Concomitant heterochromatinisation and down-regulation of gene expression unveils epigenetic silencing of RELB in an aggressive subset of chronic lymphocytic leukemia in males

<p>Abstract</p> <p>Background</p> <p>The sensitivity of chronic lymphocytic leukemia (CLL) cells to current treatments, both <it>in vitro </it>and <it>in vivo</it>, relies on their ability to activate apoptotic death. CLL cells resistant to DNA damage-induced apoptosis display deregulation of a specific set of genes.</p> <p>Methods</p> <p>Microarray hybridization (Human GeneChip, Affymetrix), immunofluorescent <it>in situ </it>labeling coupled with video-microscopy recording/analyses, chromatin-immunoprecipitation (ChIP), polymerase chain reactions (PCR), real-time quantitative PCR (RT-QPCR) and bisulfite genome sequencing were the main methods applied. Statistical analyses were performed by applying GCRMA and SAM analysis (microarray data) and Student's t-test or Mann & Whitney's U-test.</p> <p>Results</p> <p>Herein we show that, remarkably, in a resistant male CLL cells the vast majority of genes were down-regulated compared with sensitive cells, whereas this was not the case in cells derived from females. This gene down-regulation was found to be associated with an overall gain of heterochromatin as evidenced by immunofluorescent labeling of heterochromatin protein 1α (HP-1), trimethylated histone 3 lysine 9 (3metH3K9), and 5-methylcytidine (5metC). Notably, 17 genes were found to be commonly deregulated in resistant male and female cell samples. Among these, <it>RELB </it>was identified as a discriminatory candidate gene repressed in the male and upregulated in the female resistant cells.</p> <p>Conclusion</p> <p>The molecular defects in the silencing of <it>RELB </it>involve an increase in H3K9- but not CpG-island methylation in the promoter regions. Increase in acetyl-H3 in resistant female but not male CLL samples as well as a decrease of total cellular level of RelB after an inhibition of histone deacetylase (HDAC) by trichostatin A (TSA), further emphasize the role of epigenetic modifications which could discriminate two CLL subsets. Together, these results highlighted the epigenetic <it>RELB </it>silencing as a new marker of the progressive disease in males.</p

Apport des dosages des interleukines 10 et 6, de l'interféron-gamma et de l'intérféron gamma inducible protein-10 par cytométrie en flux dans le diagnostic et le suivi des lymphomes intra-oculaires et cérébraux primitifs

PARIS-BIUP (751062107) / SudocSudocFranceF

Quantification de l'apoptose des cellules souches périphériques CD34+

PARIS-BIUP (751062107) / SudocSudocFranceF

Place du dosage de l'interleukine-10, de l'interleukine-6, du CD25 soluble, de MIP-1a, de RANTES et de l'interféron-g par cytométrie en flux dans le pronostic des lymphomes intraoculaires primitifs

CHATENAY M.-PARIS 11-BU Pharma. (920192101) / SudocSudocFranceF

Site Internet "Hématobio. com" (diagnostic des hémopathies malignes)

PARIS-BIUP (751062107) / SudocSudocFranceF

Impact des cellules NK dans le traitement de la Leucémie Lymphoïde Chronique et de la maladie de Waldenström par anticorps anti-CD20 optimisés

L introduction de l anticorps monoclonal anti-CD20 rituximab a transformé le traitement de deux hémopathies du sujet âgé, la leucémie lymphoïde chronique (LLC) et la maladie de Waldenström (MW), mais ne permet pas l obtention d une guérison. Des anti-CD20 optimisés, dont le LFB-R603, ont été développés pour augmenter leur affinité pour les récepteurs Fc RIIIa et induire une plus forte cytotoxicité médiée par un anticorps (ADCC). Etant donné le rôle des cellules NK dans l ADCC, nous avons réalisé leur expertise phénotypique et fonctionnelle. Les cellules NK des sujets âgés préservent la majorité de leurs caractéristiques phénotypiques et fonctionnelles avec des signes de maturité accentués. Leur fonction ADCC est cependant parfaitement préservée. Dans la LLC, les cellules NK présentent une expression normale des récepteurs inhibiteurs et activateurs. Bien qu elles soient peu activées, leurs principales fonctions sont préservées, incluant la cytotoxicité naturelle et l ADCC induite par le rituximab contre des cellules Raji. Les activités ADCC in vitro montrent que le LFB-R603 est plus efficace que le rituximab à faibles concentrations, aussi bien contre les Raji que les cellules B de LLC. Chez les patients atteints de LLC avec délétion 17p (del17p), incluant TP53, nous observons une diminution de l ADCC in vitro, probablement en raison d une résistance des cellules de LLC avec del17p. Cependant, de faibles réponses ADCC restent détectables avec le LFB-R603. Enfin, une étude phénotypique et fonctionnelle des cellules NK a été réalisée dans la MW. Cette étude montre que des anti-CD20 optimisés peuvent avoir leur place dans l arsenal thérapeutique de la LLC et de la MWThe introduction of rituximab, an anti-CD20 monoclonal antibody (mAb), has transformed the treatment of two hematological diseases of the elderly, chronic lymphocytic leukemia (CLL) and Waldenström s macroglobulinemia (WM), but it doesn t allow obtaining a recovery. Optimized anti-CD20 mAbs, including LFB-R603, have been developed to increase their affinity to Fc RIIIa and thus induce a higher mAb-mediated cytotoxicity (ADCC). Given the role of NK cells in ADCC, we performed a phenotypic and functional expertise of these cells. NK cells from older subjects preserved most of their phenotypic and functional characteristics with accentuated maturity features. It is important to note that their ADCC function is well preserved. In patients with CLL, NK cells show normal expression of inhibitory and activating receptors. Although most of NK cells are poorly activated, their main functions are preserved, including cytotoxicity against K562 target and rituximab induced ADCC against Raji cells. ADCC activity in vitro shows that LFB-R603 is more efficient than rituximab in inducing ADCC at low doses against Raji cells and CLL cells. For patients with 17p deletion (del17p), including TP53, an anti-CD20 ADCC efficiency decrease was observed, probably linked to a potential resistance of CLL B cells with del17p. However, low ADCC responses were observed with LFB-R603. Finally, we have evaluated the phenotypic and functional capacities of NK cells in MW. In conclusion, these findings highlight that optimized anti-CD20 mAb, including LFB-R603, may participate to the control of CLL and WM, and prompt further investigations for clinical applications in combination with chemotherapiesPARIS-BIUSJ-Biologie recherche (751052107) / SudocSudocFranceF

Supporting Information Homotrimerization Approach in the Design of Thrombospondin-1 Mimetic Peptides with Improved Potency in Triggering Regulated Cell Death of Cancer Cells

International audienceIn order to optimize the potency of the first serum-stable peptide agonist of CD47 (PKHB1) in triggering regulated cell death of cancer cells, we designed a maturation process aimed to mimic the trimeric structure of the thrombospondin-1/CD47 binding epitope. For that purpose, an N-methylation scan of the PKHB1 sequence was realized to prevent peptide aggregation. Structural and pharmacological analyses were conducted in order to assess the conformational impact of these chemical modifications on the backbone structure and the biological activity. This structure–activity relationship study led to the discovery of a highly soluble N-methylated peptide that we termed PKT16. Afterward, this monomer was used for the design of a homotrimeric peptide mimic that we termed [PKT16]3, which proved to be 10-fold more potent than its monomeric counterpart. A pharmacological evaluation of [PKT16]3 in inducing cell death of adherent (A549) and nonadherent (MEC-1) cancer cell lines was also performed

Thrombospondin-1 Mimetic Agonist Peptides Induce Selective Death in Tumor Cells: Design, Synthesis, and Structure–Activity Relationship Studies

International audienceAbstract ImageThrombospondin-1 (TSP-1) is a glycoprotein considered as a key actor within the tumor microenvironment. Its binding to CD47, a cell surface receptor, triggers programmed cell death. Previous studies allowed the identification of 4N1K decapeptide derived from the TSP-1/CD47 binding epitope. Here, we demonstrate that this peptide is able to induce selective apoptosis of various cancer cell lines while sparing normal cells. A structure–activity relationship study led to the design of the first serum stable TSP-1 mimetic agonist peptide able to trigger selective programmed cell death (PCD) of at least lung, breast, and colorectal cancer cells. Altogether, these results will be of valuable interest for further investigation in the design of potent CD47 agonist peptides, opening new perspectives for the development of original anticancer therapies