Figure 2. Selective HDAC1,2 inhibition increases H3K27ac in EZH2^GOF DLBCL cells.

A: In vitro enzyme assays using recombinant HDAC proteins to determine the specificity of ACY-957 and ACY-1044 towards HDAC1, 2 and 3. Numbers in the graphs represent IC50 values obtained for an inhibitor-enzyme combination in the in vitro HDAC assays at a 95% confidence level. Compounds were diluted in DMSO to 50 fold the final concentration and a ten point three fold dilution series was made. The compounds were diluted in assay buffer (50 mM HEPES, pH 7.4, 100 mM KCl, 0.001% Tween-20, 0.05% BSA, 20 μM tris(2-carboxyethyl)phosphine) to 6 fold their final concentration. The HDAC enzymes (purchased from BPS Biosciences) were diluted to 1.5 fold their final concentration in assay buffer. The tripeptide substrate (synthesized in house) and trypsin at 0.05 μM final concentration were diluted in assay buffer at 6 fold their final concentration. Five μM compounds and 20 μM of enzyme were added to wells of a black, opaque 384 well plate in duplicate. Enzyme and compound were incubated together at room temperature for 10 minutes. Five l of substrate was added to each well, the plate was shaken for 60 seconds and placed into a Victor 2 microtiter plate reader. The development of fluorescence was monitored for 60 min and the linear rate of the reaction was calculated. The IC50 was determined using Graph Pad Prism by a four parameter curve fit. SUDHL4 (B) and Karpas-422 (C) cells were treated with increasing amounts of ACY-957 and whole cell lysates were prepared following a 24h treatment. Western blot analysis of H3K9K14ac and H4K5ac was done. Histone H3 and H4 were used as controls. D: Western blot analysis of whole cell lysate prepared from HDAC1^Fl/Fl, HDAC2^Fl/Fl or HDAC3^Fl/Fl fibrosarcoma cells following Ad-Cre infection. Lysates were prepared 72h post Ad-Cre infection. E: SUDHL4 and Karpas-422 cells were treated with DMSO, 2μM ACY-957 or 2μM SAHA for 24h and western blot analysis of whole cell lysate with anti-H3K23ac was done. Histone H3 served as the loading control. F: SUDHL4 and Karpas-422 cells were treated with either DMSO, 2μM ACY-957 or 2μM ACY-1044 and western analysis with anti-H3K23ac was performed.