FIGURE 2 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity

Effect of HDAC6 inhibitors on SIINFEKL presentation. A, Effect of HDAC6 inhibitors on SIINFEKL presentation. EL.4 lymphoma cells were transfected with the plasmid pAc-neo-Ova to generate the cell line E.G7-Ova. pAc-neo-Ova carries a complete copy of chicken OVA mRNA and the neomycin (G418) resistance gene. Proteasome degradation of Ova generates the peptide SIINFEKL that is then presented in a complex with the MHC-I molecule on E.G7-Ova cells. Shown is the relative effect of each HDAC6 inhibitor on the amount of SIINFEKL-H-2K^b molecule presented on the surface of treated cells normalized to untreated E.G7-Ova cells. Cells were also treated with bortezomib which reduced SIINFEKL-H-2K^b presentation and demonstrated that SIINFEKL presentation was proteasome dependent. B, Effect of HDAC6 inhibitors on LDH release. E.G7-Ova cells were treated with each agent at 3 µmol/L for 72 hours. Vehicle indicates E.G7-Ova cells treated with media alone. Positive control indicates E.G7-Ova cells treated with bortezomib at 10 nmol/L for 16 hours. After drug treatment, E.G7-Ova cells were incubated with B3Z cells at an E:T ratio of 2:1. Shown is the relative percent of LDH detected in culture media after 24 hours. C, Effect of HDAC6 inhibitors on CTL-mediated tumor lysis. E.G7-Ova cells were treated with each HDAC6 inhibitor at 1 µmol/L for 72 hours. Cells were also treated with bortezomib (10 nmol/L) for 16 hours. Untreated indicates E.G7-Ova cells treated with media alone. Cells were then washed, fresh media added and cells then cocultured with B3Z cells (E:T 2:1). CD8^neg/annexin-V⁺/propidium iodide⁺ cells were then quantitated by flow cytometry. D, Effect of the HDAC6-BUZ domain specific inhibitor SGC-UBD253 on SIINFEKL-H-2K^b presentation on E.G7-Ova cells. Cells were treated with SGC-UBD253 at indicated concentrations and the level of SIINFEKL-H-2K^b quantitated by flow cytometry. All bioassays were performed in triplicate. Shown are averages of multiple experiments. Error bars represent the relative SD of the mean. E, Model of the HDAC6 domain structure and function and inhibitor binding sites. A P-value ≤ 0.05 is flagged with one star (*) and P-value ≤ 0.01 is flagged with two stars (**).