Upregulation of CD38 expression on multiple myeloma cells by novel HDAC6 inhibitors is a class effect and augments the efficacy of daratumumab.

Human subjects

Peripheral blood and bone marrow aspirates were obtained from healthy donors and MM patients after we obtained their written informed consent. Patient characteristics are depicted in Supplementary Table 1 . The research protocols were approved by the Institutional Review Boards of the University Hospital of Würzburg (UKW) and University Hospital Virgen del Rocío (HUVR). All procedures conformed to the Helsinki Declaration. Isolation of primary myeloma cells, regulatory T cells, and T cells We isolated peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells by density gradient centrifugation using Ficoll–Paque (Amersham Biosciences, Uppsala, Sweden). We isolated primary MM cells from bone marrow aspirates using CD138 immunomagnetic beads (Miltenyi Biotec, Bergisch-Gladbach, Germany). Regulatory T cells were isolated from PBMCs using a two-step immunomagnetic procedure (Miltenyi). T cells were also isolated from PBMCs using negative selection with immunomagnetic beads (Miltenyi). CD8+ and CD4+ T cells were activated by anti-CD3/CD28 bead stimulation (Thermo, Waltham, MA). Immunophenotyping The expression of CD38, BCMA, SLAMF7, CD55, and CD59 on MM cells was analyzed by flow cytometry using specifically conjugated mAbs and matched isotype controls. We stained with 7-aminoactinomycin D (7-AAD) to discriminate between living and dead cells. Flow cytometry analyses were performed on a FACSCanto II (Becton Dickinson, Heidelberg, Germany) and data was analyzed using FlowJo software (Treestar, OR, USA). Treatment with HDAC inhibitors and all-trans retinoic acid (ATRA) We cultured the MM cells in RPMI-1640 (Gibco, Darmstadt, Germany) supplemented with 10% fetal bovine serum at 1 × 10e5 cells/well in 96-well flat-bottom plates (Costar, Washington, DC). We reconstituted ricolinostat, ACY-241 and WT-161 in dimethylsulfoxide and added them to the medium at final concentrations of 1, 5, and 10 µM. We likewise reconstituted ATRA and Panobinostat in dimethylsulfoxide and added these compounds to the medium at final concentrations of 10 nM.

Antibody-dependent cellular cytotoxicity

(ADCC) assay with primary myeloma cells Ricolinostat-treated (5 µM, 48 h) primary MM cells were co-cultured with autologous PBMCs at an effector-to-target ratio of 3:1 in 96-well plates in the presence of solvent control, IgG1 isotype or daratumumab. After 24 h, the percentage of viable myeloma cells was determined by flow cytometry using 7-AAD to discriminate living from dead cells.

Statistical analysis

For statistical analyses, we used Prism Software (GraphPad, San Diego, CA). Shapiro–Wilk test was used to test for normality. Unpaired Student’s t -tests were used to analyze CD38 expression level. Two-way ANOVA testing was performed to analyze the functional data. Differences with a p -value < 0.05 were considered statistically significant.

Supplementary information Supplementary Information, clean version Supplemental Table 1 Supplemental Figure 1 Supplemental Figure 2 Supplemental Figure 3 Supplemental Figure 4 Supplemental Figure 5 Supplemental Figure 6 Supplemental Figure 7 Supplemental Figure 8 Supplemental Figure 9 Supplemental Figure 10 Supplemental Figure 11