FIGURE 5 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity

Effect of HDAC6 KO on proteasome activity and antigen presentation A, SDS-PAGE of lysates from HDAC6 KO in MMCLs RPMI-8226, U266, and ARH-77. B, Effect of sgRNA targeting HDAC6 on proteasome ChT-like activity in MMCLs. C, Effect sgRNA targeting HDAC6 WT and mutant forms on proteasome ChT-like activity in HeLa cells in which endogenous HDAC6 had been knocked out by sgRNA. D, Effect of HDAC6 WT, DC, and ΔBUZ mutants on proteasome ChT-like activity in HeLa cells. HeLa-HDAC6-KO cells were transfected with pcDNA-HDAC6-FLAG (RRID: Addgene₃0482), pcDNA-HDAC6. DC-FLAG (RRID: Addgene₃0483) and pcDNA-HDAC6. ΔBuz-FLAG (RRID: Addgene₃0484), and 48 hours posttransfection, cells were incubated with the proteasome substrate LLVY-R110. Proteasome activity was determined by measuring R110 levels using a fluorometric plate reader. E, Effect of WT and mutant HDAC6 on co-immunoprecipitation of HR23B. HeLa cells were transfected with sgRNA to target HDAC6 and then transfected with plasmids that expressed full-length, WT HDAC6 (pcDNA-HDAC6-FLAG), HDAC6 catalytic active site mutant (pcDNA-HDAC6. DC-FLAG) and HDAC6-BUZ domain mutant (pcDNA-HDAC6. ΔBuz-FLAG). Cells were harvested 48 hours after transfection, washed with PBS, lysates prepared under nondenaturing conditions and immunoprecipitated using the Pierce anti-DYKDDDDK affinity resin for 2 hours at 4°C followed with elution with the Pierce 3X DYKDDDDK peptide. Samples were separated by SDS-PAGE, transferred to PVDF and immunoblots probed with anti-FLAG or HR23B antibodies. Immunoblots of input FLAG and HR23B are shown. Deletion of the C-terminal BUZ domain eliminated co-immunoprecipitation of HR23B. Note that the enhanced levels of ΔBUZ compared with pcDNA-HDAC6-FLAG and pcDNA-HDAC6. DC-FLAG in immunoprecipitates reflects increased expression of input pcDNA-HDAC6. ΔBUZ-FLAG. F, Effect of sgRNA targeting HR23A, HR23B, and HDAC6 on HR23B association with HDAC6 in RPMI-8226, U266, and ARH-77 cells. Multiple myeloma cells were transfected with scrambled (control) sgRNA or sgRNA to target HDAC6, HR23A or HR23B. Lysates were prepared and HDAC6 catalytic activity determined using α-tubulin Ac-Lys-40. HDAC6 deacetylates α-tubulin and pharmacologic or genetic inhibition of HDAC6 catalytic activity leads to an increase in α-tubulin acetylation. The effect of sgRNA targeting HDAC6 was confirmed by the increase in α-tubulin Ac-Lys-40. G, Effect of sgRNA targeting HDAC6, HR23A, and HR23B on proteasome ChT-like activity in MMCLs. Cells were electroporated with sgRNA as above. Proteasome ChT-like activity was determined using LLVY-R110. H, Effect of HDAC6 inhibitors on proteasome ChT-like activity in MMCLs. Cells were transfected with the indicated sgRNA, incubated for 72 hours and then treated with each HDAC6 inhibitor (1 µmol/L). Proteasome ChT-like activity was determined using LLVY-R110. I, Effect of HDAC6 inhibitors on pan-HLA-ABC presentation in MMCLs. Cells were transfected with the indicated sgRNA, incubated for 72 hours and then treated with each HDAC6 inhibitor (1 µmol/L). The effect of each sgRNA on pan-HLA-ABC expression on MMCLs was determined by flow cytometry. Shown is the relative change in pan-HLA-ABC expression. J, Effect of the HDAC6-BUZ domain inhibitor SG-UBD235 on pan-HLA ABC presentation in MMCLs. Cells were treated with SG-UBD253 at the indicated concentrations for 24 hours, washed, stained with a conjugated antibody to pan-HLA ABC and quantitated by flow cytometry. K, Effect of HDAC6 inhibitors on co-immunoprecipitation of HR23A and HR23B with FLAG-tagged HDAC6 in RPMI-8226 cells. Cells were transfected with a plasmid that expressed full-length, WT FLAG-tagged HDAC6, treated with HDA6 inhibitors (1 µmol/L) for 24 hours, lysates prepared and separated by SDS-PAGE. Membranes were then probed for the indicated proteins by immunoblot. L, Effect of HDAC6 inhibitors on co-immunoprecipitation of HR23A and HR23B with FLAG-tagged-PSMD14 in RPMI-8226 cells. All bioassays were performed in triplicate. Error bars represent the relative SEM. A P-value ≤ 0. 05 is flagged with one star (*) and a P-value ≤ 0. 01 is flagged with two stars (**). M, Effect of sgRNA targeting HDAC6 and HR23B in RPMI8226 cells on proteasome ChT-like activity. Cells that had been transfected with sgRNA to HDAC6, HR23B or both were transfected with a plasmid that expressed FLAG-tagged PSMD14. Lysates were prepared under nondenaturing conditions, immunoprecipitated using FLAG-sepharose, eluted using 3X-FLAG peptide and proteasome activity assay buffer added (20 mmol/L Tris-HCl, pH 7. 6, 20 mmol/L NaCl, 3 mmol/L ATP, 5 mmol/L MgCl2, 1 mmol/L DTT, 10% glycerol). Suc-LLVY-MCA was then added (100 µmol/L) and proteasome activity measured using a fluorometer at an excitation 360 nmol/L and emission 440 nmol/L. N, Effect of HDAC6 inhibitors on proteasome activity in RPMI-8226 cells. Cells were transfected with a plasmid that expressed FLAG-tagged PSMD14, lysates prepared, immunoprecipitated using FLAG-sepharose, eluted using FLAG peptide and proteasome assay buffer added (20 mmol/L Tris-HCl, pH 7. 6, 20 mmol/L NaCl, 3 mmol/L ATP, 5 mmol/L MgCl2, 1 mmol/L DTT, 10% glycerol). Suc-LLVY-MCA was then added (100 µmol/L) and proteasome activity measured using a fluorometer at an excitation 360 nmol/L and emission 440 nmol/L. All assays were performed in triplicate. A P-value ≤ 0. 05 is flagged with one star (*).