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.
©2024 The Authors; Published by the American Association for Cancer Research.

Antibody-secreting B cells have long been considered the central element of gut homeostasis; however, tumor-associated B cells in human colorectal cancer (CRC) have not been well characterized. Here, we show that the clonotype, phenotype, and immunoglobulin subclasses of tumor-infiltrating B cells have changed compared to adjacent normal tissue B cells. Remarkably, the tumor-associated B cell immunoglobulin signature alteration can also be detected in the plasma of patients with CRC, suggesting that a distinct B cell response was also evoked in CRC. We compared the altered plasma immunoglobulin signature with the existing method of CRC diagnosis. Our diagnostic model exhibits improved sensitivity compared to the traditional biomarkers, CEA and CA19-9. These findings disclose the altered B cell immunoglobulin signature in human CRC and highlight the potential of using the plasma immunoglobulin signature as a non-invasive method for the assessment of CRC.
© 2023.

Proteasome inhibitor (PI) resistance remains a central challenge in multiple myeloma. To identify pathways mediating resistance, we first mapped proteasome-associated genetic co-dependencies. We identified heat shock protein 70 (HSP70) chaperones as potential targets, consistent with proposed mechanisms of myeloma cells overcoming PI-induced stress. We therefore explored allosteric HSP70 inhibitors (JG compounds) as myeloma therapeutics. JG compounds exhibited increased efficacy against acquired and intrinsic PI-resistant myeloma models, unlike HSP90 inhibition. Shotgun and pulsed SILAC mass spectrometry demonstrated that JGs unexpectedly impact myeloma proteostasis by destabilizing the 55S mitoribosome. Our data suggest JGs have the most pronounced anti-myeloma effect not through inhibiting cytosolic HSP70 proteins but instead through mitochondrial-localized HSP70, HSPA9/mortalin. Analysis of myeloma patient data further supports strong effects of global proteostasis capacity, and particularly HSPA9 expression, on PI response. Our results characterize myeloma proteostasis networks under therapeutic pressure while motivating further investigation of HSPA9 as a specific vulnerability in PI-resistant disease.
Copyright © 2022 Elsevier Ltd. All rights reserved.

The myeloma surface proteome (surfaceome) determines tumor interaction with the microenvironment and serves as an emerging arena for therapeutic development. Here, we use glycoprotein capture proteomics to define the myeloma surfaceome at baseline, in drug resistance, and in response to acute drug treatment. We provide a scoring system for surface antigens and identify CCR10 as a promising target in this disease expressed widely on malignant plasma cells. We engineer proof-of-principle chimeric antigen receptor (CAR) T-cells targeting CCR10 using its natural ligand CCL27. In myeloma models we identify proteins that could serve as markers of resistance to bortezomib and lenalidomide, including CD53, CD10, EVI2B, and CD33. We find that acute lenalidomide treatment increases activity of MUC1-targeting CAR-T cells through antigen upregulation. Finally, we develop a miniaturized surface proteomic protocol for profiling primary plasma cell samples with low inputs. These approaches and datasets may contribute to the biological, therapeutic, and diagnostic understanding of myeloma.
© 2022. The Author(s).

h4>ABSTRACT/h4> The myeloma cell surface proteome (“surfaceome”) not only determines tumor interaction with the microenvironment but serves as an emerging arena for therapeutic development. Here, we use glycoprotein capture proteomics to first define surface markers most-enriched on myeloma when compared to B-cell malignancy models, revealing unexpected biological signatures unique to malignant plasma cells. We next integrate our proteomic dataset with existing transcriptome databases, nominating CCR10 and TXNDC11 as possible monotherapeutic targets and CD48 as a promising co-target for increasing avidity of BCMA-directed cellular therapies. We further identify potential biomarkers of resistance to both proteasome inhibitors and lenalidomide including changes in CD53, EVI2B, CD10, and CD33. Comparison of short-term treatment with chronic resistance delineates large differences in surface proteome profile under each type of drug exposure. Finally, we develop a miniaturized version of the surface proteomics protocol and present the first surface proteomic profile of a primary myeloma patient plasma cell sample. Our dataset provides a unique resource to advance the biological, therapeutic, and diagnostic understanding of myeloma.