FIGURE 7 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity

Pan-cancer cell effect of HDAC6 inhibitors on proteasomes and MHC class I antigen presentation. A, Effect of HDAC6 inhibitors on proteasome activity in cancer cell lines. Cells were treated with tubastatin-A, ACY-738 and ACY-1215 at 1 µmol/L concentration for 72 hours. Posttreatment cells were incubated for 21 hours with proteasome substrate LLVY-R110 and substrate cleavage was monitored as above. Cell lines were ranked on the basis of their effect on proteasome activity. B, Effect of HDAC6 inhibitors on pan MHC-I (HLA-ABC) in a panel of cancer cell lines. Cells were treated with tubastatin-A, ACY-738, and ACY-1215 for 72 hours at 1 µmol/L final concentration. Posttreatment the cells were washed with PBS and stained with mouse anti-human HLA-ABC (BD Pharmingen, 55555) as 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. The cells are rank ordered according to their MHC-I presentation. Error bars represent MADP of the average. C, Effect of HDAC6 inhibitors on healthy human T cells. CD8+ T cells were cultured for 18 hours with HDAC6 inhibitors, PBS washed, stained, and analyzed by flow cytometry to quantitate annexin-V+/propidium iodide+ cells. Shown is the average of triplicate measurements. D, Effect of HDAC6 inhibitors on CD8+ T-cell cytotoxicity of tumor cells. OCI-AML3 (leukemia), OV81 (ovarian), BT549 (breast), DU145 (prostate), MiaPaCa-2 (pancreatic), and PC3 (prostate) cells (1 × 106 cells/treatment) were treated with tubastatin-A, ACY-738, or ACY-1215 for 48 hours (1 µmol/L). Cells were washed and cultured alone or with healthy human T cells (E:T 2.5:1) for 24 hours. Cells were PBS washed, stained, and analyzed by flow cytometry to quantitate CD8neg/annexin-V+/propidium iodide+ cells. Shown is the average of triplicate measurements. A P-value ≤ 0.05 is flagged with one star (*) and ns indicates a difference that was not significant.