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

Abstract

[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.