American Association for Cancer Research
Browse
crc-23-0528-s21.pptx (60.35 kB)

Figure S15 from HDAC6 Inhibition Releases HR23B to Activate Proteasomes, Expand the Tumor Immunopeptidome and Amplify T-cell Antimyeloma Activity

Download (60.35 kB)
figure
posted on 2024-06-18, 14:20 authored by Priyanka S. Rana, James J. Ignatz-Hoover, Byung-Gyu Kim, Ehsan Malek, Yuriy Federov, Drew Adams, Timothy Chan, James J. Driscoll

Fig. S15. Effect of HDAC6 inhibitors on a. aggresome formation and b. autophagosome formation. RPMI8226 cells were treated with bortezomib and HDAC6 at the indicated concentrations for 16 h. Aggresomes were quantitated by flow cytometry using the cell-based Proteostat® aggresome detection kit (Enzo Life Sciences, Farmingdale, NY). The kit utilizes a molecular rotor dye which while in solution is prevented from fluorescing by free intramolecular rotation along a single central bond. Specifically intercalation of the dye into the cross-β spine of quaternary protein structures typically found in misfolded and aggregated proteins, inhibits the dye’s rotation and leads to a strong fluorescence. b. Autophagosome formation. RPMI8226 cells were treated with bortezomib and HDAC6 at the indicated concentrations for 16 h. Autophagosomes were quantitated by flow cytometry using the Cyto-ID autophagosome detection kit which measures autophagic vacuoles and monitors autophagic flux in lysosomally inhibited live cells using a novel dye that selectively labels accumulated autophagic vacuoles (Enzo Life Sciences). The 488nm-excitable green dye allows for minimal staining of lysosomes while exhibiting bright fluorescence upon incorporation into pre-autophagosomes, autophagosomes, and autolysosomes (autophagolysosomes). All assays were performed in triplicate. Error bars represent the SD of the mean.

Funding

Vinney Foundation

HHS | NIH | National Institute of Allergy and Infectious Diseases (NIAID)

History

ARTICLE ABSTRACT

Proteasomes degrade intracellular proteins to generate antigenic peptides that are recognized by the adaptive immune system and promote anticancer immunity. However, tumors subvert the antigen presentation machinery to escape immunosurveillance. We hypothesized that proteasome activation could concomitantly increase antigen abundance and diversity in multiple myeloma cells. High-throughput screens revealed that histone deacetylase 6 (HDAC6) inhibitors activated proteasomes to unmask neoantigens and amplify the tumor-specific antigenic landscape. Treatment of patient CD138+ cells with HDAC6 inhibitors significantly promoted the antimyeloma activity of autologous CD8+ T cells. Pharmacologic blockade and genetic ablation of the HDAC6 ubiquitin-binding domain released HR23B, which shuttles ubiquitinylated cargo to proteasomes, while silencing HDAC6 or HR23B in multiple myeloma cells abolished the effect of HDAC6 inhibitors on proteasomes, antigen presentation, and T-cell cytotoxicity. Taken together, our results demonstrate the paradigm-shifting translational impact of proteasome activators to expand the myeloma immunopeptidome and have revealed novel, actionable antigenic targets for T cell–directed immunotherapy. The elimination of therapy-resistant tumor cells remains a major challenge in the treatment of multiple myeloma. Our study identifies and functionally validates agents that amplify MHC class I–presented antigens and pave the way for the development of proteasome activators as immune adjuvants to enhance immunotherapeutic responses in patients with multiple myeloma.