Ags are attractive targets for immunotherapeutic strategies since they are aberrantly expressed in malignant cells and not, or in limited number, in somatic tissues, except germ cells. To identify novel CT genes in multiple myeloma, we used Affymetrix HG-U133 gene expression profiles of 5 testis, 64 primary multiple myeloma cells (MMC), and 24 normal tissue samples. A 5-filter method was developed to keep known CT genes while deleting non-CT genes. Starting from 44,928 probe sets, including probe sets for 18 previously described CT genes, we have obtained 82 genes expressed in MMC and testis and not detected in more than 6 normal tissue samples. This list includes 14 of the 18 known CT genes and 68 novel putative CT genes. Real-time RT-PCR was performed for 34 genes in 12 normal tissue samples, 5 MMC samples, and one sample of five pooled testes. It has validated the CT status of 23 of 34 genes (67%). We found one novel "testis-restricted" gene (TEX14, expression in testis and tumor only), eight "tissue-restricted" (mRNA detected in one or two nongametogenic tissues), and seven "differentially expressed" (mRNA detected in three to six nongametogenic tissues) CT genes. Further studies are warranted to determine the immunogenicity of these novel CT Ag candidates

Inhibition of DEPDC1A, a bad prognostic marker in multiple myeloma, delays growth and induces mature plasma cell markers in malignant plasma cells.

High throughput DNA microarray has made it possible to outline genes whose expression in malignant plasma cells is associated with short overall survival of patients with Multiple Myeloma (MM). A further step is to elucidate the mechanisms encoded by these genes yielding to drug resistance and/or patients' short survival. We focus here on the biological role of the DEP (for Disheveled, EGL-10, Pleckstrin) domain contained protein 1A (DEPDC1A), a poorly known protein encoded by DEPDC1A gene, whose high expression in malignant plasma cells is associated with short survival of patients. Using conditional lentiviral vector delivery of DEPDC1A shRNA, we report that DEPDC1A knockdown delayed the growth of human myeloma cell lines (HMCLs), with a block in G2 phase of the cell cycle, p53 phosphorylation and stabilization, and p21(Cip1) accumulation. DEPDC1A knockdown also resulted in increased expression of mature plasma cell markers, including CXCR4, IL6-R and CD38. Thus DEPDC1A could contribute to the plasmablast features of MMCs found in some patients with adverse prognosis, blocking the differentiation of malignant plasma cells and promoting cell cycle

Hypoxia favors the generation of human plasma cells

<p>Plasma cells (PCs) generation occurs in hypoxic conditions <i>in vivo</i>, whereas the relevance of O₂ pressure in PC differentiation remains unknown.</p> <p>Using our <i>in vitro</i> PC differentiation model, we investigated the role of hypoxia in PC generation.</p> <p>Hypoxia increases the generation of plasmablasts (PBs) starting from memory B cells, by increasing cell cycle and division number. Reactome analysis demonstrated a significant enrichment of genes involved in HIF1α and HIF2α transcription factor network, metabolism and MYC related pathways in hypoxic compared with normoxic PBs. Hypoxia-induced metabolism alteration and MYC pathway are involved in malignant PC pathophysiology. Therefore, the expression of 28 out of the 74 genes overexpressed in hypoxic PBs compared with normoxic ones was found to be associated with an adverse prognosis (event free survival and overall survival) in newly diagnosed multiple myeloma patients.</p> <p>According to the role of hypoxia in supporting PBs generation through cell cycle induction, c-MYC activation and metabolism alteration, it could be involved in plasma cell tumorigenesis.</p