Elucidating the mechanism of action of domatinostat (4SC-202) in cutaneous T cell lymphoma cells

Abstract

BackgroundTargeting epigenetic modifiers is effective in cutaneous T cell lymphoma (CTCL). However, there is a need for further improvement of this therapeutic approach. Here, we compared the mode of action of romidepsin (FK228), an established class I histone deacetylase inhibitor, and domatinostat (4SC-202), a novel inhibitor of class I HDACs, which has been reported to also target the lysine-specific histone demethylase 1A (LSD1).MethodsWe performed MTS assays and flow cytometric analyses of propidium iodide or annexin V-stained cells to assess drug impact on cellular proliferation, cell cycle distribution, and survival. Histone acetylation and methylation as well as caspase activation was analyzed by immunoblot. Gene expression analysis was performed using NanosString technology. Knockdown and knockout of LSD1 was achieved with shRNA and CRISPR/Cas9, respectively, while the CRISPR/Cas9 synergistic activation mediator system was used to induce expression of endogenous HDACs and LSD1. Furthermore, time-lapse fluorescence microscopy and an in vitro tubulin polymerization assay were applied.ResultsWhile FK228 as well as 4SC-202 potently induced cell death in six different CTCL cell lines, only in the case of 4SC-202 death was preceded by an accumulation of cells in the G2/M phase of the cell cycle. Surprisingly, apoptosis and accumulation of cells with double DNA content occurred already at 4SC-202 concentrations hardly affecting histone acetylation and methylation, and provoking significantly less changes in gene expression compared to biologically equivalent doses of FK228. Indeed, we provide evidence that the 4SC-202-induced G2/M arrest in CTCL cells is independent of de novo transcription. Furthermore, neither enforced expression of HDAC1 nor knockdown or knockout of LSD1 affected the 4SC-202-induced effects. Since time-lapse microscopy revealed that 4SC-202 could affect mitotic spindle formation, we performed an in vitro tubulin polymerization assay revealing that 4SC-202 can directly inhibit microtubule formation.ConclusionsWe demonstrate that 4SC-202, a drug currently tested in clinical trials, effectively inhibits growth of CTCL cells. The anti-cancer cell activity of 4SC-202 is however not limited to LSD1-inhibition, modulation of histone modifications, and consecutive alteration of gene expression. Indeed, the compound is also a potent microtubule-destabilizing agent.Electronic supplementary materialThe online version of this article (10.1186/s13045-019-0719-4) contains supplementary material, which is available to authorized users.

Analysis of Dual Class I Histone Deacetylase and Lysine Demethylase Inhibitor Domatinostat (4SC-202) on Growth and Cellular and Genomic Landscape of Atypical Teratoid/Rhabdoid

“…Indeed, proliferation and viability assays using several different techniques suggests that 4SC-202 is both cytotoxic and cytostatic in two-and three-dimensional cell culture models. In a number of different cancer models studies, 4SC-202 exerts both cytotoxic and cytostatic effects and arrested cell growth in the G2M phase [46]. In this study, 4SC-202 exerted cytostatic properties in the ATRT three-dimensional scaffold model, confirming results obtained in the two-dimensional cell culture.…”

LSD1/KDM1A inhibitors in clinical trials: advances and prospects

“…Domatinostat (4SC-202), a class I histone deacetylase/LSD1 dual inhibitor, is currently under assessment in phase I trial in patients with advanced hematological malignancies [118]. Wobser et al recently reported that 4SC-202 directly inhibited microtubule formation and effectively suppressed growth of cutaneous T cell lymphoma (CTCL) cells [119]. Studies have showed that LSD1 mediates epidermal growth factor (EGF) signaling, LSD1 knockdown or inhibition suppressed both intrinsic and EGF-induced cell migration in SKOV3 and HO8910 cells [120].…”

Anticancer Therapy with HDAC Inhibitors: Mechanism-Based Combination Strategies and Future Perspectives

“…Originally believed to impair the function of the epigenetic eraser lysine-specific demethylase 1 (LSD1) which participates in the CoREST complex formation alongside HDACs 1 and 2, the class I HDACi unexpectedly turned out to inhibit tubulin polymerisation as well. Ongoing clinical trials in Phases I and II investigate its efficacy in hematological and gastrointestinal cancers [258,259].…”