FIGURE 1 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity

Cell-based HTS to identify pharmacologics that increased proteasome ChT-like activity. A, HTS scheme using the cell-permeable substrate LLVY-R110 to measure proteasome activity. LLVY-R110 is specifically cleaved by the proteasome to release the fluorogenic substrate R110 which fluoresces at an excitation of 490 nmol/L and emission 525 nmol/L. RPMI-8226 cells were incubated under standard culture conditions in 96-well plates in complete RPMI media that lacked phenol red. Pharmacologics (3,400) were then individually injected into each well at a final concentration of 3 µmol/L. Cells were incubated with pharmacologics for 21 hours. LLVY-R110 at a final concentration of 100 µmol/L was then added and incubated with cells for 18 hours under the same conditions. B, Rank-order effect of the top pharmacologics detected in the HTS that increased proteasome ChT-like activity. RPMI-8226 cells were incubated in 96-well plates with each pharmacologic at 1 µmol/L for 72 hours. Bioassays were performed in triplicate. Shown is the arithmetic average of triplicate measurements. Error bars indicate the SD of the mean. C, Effect of the top pharmacologics identified in the HTS on cell viability. Pharmacologics were added at 1 µmol/L for 16 hours and cell viability determined using the XTT assay (53). Shown is the arithmetic average of proteasome hydrolysis of LLVY-R110. D, Plot of the effect of pharmacologics on proteasome activity versus the effect on cell viability. Pharmacologics were prioritized based upon arbitrary cutoffs of >30% increase in proteasome activity and 75% cell viability. E, Chemical structure of clinically relevant HDAC6 inhibitors. F, Effect of HDAC6 inhibitors on proteasome ChT-like activity in MMCLs. Cells were incubated in 96-well plates with pharmacologics for 72 hours. Lysates were prepared in CelLyticM (Sigma) containing 20 mmol/L Tris-HCl (pH 7.6), 20 mmol/L NaCl, 10% glycerol, 3 mmol/L ATP, 5 mmol/L MgCl2, and 1 mmol/L DTT. Shown is the arithmetic average of triplicate measurements. Error bars indicate the SD of the mean. G, Effect of HDAC6 inhibitors on individual proteasome peptide-hydrolyzing activities. RPMI-8226 cells were incubated with HDAC6 inhibitors at 1 µmol/L for 72 hours, pelleted, washed thrice in cold PBS and lysed in CelLyticM containing 20 mmol/L Tris-HCl (pH 7.6), 20 mmol/L NaCl, 10% glycerol, 3 mmol/L ATP, 5 mmol/L MgCl2, and 1 mmol/L DTT. Fluorogenic peptide substrates were incubated with 5 µg of lysate for 1 hour at 37°C. All assays were performed in triplicate. Error bars indicate the relative SD.