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

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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

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-2Kb 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-2Kb 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). CD8neg/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-2Kb presentation on E.G7-Ova cells. Cells were treated with SGC-UBD253 at indicated concentrations and the level of SIINFEKL-H-2Kb 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 (**).


Vinney Foundation

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



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.