Novel CTCF binding at a site in exon1A of BCL6 is associated with active histone marks and a transcriptionally active locus

BCL6 is a zinc-finger transcriptional repressor, which is highly expressed in germinal centre B-cells and is essential for germinal centre formation and T-dependent antibody responses. Constitutive BCL6 expression is sufficient to produce lymphomas in mice. Deregulated expression of BCL6 due to chromosomal rearrangements, mutations of a negative autoregulatory site in the BCL6 promoter region and aberrant post-translational modifications have been detected in a number of human lymphomas. Tight lineage and temporal regulation of BCL6 is, therefore, required for normal immunity, and abnormal regulation occurs in lymphomas. CCCTC-binding factor (CTCF) is a multi-functional chromatin regulator, which has recently been shown to bind in a methylation-sensitive manner to sites within the BCL6 first intron. We demonstrate a novel CTCF-binding site in BCL6 exon1A within a potential CpG island, which is unmethylated both in cell lines and in primary lymphoma samples. CTCF binding, which was found in BCL6-expressing cell lines, correlated with the presence of histone variant H2A.Z and active histone marks, suggesting that CTCF induces chromatin modification at a transcriptionally active BCL6 locus. CTCF binding to exon1A was required to maintain BCL6 expression in germinal centre cells by avoiding BCL6-negative autoregulation. Silencing of CTCF in BCL6-expressing cells reduced BCL6 mRNA and protein expression, which is sufficient to induce B-cell terminal differentiation toward plasma cells. Moreover, lack of CTCF binding to exon1A shifts the BCL6 local chromatin from an active to a repressive state. This work demonstrates that, in contexts in which BCL6 is expressed, CTCF binding to BCL6 exon1A associates with epigenetic modifications indicative of transcriptionally open chromatin

Suppression of BCL6 Function by HDAC Inhibitor Mediated Acetylation and Chromatin Modification Enhances BET Inhibitor Effects in B-cell Lymphoma Cells

Multiple genetic aberrations in the regulation of BCL6, including in acetyltransferase genes, occur in clinically aggressive B-cell lymphomas and lead to higher expression levels and activity of this transcriptional repressor. BCL6 is, therefore, an attractive target for therapy in aggressive lymphomas. In this study romidepsin, a potent histone deacetylase inhibitor (HDACi), induced apoptosis and cell cycle arrest in Burkitt and diffuse large B-cell lymphoma cell lines, which are model cells for studying the mechanism of action of BCL6. Romidepsin caused BCL6 acetylation at early timepoints inhibiting its function, while at later timepoints BCL6 expression was reduced and target gene expression increased due to chromatin modification. MYC contributes to poor prognosis in aggressive lymphoma. MYC function is reduced by inhibition of chromatin readers of the bromodomain and extra-terminal repeat (BET) family, which includes BRD4. The novel combination of romidepsin and JQ1, a BRD4 inhibitor was investigated and showed synergy. Collectively we suggest that the combination of HDACi and BRD4i should be pursued in further pre-clinical testing.Funding: The work was supported by grants SAF2014-53526-R and SAF2017-88026-R from MINECO, Spanish Government, to M.D.D. and J.L. (partially funded by FEDER program from European Union). M.G.C. was recipient of a “Marcos Fernández” fellowship from Leukemia and Lymphoma foundation. L.G.G. was recipient of a FPI fellowship from Spanish Government

Effects of the histone deacetylase inhibitor romidepsin in lymphoma B-cells and BCL6 regulation

Resumen del póster presentado en el 23rd Biennial Congress of the European Association for Cancer Research, celebrado del 5 al 8 de julio de 2014 en Munich (Alemania). Abstract publicado en: European Journal of Cancer 50(Suppl. 5): S106 (2014). ISSN: 0959-8049 DOI: 10.1016/S0959-8049(14)50393-0[Introduction]: Histone deacetylase inhibitors (HDACi) have enormous potential in the treatment of hematologic malignancies. Romidepsin is a HDACi that is effective in T-cell lymphomas, but its potential role in B-cell lymphoma is largely unknown. We have previously shown that BCL6 gene is regulated through epigenetic mechanisms in lymphoma cells. BCL6 is a transcriptional repressor highly expressed within germinal centres B-cells, being required for the germinal centre formation and T-dependent antibody responses. BCL6 deregulation has been implicated in B-cell lymphoma pathogenesis but the molecular mechanisms involved in its regulation remain elusive. In this study we have tested the effect of romidepsin on proliferation, cell death and differentiation of B-cell lymphoma cells with different origins as well as the effect of this HDACi on BCL6 gene regulation. [Objectives]: To analyze the effects of the HDACi romidepsin in the cell cycle, apoptosis and differentiation of B-cell lymphoma cell lines through the regulation of BCL6 gene. To study the romidepsin effect on BCL6 gene epigenetic regulation in germinal centre B-cell lymphoma cells. [Methods]: We screened lymphoma-B cells derived from Burkitt’s lymphoma or from diffuse large B cell lymphoma (DLBCL) using a short-term treatment with romidepsin. Cell metabolic activity (WST1 method), cell cycle (propidium iodide staining) and apoptosis (Annexin V binding, PARP cleavage) analysis were performed. mRNA and protein expression were analysed by RT-PCR and western blot methods, respectively. Flow cytometry was used to measure differentiation by analyzing plasmatic cells surface markers. [Results and Conclusions]: In this study we analyzed the effects of romidepsin in human B-cell lymphomas from different origins. Cytotoxic effect of romidepsin was found to be cell-specific and dose dependent. Some cell lines were sensitive showing clear PARP cleavage with 2nM of romidepsine while others were resistant to high dose of romidepsin showing that PARP was uncleaved. These results were consistent with the results observed by annexing staining. Depending on the cell line, we observed cell cycle arrest in G0/G1 phase followed by either apoptosis or followed by differentiation to plasmatic cells. BCL6 downregulation at mRNA and protein levels was found to be a common feature in BCL6 expressing germinal centre derived cell lines upon treatment with the HDACi. The expression of genes involved in cell cycle arrest, apoptosis and markers of the plasma cell transcriptional program were also analyzed. An increase in p27 and/or p21 (cyclin-dependent kinase inhibitors) was accompanied by an induction of genes involved in plasmatic cell differentiation such as BLIMP-1 and IRF4. Our results indicate differential effects of the HDACi romidepsin in lymphoma B cells, in a cell type and dose dependent manner. Histone acetylation may play a role in the treatment of B Lymphomas by epigenetically regulating the BCL6 oncogene.Authors are grateful to ISCIII-FIS, Celgene, Vistare and Leucemia-Linfoma Foundations.Peer Reviewe

Regulation of BCL6 in aggressive B-cell lymphoma: Effects of epigenetic drugs

Resumen del póster presentado aI 1er Simposio Educacional de la Asociación Española de Investigación sobre el Cáncer (ASEICA), celebrado en la Universidad Autónoma de Madrid del 14 al 15 de septiembre de 2017.[Introduction]: BCL6 is an important transcriptional repressor considered one of the master regulators of the germinal center reaction. BCL6 controls the exit of the B-cells from the germinal center in order to differentiate toward plasma cells. In some lymphomas deregulated expression of BCL6 is detected. This deregulation is frequently caused by genetic modifications like translocations or point mutations and epigenetic mechanisms are also involved. Previous results of our group have demonstrated that CTCF regulates BCL6 expression through epigenetic mechanisms in lymphoma cells (Batlle-López et al. Oncogene 2015). Therapy with epigenetic drugs has an enormous potential for cancer treatment. Romidepsin is an inhibitor of histone deacetylases (HDACi) approved for the treatment of some Tcell lymphomas, but its role on B-cell lymphoma has not been thoroughly investigated. JQ1 is a BET bromodomain inhibitor that represses the expression of some genes as MYC, often found deregulated in lymphomas. In this study, we analyze the effects of Romidepsin and/or JQ1 treatment in different aggressive B-cell lymphoma cells where BCL6 and/or MYC are deregulated. [Objectives]: To analyze the effects of the HDACi Romidepsin and the BET bromodomain inhibitor JQ1 on cell cycle, apoptosis and differentiation of B-cell lymphoma cell lines. To study the regulation of BCL6 mediated by these drugs and the epigenetic regulation of BCL6 by CTCF. [Methods]: Lymphoma-B cells derived from Burkitt Lymphoma or from Diffuse Large B-Cell Lymphoma (DLBCL) were treated with Romidepsin and/or JQ1 drugs. BCL6 and MYC loci status were analyzed by Fluorescence in situ hybridization (FISH). Cell metabolic activity (WST-1 assay), cell proliferation (cell counting), cell cycle (propidium iodide staining and p27 and/or p21 protein expression) and apoptosis (Annexin-V binding and PARP cleavage protein expression) analysis were performed. B-cell differentiation was studied by analyzing BCL6 protein expression by Western-Blot and plasmatic cell surface markers expression measured by flow cytometry. Luciferase assays were used to measure the changes on the BCL6 repression activity upon HDACi treatment. BCL6 acetylation after Romidepsin treatment was assessed by protein immunoprecipitation. Chromatin immunoprecipitation (ChIP) experiments were performed to study the role of CTCF on BCL6 epigenetic regulation.[Results and Conclusions]: In this study, we analyzed the effects of the treatment with Romidepsin and JQ1 alone or in combination in different human B-cell lymphoma cell lines. A decrease in the metabolic activity and inhibition on cell proliferation were found with the different treatments. Romidepsin treatment alone and in combination with JQ1 induced apoptosis in lymphoma cells. Apoptosis was demonstrated by PARP cleavage and an increase in the number of annexin-V positive cells measured by flow cytometry. All cell lines analyzed treated with JQ1 alone showed cell cycle arrest in G0/G1 phase and an increase in p21 and 27 protein expression, which is consistent with the cell cycle arrest. Protein levels of BCL6 were found downregulated in BCL6 expressing cell lines upon Romidepsin and/or JQ1 treatments. Simultaneously, an increase in genes of the plasmatic differentiation program as PRMD1/Blimp1 was observed. This effect was accompanied by the increase of plasmatic surface markers. In presence of Romidepsin an increase of BCL6 acetylation and a decrease of its repressor activity was observed. CTCF positively regulates BCL6 by its binding to the exon1 of BCL6, accompanied by active chromatin marks. In the presence of Romidepsin, this binding is reverted and repressive chromatin marks are incorporated. Altogether, our results show differential effects of the treatment with Romidepsin and/or JQ1 in lymphoma B cells. Finally, histone acetylation is important in the epigenetic regulation of BCL6 mediated by CTCF.Peer Reviewe

El inhibidor de histonas deacetilasa romidepsina induce diferenciación en linfomas de origen centrogerminal a través de la regulación BCL6

Resumen del póster presentado al LVIII Congreso Nacional de la Sociedad Española de Hematología y Hemoterapia, celebrado en Santiago de Compostela del 20 al 22 de octubre de 2016.[Introducción]: BCL6 es un represor transcripcional indispensable para la generación del centro germinal linfoide. La expresión desregulada de BCL6 está implicada en el desarrollo de linfomas. Nuestro grupo ha demostrado que BCL6 se regula epigenéticamente en células de linfoma. La romidepsina es un inhibidor de histona deacetilasas aprobado para el tratamiento de algunos tipos de linfoma de células T, pero su potencial frente a los linfomas de células B no ha sido investigado en profundidad. El objetivo de este trabajo es analizar los efectos de la romidepsina en apoptosis, ciclo celular y diferenciación, así como estudiar la regulación epigenética de BCL6 mediada por este fármaco en líneas celulares de linfoma de origen centro germinal. [Métodos]: Los efectos de distintas concentraciones de romidepsina y a distintos tiempos se evaluaron en una variedad líneas celulares derivadas de pacientes con linfoma de Burkitt o de linfoma difuso de célula grande (LBDCG). Se llevaron a cabo estudios de proliferación, análisis de apoptosis (unión de Annexina V) y ciclo celular (ioduro de propidio). Los niveles de RNA y proteína se analizaron mediante RT-PCR y westem blot, respectivamente. Se estudiaron marcadores de superficie propios de los diferentes estadios de diferenciación de células B por citometría de flujo. El efecto directo de la romidepsina sobre la regulación de la actividad represora de BCL6 se midió mediante ensayos luciferasa y el análisis de la acetilación de BCL6 por inmunoprecipitación de proteína. Finalmente, se analizó la regulación de BCL6 en presencia de romidepsina, mediada por el factor de transcripción CTCF, utilizando la técnica de inmunoprecipitación de cromatina (ChiP). [Resultados]: El tratamiento con romidepsina induce apoptosis en la mayoría de las líneas de linfoma de origen centrogerminal, aunque con un grado variable de respuesta y de forma dosis y tiempo dependiente. Todas las líneas analizadas muestran una parada del ciclo celular en fase GO/G 1 seguido de apoptosis y/o diferenciación. Las líneas de linfoma de Burkitt mostraban una disminución en la expresión de BCL6, acompañada del aumento de alguno de sus genes diana (CCND2, P21 y P27), junto con la bajada de los niveles de otros genes de centro germinal como PAX5 y BACH2. Al mismo tiempo se observó un incremento en genes propios del programa de diferenciación a célula plasmática como PRMD1/BLIMP1. En las líneas de linfoma difuso de célula grande, se produce disminución de BCL6, sin cambios importantes en los genes del programa de diferenciación plasmática. Este efecto va acompañado de un aumento del marcador de superficie CD138 indicador de diferenciación plasmática. Por otro lado, en presencia de romidepsina se observa un aumento en la acetilación de BCL6 y una disminución su actividad represora. CTCF regula positivamente a BCL6 mediante su unión al exon 1 del gen, acompañado de marcas de cromatina activa. En presencia de romidepsina, esta unión es revertida y se incorporan marcas de cromatina represiva. [Conclusiones]: Nuestros resultados indican que la romidepsina induce apoptosis en los LNH de origen centrogerminal y desencadena el programa de célula plasmática. La regulación epigenética de BCL6 está implicada en este proceso.Peer Reviewe