FIGURE 6 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity

Effect of HDAC6 inhibitors on the antimyeloma effect of autologous T cells. A, Effect of HDAC6 inhibitors on the viability of myeloma patient CD138+ cells. Cells were treated with the pharmocologics at 1 µmol/L for 18 hours and cell viability measured using the XTT assay. Assays were performed in triplicate. B, Effect of HDAC6 inhibitors on ChT-like proteasome activity in CD138+ myeloma patient cells. Cells were treated with tubastatin-A, ACY-738, and ACY-1215 for 18 hours. Posttreatment cells were then incubated for 21 hours with LLVY-R110. Cleavage of LLVY-R110 was determined as above. C, Effect of HDAC6 inhibitors on pan MHC-I (HLA-ABC) presentation on CD138+ cells. Cells were treated with tubastatin-A, ACY-738 and ACY-1215 for 18 hours at 1 µmol/L, PBS washed, and stained with a mouse anti-human HLA-ABC (BD Pharmingen, 55555) and CD138 antibody per manufacturer's guidelines. Cells that stained positively with an anti-human HLA-ABC were analyzed on Attune Nxt and quantified using Flow Jo version 10.9.0. Error bars represent the median absolute deviation percentile (MADP) of the average. D, Effect of HDAC6 inhibitors on autologous T cell–mediated myeloma cytotoxicity. CD138+ cells were treated with tubastatin-A, ACY-738, and ACY-1215 for 18 hours and cocultured with T cells for 18 hours. After treatment, cells were then PBS washed and stained with CD138, FITC annexin-V in buffer containing propidium iodide for 20 minutes at room temperature. Cells were then analyzed on an Attune NxT and quantified using Flow Jo version 10.9.0. Error bars represent the MADP of the average. A P-value ≤ 0.05 is flagged with one star (*) and a P-value ≤ 0.001 is flagged with three stars (***).