Fig. 1: Ricolinostat treatment leads to enhanced CD38 expression on myeloma cells.

a CD38 expression on MM.1S cells (n = 13 experiments) before and after treatment with ricolinostat at the given final concentrations (fc) for the given time intervals. Bar diagram shows CD38 expression as normalized MFI of treated vs. untreated MM.1S cells. b The overlay histogram (left) shows flow cytometric analysis of CD38 expression on MM.1S cells cultured in the absence or presence of ricolinostat for 72 h. The overlay histogram (right) shows change in CD38 expression on MM.1S cells during ricolinostat treatment at a fc of 5 µM. c The overlay histogram shows CD38 expression on untreated MM.1S cells, 24 h after ricolinostat treatment at a fc of 5 µM, and 72 h after subsequent removal of the drug. d Bar diagram shows CD38 expression level as normalized MFI on untreated MM.1S cells (n = 3 experiments), 24 h after ricolinostat treatment at a fc of 5 µM, and 72 h after subsequent removal of the drug. e CD38 RNA levels on MM.1S cells (n = 4 experiments) by quantitative reverse transcription PCR (qRT-PCR) were quantified after incubation with ricolinostat at a fc of 5 µM for 48 h. f ChIP of histone 3 lysine 27 acetylation in the CD38 promoter. Chromatin immunoprecipitation was performed on MM.1S cells treated with 5 µM of  ricolinostat for 24 h or untreated control cells, using anti H3K27Ac antibodies, or with no antibodies as control. Immunoprecipitated chromatin was quantified using RT-qPCR with primer pairs for the promoters of Actin (technique control), CD55 (internal control), and three different pairs for CD38. CD55 was used as an internal control because its expression level is not affected by ricolinostat treatment. The immunoprecipitation was calculated normalizing to the input of each sample, the Actin promoter signal and the average of the control samples. Mean with SD of two biological replicates is shown. g Bar diagram shows CD38 expression on primary myeloma cells (n = 9 patients) before and after ricolinostat treatment. h The overlay histogram shows flow cytometric analysis of CD38 expression on primary MM cells cultured in the absence or presence of ricolinostat for 72 h. i The overlay histogram shows CD38 expression on untreated primary MM cells, 72 h after ricolinostat treatment at a fc of 5 µM, and 24 h after subsequent removal of the drug. j Bar diagram shows CD38 expression of newly diagnosed (ND, n = 4 patients) and relapsed/refractory (R/R, n = 5 patients) MM patients after 48 h treatment with ricolinostat at a fc of 5 µM. Shaded histograms show staining with anti-CD38 mAb, white histograms show staining with isotype control antibody. 7-AAD was used to exclude dead cells from analysis. Depicted are mean values with SD. p-Values between indicated groups were calculated using Student’s t-test. n.s. = not significant, *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.