Pb1856: demethylating agents and histone deacetylase inhibitors as new therapeutic strategies in chronic lymphocytic leukemia

Background: Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in Western countries and is characterized by the accumulation of long-lived resting clonal B lymphocytes in peripheral blood, bone marrow and secondary lymphoid organs. This disease has a notable heterogeneous clinical course, with different prognosis and distinct treatment approaches. However, the exact etiology is still unknown. From global hypomethylation and hypermethylation of single-gene promoters, to altered regulation of histone modifying enzymes, epigenetic dysregulation plays an important role in CLL development and progression. Due to reversible nature of epigenetic alteration, modulation of these mechanisms might constitute a successful therapeutic application in CLL. Aims: With this work we intended to evaluate the therapeutic potential of four epigenetic modulators, two demethylating agents (DNMTis), azacytidine (AZA) and decitabine (DAC), and two histone deacetylase inhibitors (HDACis), vorinostat (SAHA) and Panobinostat (PAN), in monotherapy and in combination, in an in vitro model of CLL. Methods: To this propose, the CLL cell line HG-3 was incubated in the absence and presence of epigenetic modulators, in monotherapy and in combination (0.1µM AZA+0.1µM SAHA, 0.1µM AZA+0.1nM PAN, 0.5µM DAC+0.1µM SAHA and 0.5µM DAC+0.1nM PAN) for 72h. Metabolic activity was evaluated by resazurin assay. Cell death was assessed by flow cytometry (FC), using the Annexin V and 7-AAD double staining, and by optic microscopy using May-Grünwald-Giemsa staining. Cell cycle analysis was evaluated by FC using a PI/RNAse solution. The statistical analysis was performed, considering a significance level of 95%. Results: Our results showed a decreased metabolic activity induced by all the epigenetic modulators in a time- and dose-dependent manner. After 48h, the IC50 was 2.1μM for AZA, 2.9μM for DAC, 0.5μM for SAHA and 5.5nM for PAN. The metabolic activity decrease was even more relevant in the combination scheme of small doses of DNMTis plus HDACis (0.1µM AZA+0.1µM SAHA, 0.1µM AZA+0.1nM PAN, 0.5µM DAC+0.1µM SAHA and 0.5µM DAC+0.1nM PAN), reaching a 75% decrease in metabolic activity. DNMTis induced a cytotoxic effect mediated by apoptosis, confirmed by morphology analysis, namely the presence of apoptotic blebbings. When in combination with HDACis, DAC induced a higher percentage of apoptotic cells comparing to combinations with AZA. All epigenetic modulators, excepting PAN, induced a cytostatic effect, with cell cycle arrest in G0/G1 phase. Therapeutic combinations also induced cell cycle arrest in G0/G1 phase, with exception to AZA+SAHA. Summary/Conclusion: These results suggest that epigenetic modulators may constitute a new therapeutic strategy for chronic lymphocytic leukemia treatment. This is even more relevant when combining DNA methyltransferase inhibitors with histone deacetylase inhibitors. Studies including primary cell culture from CLL patients should be performed to confirm these results. # equally contributing authors.