Contrôle de l'expression du gène HOXA9 dans les cellules souches/progénitrices hématopoïétiques : rôle des enzymes épigénétiques MOZ et MLL, et du facteur de polyadénylation Symplekin

My thesis project has consisted of the study of MOZ, and MLL. They are epigenetic regulators. MOZ and MLL activate transcription of HOX genes, which are transcription factors essential during haematopoiesis. MOZ and MLL have some target genes in common. In our study, we characterised a cooperation between MOZ and MLL in human haematopoietic stem/progenitor cells CD34+. They are both recruited onto HOX promoters. MOZ is essential for MLL recruitment, and this is reciprocal. In conclusion, we provided an example of a mechanism involving a direct cross-talk between two histone modifying enzymes.In order to dissect the mechanism of action of this complex, we decided to identify novel proteins interacting with both MOZ and MLL. A member of the RNA polyadenylation machinery has been isolated: Symplekin. We confirmed the interaction between MOZ, MLL and Symplekin in the human haematopoietic immature cell line KG1. We showed that Symplekin is co-recruited to HOXA9 promoter along with MOZ and MLL. We demonstrated the dual role of this member of the polyadenylation machinery. Indeed, besides the fact that Symplekin is important for Hoxa9 polyadenylation, thus its stability, it prevents MOZ and MLL recruitment onto HOXA9 promoter, leading to a decrease of HOXA9 transcription.Our work improved the understanding of the mechanism of action of MOZ and MLL in HOX control.Mon travail de thèse porte sur l’étude du rôle de l’histone acétyl-transférase MOZ et de l’histone méthyle-transférase MLL dans l’hématopoïèse. Elles contrôlent l’expression de nombreux gènes, nottament des gènes HOX, des facteurs de transcription connus pour leur rôle dans l’hématopoïèse normale et pathologique. Les deux protéines ont des gènes cibles communs tel qu'HOXA9. Ces observations nous ont conduit à rechercher une coopération fonctionnelle entre MOZ et MLL. Nous avons montré que MOZ était associée avec MLL dans les cellules souches/progénitrices humaines CD34+ afin d’activer la transcription des gènes HOXA5, HOXA7 et HOXA9. En effet, les deux protéines interagissent et sont recrutées au niveau de leur promoteur. Nous avons mis en évidence une interférence fonctionnelle entre ces deux facteurs épigénétiques, puisque MOZ est nécessaire au recrutement et à l’activité enzymatique de MLL au niveau des gènes HOXA5, HOXA7 et HOXA9 et réciproquement.Afin de caractériser le mécanisme d’action impliquant la coopération entre MOZ et MLL, nous avons recherché d’autres partenaires associés à ce duo. Nous avons identifié la Symplekin, un membre de la machinerie de polyadénylation. Nous avons mis en évidence l’interaction de la Symplekin avec MOZ et MLL dans les cellules de la lignée hématopoïétique humaine KG1. Les trois protéines sont co-recrutées sur le promoteur du gène HOXA9. Nous avons démontré le rôle ambivalent de la Symplekin. Bien qu’elle soit importante pour la polyadénylation et par conséquent pour la stabilité de l’ARN Hoxa9, la Symplekin empêche le recrutement de MOZ et de MLL au niveau du gène HOXA9, conduisant ainsi à une diminution de sa transcription

Diagnostic and Therapeutic Potential of Extracellular Vesicles in B-Cell Malignancies

Extracellular vesicles (EV), comprising microvesicles and exosomes, are particles released by every cell of an organism, found in all biological fluids, and commonly involved in cell-to-cell communication through the transfer of cargo materials such as miRNA, proteins, and immune-related ligands (e.g., FasL and PD-L1). An important characteristic of EV is that their composition, abundance, and roles are tightly related to the parental cells. This translates into a higher release of characteristic pro-tumor EV by cancer cells that leads to harming signals toward healthy microenvironment cells. In line with this, the key role of tumor-derived EV in cancer progression was demonstrated in multiple studies and is considered a hot topic in the field of oncology. Given their characteristics, tumor-derived EV carry important information concerning the state of tumor cells. This can be used to follow the outset, development, and progression of the neoplasia and to evaluate the design of appropriate therapeutic strategies. In keeping with this, the present brief review will focus on B-cell malignancies and how EV can be used as potential biomarkers to follow disease progression and stage. Furthermore, we will explore several proposed strategies aimed at using biologically engineered EV for treatment (e.g., drug delivery mechanisms) as well as for impairing the biogenesis, release, and internalization of cancer-derived EV, with the final objective to disrupt tumor–microenvironment communication.Fil: Gargiulo, Ernesto. Luxembourg Institute of Health; LuxemburgoFil: Morande, Pablo Elías. Luxembourg Institute of Health; Luxemburgo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Largeot, Anne. Luxembourg Institute of Health; LuxemburgoFil: Moussay, Etienne. Luxembourg Institute of Health; LuxemburgoFil: Paggetti, Jérôme. Luxembourg Institute of Health; Luxemburg

Expression of the MOZ-TIF2 oncoprotein in mice represses senescence

The MOZ-TIF2 translocation, which fuses monocytic leukemia zinc finger protein (MOZ) histone acetyltransferase (HAT) with the nuclear co-activator TIF2, is associated with the development of acute myeloid leukemia. We recently found that in the absence of MOZ HAT activity, p16INK4a transcriptional levels are significantly increased, triggering an early entrance into replicative senescence. Because oncogenic fusion proteins must bypass cellular safeguard mechanisms, such as senescence and apoptosis, to induce leukemia, we hypothesized that this repressive activity of MOZ over p16INK4a transcription could be preserved, or even reinforced, in MOZ leukemogenic fusion proteins, such as MOZ-TIF2. We describe here that, indeed, MOZ-TIF2 silences expression of the CDKN2A locus (p16INK4a and p19ARF), inhibits the triggering of senescence and enhances proliferation, providing conditions favorable to the development of leukemia. Furthermore, we describe that abolishing the MOZ HAT activity of the fusion protein leads to a significant increase in expression of the CDKN2A locus and the number of hematopoietic progenitors undergoing senescence. Finally, we report that inhibition of senescence by MOZ-TIF2 is associated with increased apoptosis, suggesting a role for the fusion protein in p53 apoptosis-versus-senescence balance. Our results underscore the importance of the HAT activity of MOZ, preserved in the fusion protein, for repression of the CDKN2A locus transcription and the subsequent block of senescence, a necessary step for the survival of leukemic cells.Work in our laboratory is supported by the Leukemia and Lymphoma Research Foundation (LLR), Cancer Research UK (CRUK), and the Biotechnology and Biological Sciences Research Council (BBSRC)

The Tumor Microenvironment-Dependent Transcription Factors AHR and HIF-1α Are Dispensable for Leukemogenesis in the Eµ-TCL1 Mouse Model of Chronic Lymphocytic Leukemia.

peer reviewedChronic lymphocytic leukemia (CLL) is the most frequent leukemia in the elderly and is characterized by the accumulation of mature B lymphocytes in peripheral blood and primary lymphoid organs. In order to proliferate, leukemic cells are highly dependent on complex interactions with their microenvironment in proliferative niches. Not only soluble factors and BCR stimulation are important for their survival and proliferation, but also the activation of transcription factors through different signaling pathways. The aryl hydrocarbon receptor (AHR) and hypoxia-inducible factor (HIF)-1α are two transcription factors crucial for cancer development, whose activities are dependent on tumor microenvironment conditions, such as the presence of metabolites from the tryptophan pathway and hypoxia, respectively. In this study, we addressed the potential role of AHR and HIF-1α in chronic lymphocytic leukemia (CLL) development in vivo. To this end, we crossed the CLL mouse model Eµ-TCL1 with the corresponding transcription factor-conditional knock-out mice to delete one or both transcription factors in CD19+ B cells only. Despite AHR and HIF-1α being activated in CLL cells, deletion of either or both of them had no impact on CLL progression or survival in vivo, suggesting that these transcription factors are not crucial for leukemogenesis in CLL

Control of the HOXA9 gene expression in the hematopoietic stem/progenitor cells : role of the epigenetic factors MOZ, MLL and of the polyadenylation factor Symplekin

Mon travail de thèse porte sur l’étude du rôle de l’histone acétyl-transférase MOZ et de l’histone méthyle-transférase MLL dans l’hématopoïèse. Elles contrôlent l’expression de nombreux gènes, nottament des gènes HOX, des facteurs de transcription connus pour leur rôle dans l’hématopoïèse normale et pathologique. Les deux protéines ont des gènes cibles communs tel qu'HOXA9. Ces observations nous ont conduit à rechercher une coopération fonctionnelle entre MOZ et MLL. Nous avons montré que MOZ était associée avec MLL dans les cellules souches/progénitrices humaines CD34+ afin d’activer la transcription des gènes HOXA5, HOXA7 et HOXA9. En effet, les deux protéines interagissent et sont recrutées au niveau de leur promoteur. Nous avons mis en évidence une interférence fonctionnelle entre ces deux facteurs épigénétiques, puisque MOZ est nécessaire au recrutement et à l’activité enzymatique de MLL au niveau des gènes HOXA5, HOXA7 et HOXA9 et réciproquement.Afin de caractériser le mécanisme d’action impliquant la coopération entre MOZ et MLL, nous avons recherché d’autres partenaires associés à ce duo. Nous avons identifié la Symplekin, un membre de la machinerie de polyadénylation. Nous avons mis en évidence l’interaction de la Symplekin avec MOZ et MLL dans les cellules de la lignée hématopoïétique humaine KG1. Les trois protéines sont co-recrutées sur le promoteur du gène HOXA9. Nous avons démontré le rôle ambivalent de la Symplekin. Bien qu’elle soit importante pour la polyadénylation et par conséquent pour la stabilité de l’ARN Hoxa9, la Symplekin empêche le recrutement de MOZ et de MLL au niveau du gène HOXA9, conduisant ainsi à une diminution de sa transcription.My thesis project has consisted of the study of MOZ, and MLL. They are epigenetic regulators. MOZ and MLL activate transcription of HOX genes, which are transcription factors essential during haematopoiesis. MOZ and MLL have some target genes in common. In our study, we characterised a cooperation between MOZ and MLL in human haematopoietic stem/progenitor cells CD34+. They are both recruited onto HOX promoters. MOZ is essential for MLL recruitment, and this is reciprocal. In conclusion, we provided an example of a mechanism involving a direct cross-talk between two histone modifying enzymes.In order to dissect the mechanism of action of this complex, we decided to identify novel proteins interacting with both MOZ and MLL. A member of the RNA polyadenylation machinery has been isolated: Symplekin. We confirmed the interaction between MOZ, MLL and Symplekin in the human haematopoietic immature cell line KG1. We showed that Symplekin is co-recruited to HOXA9 promoter along with MOZ and MLL. We demonstrated the dual role of this member of the polyadenylation machinery. Indeed, besides the fact that Symplekin is important for Hoxa9 polyadenylation, thus its stability, it prevents MOZ and MLL recruitment onto HOXA9 promoter, leading to a decrease of HOXA9 transcription.Our work improved the understanding of the mechanism of action of MOZ and MLL in HOX control

The B-Side of Cancer Immunity: The Underrated Tune

Tumor-infiltrating lymphocytes are known to be critical in controlling tumor progression. While the role of T lymphocytes has been extensively studied, the function of B cells in this context is still ill-defined. In this review, we propose to explore the role of B cells in tumor immunity. First of all we define their dual role in promoting and inhibiting cancer progression depending on their phenotype. To continue, we describe the influence of different tumor microenvironment factors such as hypoxia on B cells functions and differentiation. Finally, the role of B cells in response to therapy and as potential target is examined. In accordance with the importance of B cells in immuno-oncology, we conclude that more studies are required to throw light on the precise role of B cells in the tumor microenvironment in order to have a better understanding of their functions, and to design new strategies that efficiently target these cells by immunotherapy

Interplay between SOX7 and RUNX1 regulates hemogenic endothelial fate in the yolk sac

Endothelial to hematopoietic transition (EHT) is a dynamic process involving the shutting down of endothelial gene expression and switching on of hematopoietic gene transcription. Whilst the factors regulating EHT in hemogenic endothelium (HE) of the dorsal aorta have been relatively well studied, the molecular regulation of yolk sac HE remains poorly understood. Here, we show that SOX7 inhibits the expression of RUNX1 target genes in HE, whilst having no effect on RUNX1 expression itself. We establish that SOX7 directly interacts with RUNX1 and inhibits its transcriptional activity. Through this interaction we demonstrate that SOX7 hinders RUNX1 DNA binding as well as the interaction between RUNX1 and its cofactor CBFβ. Finally, we show by single cell expression profiling and immunofluorescence that SOX7 is broadly expressed across the RUNX1+ yolk sac HE population compared with SOX17. Collectively, these data demonstrate for the first time how direct protein-protein interactions between endothelial and hematopoietic transcription factors regulate contrasting transcriptional programs during HE differentiation and EHT.</jats:p

Method for the Analysis of the Tumor Microenvironment by Mass Cytometry: Application to Chronic Lymphocytic Leukemia.

peer reviewedIn the past 20 years, the interest for the tumor microenvironment (TME) has exponentially increased. Indeed, it is now commonly admitted that the TME plays a crucial role in cancer development, maintenance, immune escape and resistance to therapy. This stands true for hematological malignancies as well. A considerable amount of newly developed therapies are directed against the cancer-supporting TME instead of targeting tumor cells themselves. However, the TME is often not clearly defined. In addition, the unique phenotype of each tumor and the variability among patients limit the success of such therapies. Recently, our group took advantage of the mass cytometry technology to unveil the specific TME in the context of chronic lymphocytic leukemia (CLL) in mice. We found the enrichment of LAG3 and PD1, two immune checkpoints. We tested an antibody-based immunotherapy, targeting these two molecules. This combination of antibodies was successful in the treatment of murine CLL. In this methods article, we provide a detailed protocol for the staining of CLL TME cells aiming at their characterization using mass cytometry. We include panel design and validation, sample preparation and acquisition, machine set-up, quality control, and analysis. Additionally, we discuss different advantages and pitfalls of this technique

Trim33/Tif1γ is involved in late stages of granulomonopoiesis in mice.

IF 2.303International audienceTrim33/Tif1γ (Trim33) is a member of the tripartite motif family. Using a conditional hematopoietic-specific Trim33 knock-out (Trim33(Δ/Δ)) mouse, we showed previously that Trim33 deficiency in hematopoietic stem cells leads to severe defects in hematopoiesis, resembling the main features of human chronic myelomonocytic leukemia. We also demonstrated that Trim33 is involved in hematopoietic aging through TGFβ signaling. Nevertheless, how Trim33 contributes to the terminal stages of myeloid differentiation remains to be clarified. We reveal here the crucial role of Trim33 expression in the control of mature granulomonocytic differentiation. An important component of Trim33-deficient mice is the alteration of myeloid differentiation, as characterized by dysplastic features, abnormal granulocyte and monocyte maturation, and the expansion of CD11b(+)Ly6G(high)Ly6C(low) myeloid cells, which share some features with polymorphonuclear-myeloid-derived suppressor cells. Moreover, in Trim33(Δ/Δ) mice, we observed the alteration of CSF-1-mediated macrophage differentiation in association with the lack of Csf-1 receptor. Altogether, these results indicate that Trim33 deficiency leads to the expansion of a subset of myeloid cells characterizing the myelodysplastic/myeloproliferative neoplasm

Symplekin, a polyadenylation factor, prevents MOZ and MLL activity on HOXA9 in hematopoietic cells

International audienceMOZ and MLL encoding a histone acetyltransferase and a histone methyltransferase, respectively, are targets for recurrent chromosomal translocations found in acute myeloblastic or lymphoblastic leukemia. We have previously shown that MOZ and MLL cooperate to activate HOXA9 gene expression in hematopoietic stem/progenitors cells. To dissect the mechanism of action of this complex, we decided to identify new proteins interacting with MOZ. We found that the scaffold protein Symplekin that supports the assembly of polyadenylation machinery was identified by mass spectrometry. Symplekin interacts and co-localizes with both MOZ and MLL in immature hematopoietic cells. Its inhibition leads to a decrease of the HOXA9 protein level but not of Hoxa9 mRNA and to an over-recruitment of MOZ and MLL onto the HOXA9 promoter. Altogether, our results highlight the role of Symplekin in transcription repression involving a regulatory network between MOZ, MLL and Symplekin