Figure 1.
Augmented CD38 expression and daratumumab-mediated ADCC against primary myeloma and myeloma cell lines (MM1.S) after panobinostat treatment. (A) CD38 expression on primary myeloma cells (n = 12 patients) before and after panobinostat treatment. Left bar diagram shows CD38 expression as normalized MFI of panobinostat-treated vs untreated myeloma after 24, 48, and 72 hours of culture. Normalized MFI values were calculated as follows: MFIs 10 nM or 25 nM × 100/MFI untreated cells. Right bar diagram shows CD38 expression of new diagnosed (ND) vs R/R myeloma patients after 48 hours of treatment with 10 nM of panobinostat. Upper and bottom overlay histograms show CD38 expression from the patient with the highest and the patient with the lowest increase in CD38 expression after 48 hours of treatment with panobinostat at the indicated dose, respectively. Shaded histograms show staining with anti-CD38 mAb, and the white histogram shows staining with isotype control antibody. (B) CD38 expression on MM1.S myeloma cells (n = 5 experiments) before and after panobinostat treatment, in experiments performed analogous to panel A. The upper overlay histogram shows CD38 expression after 48 hours of treatment with panobinostat at the indicated dose in 1 representative case. The bottom overlay histogram shows CD38 expression on untreated MM1.S cells (off), 72 hours after panobinostat treatment (10 nM, on), 24 hours after subsequent removal of the drug (off), and after 72 hours of reexposition (on). Shaded histograms show staining with anti-CD38 mAb, and the white histogram shows staining with isotype control antibody. (C) ADCC against primary myeloma cells (n = 4 patients) with and without panobinostat treatment. Panobinostat pretreatment was performed for 48 hours at a dose of 10 nM, and then autologous PBMCs (effector/target ratio of 3:1) and daratumumab (0.1 µg/mL) or control antibody (1µg/mL) were added to induce ADCC. The percentage of live myeloma cells was determined after 4 hours by flow cytometry. The bar diagram shows the percentage of viable (7-AAD–) myeloma cells (CD38+/CD138+). (D) ADCC against MM1.S myeloma cells (n = 3 experiments) with and without panobinostat treatment. Panobinostat pretreatment was performed for 48 hours at a dose of 10 nM. PBMCs from healthy donors (effector/target ratio of 25:1) and control antibody or daratumumab were added at the indicated concentrations. MM1.S stably expressed firefly luciferase, and viability was analyzed after the addition of luciferin substrate by bioluminescence measurements after 20 hours. (A-D) Values are mean ± SD. P values between indicated groups were calculated by using a paired Student t test for expression levels and 2-way analysis of variance for ADCC assays. *P < .05; **P < .01; ***P < .001; ****P < .0001. Dara, daratumumab; n.s., not significant; Pano, panobinostat.

Augmented CD38 expression and daratumumab-mediated ADCC against primary myeloma and myeloma cell lines (MM1.S) after panobinostat treatment. (A) CD38 expression on primary myeloma cells (n = 12 patients) before and after panobinostat treatment. Left bar diagram shows CD38 expression as normalized MFI of panobinostat-treated vs untreated myeloma after 24, 48, and 72 hours of culture. Normalized MFI values were calculated as follows: MFIs 10 nM or 25 nM × 100/MFI untreated cells. Right bar diagram shows CD38 expression of new diagnosed (ND) vs R/R myeloma patients after 48 hours of treatment with 10 nM of panobinostat. Upper and bottom overlay histograms show CD38 expression from the patient with the highest and the patient with the lowest increase in CD38 expression after 48 hours of treatment with panobinostat at the indicated dose, respectively. Shaded histograms show staining with anti-CD38 mAb, and the white histogram shows staining with isotype control antibody. (B) CD38 expression on MM1.S myeloma cells (n = 5 experiments) before and after panobinostat treatment, in experiments performed analogous to panel A. The upper overlay histogram shows CD38 expression after 48 hours of treatment with panobinostat at the indicated dose in 1 representative case. The bottom overlay histogram shows CD38 expression on untreated MM1.S cells (off), 72 hours after panobinostat treatment (10 nM, on), 24 hours after subsequent removal of the drug (off), and after 72 hours of reexposition (on). Shaded histograms show staining with anti-CD38 mAb, and the white histogram shows staining with isotype control antibody. (C) ADCC against primary myeloma cells (n = 4 patients) with and without panobinostat treatment. Panobinostat pretreatment was performed for 48 hours at a dose of 10 nM, and then autologous PBMCs (effector/target ratio of 3:1) and daratumumab (0.1 µg/mL) or control antibody (1µg/mL) were added to induce ADCC. The percentage of live myeloma cells was determined after 4 hours by flow cytometry. The bar diagram shows the percentage of viable (7-AAD–) myeloma cells (CD38+/CD138+). (D) ADCC against MM1.S myeloma cells (n = 3 experiments) with and without panobinostat treatment. Panobinostat pretreatment was performed for 48 hours at a dose of 10 nM. PBMCs from healthy donors (effector/target ratio of 25:1) and control antibody or daratumumab were added at the indicated concentrations. MM1.S stably expressed firefly luciferase, and viability was analyzed after the addition of luciferin substrate by bioluminescence measurements after 20 hours. (A-D) Values are mean ± SD. P values between indicated groups were calculated by using a paired Student t test for expression levels and 2-way analysis of variance for ADCC assays. *P < .05; **P < .01; ***P < .001; ****P < .0001. Dara, daratumumab; n.s., not significant; Pano, panobinostat.

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