Epigenetics in acute and chronic lymphocytic leukaemia: therapeutic potential and methylation profile evaluation

• Autor(a) principal: Ribau, Beatriz Baptista
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10773/24277
• topic: Acute Lymphocytic Leukaemia (ALL); Chronic Lymphocytic Leukaemia (CLL); DNA methylation; Tumour supressor genes; DNA hypomethylating agents; Azacytidine; Decitabine; Histone deacetylase inhibitors; Panobinostat; Vorinostat

Cancer is a multifactorial disease caused by multiple unrepaired DNA mutations and/or epigenetic changes, occurred during cell life cycle that the organism cannot detect and reverse. Acute lymphocytic leukaemia (ALL) is a disease characterized by a rapidly proliferation of early lymphoid precursors (namely lymphoblast), arrested in an early stage of its development, that replace normal hematopoietic cells in the bone marrow and cause decline in the production of normal marrow cells. Chronic lymphocytic leukaemia (CLL) is a disorder characterized by a progressive accumulation of lymphocytes that are apoptosis resistant, being the most common type of chronic leukaemia found in adults and among men over 55 years, hardly affecting children. Each type of cancer not only has its own recurrent genomic aberrations but also has its characteristic epigenetic changes which demonstrate its importance to cancer pathogenesis. Epigenetic modifications participate in the silencing and/or activation of genes that are the main key to cell and tissue differentiation. The most studied epigenetic changes in cancer are DNA methylation of CpG islands in regulatory regions of gene and post- translational modifications of histones. In ALL and CLL, aberrant DNA methylation, such as hypermethylation of CpG islands of gene promoters and abnormal histone deacetylation have been associated with cancer. Thus, DNA hypomethylating agents and histone deacetylase inhibitors are two categories of epigenetic therapies that are being studied as possible new therapies for ALL and CLL. The present study has two main goals. First, evaluate the therapeutic potential of two DNA methyltransferase inhibitors (Azacytidine and Decitabine) and two histone deacetylase inhibitors (Panobinostat and Vorinostat), in monotherapy and in combination, in two B-ALL cell lines (697 and KOPN8 cells) and in peripheral blood mononuclear cells (PBMC) obtained from CLL patients. Second, evaluate the methylation profile of the B-ALL cell lines and CLL samples. To this end, two B-ALL cell lines (697 and KOPN8 cells) and 31 individuals participated in this study, 21 patients diagnosed with CLL and 10 non¬neoplastic controls. ALL cell lines (KOPN8 and 697 cells) and PBMCs from CLL patients were incubated with 5-AC, DAC, LBH589, and SAHA, in monotherapy (single dose and daily administration) and in combination, during 72h and 48h, respectively. The cytotoxic effect of drugs was evaluated by fluorometric microculture cytotoxicity assay (FMCA). Cell death and cell cycle were analysed by flow cytometry through annexin V/Propidium iodide and PI/RNAse assays, respectively. Methylation patterns were determined by MS-MLPA and levels of 5-mC were determined by intracellular labelling with anti-5-mC antibodies by flow cytometry. CD5 and CD19 antibodies were used to identify normal B cells (CD5-/CD19+), neoplastic B cells (CD5+/CD19+), normal T cells (CD5+/CD19-) and other mononuclear cells (CD5"/CD19-). Methylation study was determined by MS-MLPA. For the methylation study, DNA was extracted from samples and controls by salting out protocol. The evaluation of the differences between monotherapy doses, combination schemes, cell cycle and cell death data were determined by applying the nonparametric ANOVA Kruskal-Wallis test (Dunn's multiple comparisons test). MS-MLPA obtained data were analysed using nonparametric ANOVA Kruskal- Wallis test (Dunn's multiple comparisons test) for cell lines and chi-squared (X2) test for patients. A probability value of p<0.05 was considered statistically significant for both cell lines and patient studies. The in vitro studies showed that epigenetic modulators have a cytotoxic effect, being able to reduce cell viability in B-ALL cell lines, 697 and KOPN8. All four therapies demonstrated to have effect on cell viability that is concentration, incubation time and cell type dependent. 697 cells seem to be more sensitive to all four therapies, since the IC50 dose was reached with lower concentrations compared with KOPN8 cell line, where the IC50 was not reached with tested doses. In 697 cells, DAC demonstrated to have more effect than 5-AC, when comparing same doses. For KOPN8 cells, the combinations studied do not show beneficial results compared to those obtained in monotherapy. On the other hand, for 697 cells, the combination of LBH589 and SAHA with DAC leads to a higher reduction in cell viability compared to those observed in cells treated with drugs in monotherapy, independent of the administration scheme. The best administration scheme was the administration of LBH589 and SAHA, 3 hours after the administration of DAC. All four tested drugs induced cell death by apoptosis, confirmed by changes in morphologic aspects. It was also observed that those drugs, in a global way, they showed antiproliferative effect, inducing cell cycle arrest at G0/G1 phase. Moreover, 5-AC and DAC in monotherapy and in therapeutic combination, induced a decrease in 5-mC levels. Methylation data showed that none condition tested caused an alteration on gene promoter methylation levels, compared with control. CLL studies also demonstrated that DNA hypomethylating agents and histone deacetylase inhibitors induced similar effects and all four therapies reduced cell viability in a dose-dependent manner. For the combination of therapies, none demonstrated to be statistically significant in comparison to monotherapy. 5-AC in combination with LBH589 obtained better results on cell viability than the ones obtained for monotherapy, causing an increased reduction on cell viability. However, this reduction was not significant. The results obtained for cells treated with the epidrugs, in a daily dose administration scheme, were very similar to the combination therapy, where none demonstrated to be statistically significant in comparison to monotherapy. DNA hypomethylating agent therapies and histone deacetylase inhibitors arrested cell cycle in S phase. Neoplastic B lymphocytes demonstrated to be more affected than normal B and T cells, which was expected. Only LBH589 and SAHA IC50 doses were considered statistically significant, compared with control, with p<0.01 for both conditions. However, all four therapies demonstrated to induce much more apoptosis on neoplastic B lymphocytes than on normal lymphocytes and other mononuclear cells. Methylation data showed that CLL patients had a significant higher methylation frequency of MSH6 (86%, 18/21), KLLN (67%, 14/21), WT1 (86%, 18/21) and GATA5 (71%, 15/21) gene promoters, when compared with controls, suggesting the involvement of DNA methylation on CLL development. Results also suggest that the methylation of tumour suppressor genes is a common event in CLL patients and that epigenetic modulators induce a cytotoxic effect in cells, reducing cell viability in a time- and dose-dependent manner. Therefore, these results are promising and encourage further studies