Proteasomes generate antigenic peptides that are presented on the tumor surface to cytotoxic T-lymphocytes. Immunoproteasomes are highly specialized proteasome variants that are expressed at higher levels in antigen-presenting cells and contain replacements of the three constitutive proteasome catalytic subunits to generate peptides with a hydrophobic C-terminus that fit within the groove of MHC class I (MHC-I) molecules. A hallmark of cancer is the ability to evade immunosurveillance by disrupting the antigen presentation machinery and downregulating MHC-I antigen presentation. High-throughput screening was performed to identify compound A, a novel molecule that selectively increased immunoproteasome activity and expanded the number and diversity of MHC-I–bound peptides presented on multiple myeloma cells. Compound A increased the presentation of individual MHC-I–bound peptides by >100-fold and unmasked tumor-specific neoantigens on myeloma cells. Global proteomic integral stability assays determined that compound A binds to the proteasome structural subunit PSMA1 and promotes association of the proteasome activator PA28α/β (PSME1/PSME2) with immunoproteasomes. CRISPR/Cas9 silencing of PSMA1, PSME1, or PSME2 as well as treatment with immunoproteasome-specific suicide inhibitors abolished the effects of compound A on antigen presentation. Treatment of multiple myeloma cell lines and patient bone marrow–derived CD138+ cells with compound A increased the anti-myeloma activity of allogenic and autologous T cells. Compound A was well-tolerated in vivo and co-treatment with allogeneic T cells reduced the growth of myeloma xenotransplants in NOD/SCID gamma mice. Taken together, our results demonstrate the paradigm shifting impact of immunoproteasome activators to diversify the antigenic landscape, expand the immunopeptidome, potentiate T-cell–directed therapy, and reveal actionable neoantigens for personalized T-cell immunotherapy.

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©2024 The Authors; Published by the American Association for Cancer Research
2024
American Association for Cancer Research
This open access article is distributed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0) license.
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Supplementary data

Supplementary Table 1

Supplementary Table 1

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Supplementary Table 2

Supplementary Table 2

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Supplementary Table 3

Supplementary Table 3

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Supplementary Table 4

Supplementary Table 4

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Figure S1

Figure S1. Chemical structure of the eight hits, designated as Compounds A-H, detected in the high throughput screen. Also shown are the known HDAC6 inhibitors tubastatin-A, ACY-1215, ACY-738, and nexturastat A.

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Figure S2

Figure S2. Methods of synthesis for Compounds A-H and tubastatin-A, ACY-738, ACY-1215 and nexturastat. Compounds A through H were synthesized by ChemDiv as indicated. The step-by-step details for the synthesis of Compounds A through H are shown. ChemDiv custom synthesis products were then subjected to quality control by NMR and LC-MS and purity was guaranteed to be >90%.

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Figure S3

Figure S3. Time and dose-dependent effect of the top eights compounds (A-H) identified in the HTS on (a.) proteasome ChT-like activity and (b.) cell viability.

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Figure S4

Figure S4. Effect of Compound A on murine H2Kb MHC-I-SIINFEKL presentation and B3Z tumor lysis. a. Effect of the eight hits on proteasomal ChT-like activity at increasing concentrations in E.G7-ova cells. Compounds that increased proteasome ChT-like activity >1.5-fold and maintained cell viability >50% are represented by red boxes. b. Effect of the eight hits on proteasomal ChT-like activity and SIINFEKL-MHC-I complex presentation in E.G7-ova cells. E.G7-Ova cells were pre-treated with the indicated compounds for 72 h after which they were stained with a monoclonal antibody to SIINFEKL-bound to H2Kb. Fluorescence was quantitated by flow cytometry. Red boxes indicate compounds that increased proteasomal activity >1.5-fold, and increased SIINFEKL-H2Kb presentation >2-fold. c. Effect of the eight hits that increased proteasomal ChT-like activity with SIINFEKL-MHC-I complex presentation on lysis of E.G7-Ova cells by the B3Z T-cells. E.G7-Ova cells were pretreated with compounds (1 uM) after which cells were washed and co-cultured with the H-2Kb-restricted B3Z T-cells genetically engineered to express a TCR that specifically recognizes the SIINFEKL-H2Kb complex. E.G7-Ova cells were co-cultured with B3Z cells at E:T ratios of 1:1, 2:1, and 3:1.

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Figure S5

Figure S5. Effect of Compound A on pan-MHC-I expression. a. ARH77 and U266 cells were treated with Compound A and probed with pan-MHC-I or MHC-I subtype allele-specific antibodies after which the samples were analyzed by flow cytometry. Pretreatment with Compound A significantly increased pan-MHC class-I HLA-ABC and HLA-B surface presentation in all MMCLs assayed. b. ARH77 and U266 cells were pre-treated with Compound A for 48 h after which they were washed and co-cultured with healthy CD8+ cytotoxic T-cells for 18 h at the E:T ratios of 1:1, 2:1and 3:1. Compound A increased CTL-mediated cytotoxicity of tumor cells which was measured by gating for CD138, Annexin-V and PI positive cells at E:T 2:1 and 3:1. Pretreatment of MMCLs and T-cells alone with Compound A resulted in ∼5-10% cell death which was enhanced further when MM cells were co-cultured at E:T ratios of 2:1 and 3:1. T-cells alone also did not show significant cell death when pre-treated with Compound A. There was <5% MM cell death detected with or without Compound A treatment. Similar results were observed with CD8+ T-cells alone. c. Effect of Compound A on the viability of ARH77 and U266 cells alone or combined with ICIs and CD8+ T-cells. MM cells and CD8+ T-cells were separately treated with or without Compound A and the indicated agents. Following co-culture, the relative percent of indicated viable cells was quantitated by annexin-V and propidium iodide staining and flow cytometry. c. Effect of Compound A on T-cell exhaustion markers. CD8+ T-cells were treated with compound A for 24 h after which they were assessed for surface expression of PD-1, TIM-3, CTLA-4, and LAG-3. Compound A induced a marked decrease in PD-1 surface expression, while no significant change was observed in TIM-3, CTLA-4, and LAG-3.

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Figure S6

Figure S6. Effect of Compound A on the viability of ARH77 and U266 cells alone or combined with ICIs and CD8+ T-cells. Similar to as shown in Fig. 7f, MM and CD8+ T-cells were separately treated with or without Compound A and the indicated agents (BTZ, TGFβ, ICIs). Following co-culture, the relative percent of viable MM cells was quantitated by annexin-V and propidium iodide staining and flow cytometry. Assays were performed in triplicate.

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Figure S7

Figure S7. Effect of Compound A pretreatment in vitro on the growth of E.G7-Ova-GFP-Luc tumors in vivo. a. Schematic representation of the experimental details with a timeline of treatments and tumor measurements. b. Phase contrast and FITC images of E.G7-Ova and E.G7-Ova-GFP-Luc cells. Scale bar = 100µm c. Relative luciferase activity in E.G7-Ova and E.G7-Ova-GFP-Luc cells. d. TBW for individual mice treated with either vehicle or Compound A. e. Average total flux as a measure of tumor burden in mice injected with vehicle or Compound A pretreated E.G7-Ova-Luc-GFP cells. Tumor burden (y-axis) was quantified as total flux (p/s) and monitored post-implantation (x-axis). Total flux represents a measure of the radiance (photons/sec) in each pixel integrated over the ROI area (cm2) x 4π. Average radiance was calculated by summing the radiance from each pixel within the ROI and then dividing by the number of pixels, resulting in units of photons/second/cm²/steradian. f. BLI of E.G7-Ova-GFP-Luc tumors from vehicle and Compound A-treated cells. Negative control represents an OT-1 mouse that was not injected with E.G7-Ova-GFP-Luc cells nor treated. Scale bar = 2 cm g. Percent survival of mice following injection of E.G7-Ova-GFP-Luc cells pretreated with either vehicle or Compound A.

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Figure S8

Figure S8. Effect of in vivo administration of Compound A on myeloma xenotransplants in NSG mice. a. Schematic representation of the experimental details with a timeline of treatments and tumor measurements. b. Relative luciferase activity in MM1.S and MM1.S-Luc cells. c. Average TBW of mice from each treatment group. d. In vivo effect of Compound A on tumor volume of MM1.S-Luc cells in OT-1 mice. Shown is the average tumor volume (y-axis) vs. time from implantation (x-axis) for each treatment group. e. Tumor monitoring was performed using the IVIS spectrum imaging system where BLI was quantified as total flux (p/s) as a measure of total tumor burden (y-axis) relative to days post-implantation (x-axis). Total flux is a measure of the radiance (photons/sec) in each pixel summed or integrated over the ROI area (cm2) x 4π. Average radiance, a measure of luminescence, was calculated by summing the radiance from each pixel within the ROI and dividing it by the number of pixels or super pixels, resulting in units of photons/second/cm²/steradian.

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Figure S9

Figure S9. Effect of Btz and Compound A alone and combined in a. RPMI-8226, b. ARH77, c. U266 and d. E.G7-Ova cells. Cell viability was determined using the XTT assay. Assays were performed in triplicate. Staurosporine (ST, 2 uM) represents a positive control for cell death.

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Supp Figure Legends

Supplementary Figure Legends

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