Antigen Processing Machinery Research Articles

Altered ACE2 and interferon landscape in the COVID-19 microenvironment correlate with the anti-PD-1 response in solid tumors

Angiotensensin-converting enzyme-2 (ACE2) is a receptor for SARS-CoV-2, allowing the virus to enter cells. Although tumor patients infected by SARS-CoV-2 often have a worse outcome, the expression, function and clinical relevance of ACE2 in tumors has not yet been thoroughly analyzed. In this study, RNA sequencing (RNA-seq) data from tumors, adjacent tissues and whole blood samples of COVID-19 patients from genome databases and from tumor cell lines and endothelial cells infected with different SARS-CoV-2 variants or transfected with an ACE2 expression vector (ACE2high) or mock (ACE2low) were analyzed for the expression of ACE2 and immune response relevant molecules in silico or by qPCR, flow cytometry, Western blot and/or RNA-seq. The differential expression profiles in ACE2high vs. ACE2low cells correlated with available SARS-CoV-2 RNA-seq datasets. ACE2high cells demonstrated upregulated mRNA and/or protein levels of HLA class I, programmed death ligand 1 (PD-L1), components of the antigen processing machinery (APM) and the interferon (IFN) signaling pathway compared to ACE2low cells. Co-cultures of ACE2high cells with peripheral blood mononuclear cells increased immune cell migration and infiltration towards ACE2high cells, apoptosis of ACE2high cells, release of innate immunity-related cytokines and altered NK cell-mediated cytotoxicity. Thus, ACE2 expression was associated in different model systems and upon SARS-CoV-2 infection with an altered host immunogenicity, which might influence the efficacy of immune checkpoint inhibitors. These results provide novel insights into the (patho)physiological role of ACE2 on immune response-relevant mechanisms and suggest an alternative strategy to reduce COVID-19 severity in infected tumor patients targeting the ACE2-induced IFN-PD-L1 axis.

IMMU-07. THE IMMUNE LANDSCAPE OF PEDIATRIC BRAIN TUMORS: A TRANSCRIPTOMIC ANALYSIS

Abstract Despite standard-of-care therapy, pediatric brain tumors are now the leading cause of cancer-related death in children, highlighting an urgent need for the development of new treatments. Immunotherapy is actively being evaluated as a therapeutic approach to pediatric brain tumors, however, more research is needed to reveal additional antigenic targets that exist in these malignancies. In this study, our team performed a transcriptomic analysis of pediatric brain tumor RNA sequencing data within the Children’s Brain Tumor Network (CBTN) to identify intra and extracellular antigens that are highly expressed across major tumor types. Interestingly, our team observed that most tumors had high gene expression of a group of antigens that were unique to that specific tumor type. Specifically, our analysis revealed that diffuse midline gliomas (DMGs) highly expressed the extracellular antigens CA9, CTCFL, and ST8SIA1, whereas atypical rhabdoid tumors (ATRTs) highly expressed MUC1 and TEK. Other tumors that displayed high gene expression for a variety of antigenic targets included H3-wildtype high-grade gliomas (HGGs), and ependymomas (EPNs). Strikingly, we observed minimal difference in antigen expression in primary tumors when compared to patient-matched recurrences. Beyond antigen expression, we evaluated the expression of antigen processing machinery (APM) which includes major histocompatibility complexes (MHCs). Our findings revealed that DMGs had the lowest level of APM expression, however, these tumors uniquely expressed high levels of the immune checkpoints ADORA2A, CD276, and KLRC1. Contrarily, ATRTs showed a high expression of immune checkpoints CD274 (PD-L1), PVR, and CD80. Other tumors included in our analysis had unique expression patterns of antigen, as well as APM and immune checkpoints. Our findings illustrate that pediatric brain tumors have distinct expression patterns of antigens, APM, and immune checkpoints that are specific to tumor type, emphasizing diverse, rather than general, immunotherapeutic approaches must be considered for childhood brain tumors.

Adoptive transfer of membrane-restricted IL-12-TCR T cells promotes antigen spreading and elimination of antigen-negative tumor variants

BackgroundAdoptive T-cell therapy has demonstrated clinical activity in B-cell malignancies, offering hope for its application to a broad spectrum of cancers. However, a significant portion of patients with solid tumors...

Integrating single cell and bulk RNA sequencing data identifies RBM17 as a novel response biomarker for immunotherapy in bladder cancer.

Checkpoint inhibitors (CPIs) have been widely applied in the treatment of patients with bladder cancer (BLCA). However, there is still unmet need to dissect response predict biomarkers. To uncover CPI response-related marker genes in cancer cells, we utilized SCISSOR, integrating single-cell RNA and bulk RNA sequencing data. Transcriptomic and clinical data from IMvigor210, UNC-108, and BCAN/HCRN datasets were collected to evaluate and validate the identified biomarkers and signatures. Additionally, we analyzed TCGA-BLCA and local-BLCA RNA-seq data to investigate alternative splicing events (ASEs). Cell viability was assessed in T24 and UMUC3 cells with RBM17 upregulation or downregulation. Through SCISSOR analysis, we discovered that the expression levels of RBM17, TAP1, and PSMB8 were significantly associated with CPI response. Since PSMB8 displayed a highly positive correlation with TAP1, we developed a CPI response score (CRS) signature based on the expression profiles of RBM17 and TAP1. The CRS demonstrated robust predictive capacity in IMvigor210, UNC-108, and BCAN/HCRN datasets and was associated with higher tumor mutational burden (TMB), PD-L1 expression, and unique genomic features. Notably, RBM17 was not linked to the clinical outcomes of BLCA patients but positively correlated with BLCA cell proliferation in vitro. In the meantime, RBM17 was correlated with higher activity in core biological pathways, including antigen processing machinery, CD8 + T effector cells, cell cycle, DNA damage repair, epithelial-mesenchymal transition, histone regulation, and immune checkpoints. Moreover, the high-RBM17 group showed enrichment of LumU/Ba/sq subtypes but fewer FGFR3 alterations. Lastly, RBM17 significantly upregulated ASEs in BLCA samples, leading to higher neoantigen levels, a more inflamed tumor microenvironment, and improved CPI response. RBM17 is associated with higher ASEs and neoantigen levels, thereby potentiating the efficacy of CPI in BLCA. The established predictive signature, utilizing only two genes, has the potential to streamline clinical applications, providing a cost-effective alternative to expensive genomic, transcriptomic, and biological feature tests.

Disruptions in antigen processing and presentation machinery on sarcoma

BackgroundThe antigen processing machinery (APM) plays a critical role in generating tumor-specific antigens that can be recognized and targeted by the immune system. Proper functioning of APM components is essential for presenting these antigens on the surface of tumor cells, enabling immune detection and destruction. In many cancers, defects in APM can lead to immune evasion, contributing to tumor progression and poor clinical outcomes. However, the status of the APM in sarcomas is not well characterized, limiting the development of effective immunotherapeutic strategies for these patients.MethodsWe investigated 126 patients with 8 types of bone and soft tissue sarcoma operated between 2001–2021. Tissue microarrays mapped 11 specific areas in each case. The presence/absence of APM protein was determined through immunohistochemistry. Bayesian networks were used.ResultsAll investigated sarcomas had some defects in APM. The least damaged component was HLA Class I subunit β2-microglobulin and HLA Class II. The proteasome LMP10 subunit was defective in leiomyosarcoma (LMS), myxoid liposarcoma (MLPS), and dedifferentiated liposarcoma (DDLPS), while MHC I transporting unit TAP2 was altered in undifferentiated pleomorphic sarcoma (UPS), gastrointestinal stromal tumor (GIST), and chordoma (CH). Among different neoplastic areas, high-grade areas showed different patterns of expression compared to high lymphocytic infiltrate areas. Heterogeneity at the patient level was also observed. Loss of any APM component was prognostic of distant metastasis (DM) for LMS and DDLPS and of overall survival (OS) for LMS.ConclusionSarcomas exhibit a high degree of defects in APM components, with differences among histotypes and tumoral areas. The most commonly altered APM components were HLA Class I subunit β2-microglobulin, HLA Class I subunit α (HC10), and MHC I transporting unit TAP2. The loss of APM components was prognostic of DM and OS and clinically relevant for LMS and DDLPS. This study explores sarcoma molecular mechanisms, enriching personalized therapeutic approaches.

Transcriptomic observations of intra and extracellular immunotherapy targets for pediatric brain tumors

ABSTRACT Objectives Despite surgical resection, chemoradiation, and targeted therapy, brain tumors remain a leading cause of cancer-related death in children. Immunotherapy has shown some promise and is actively being investigated for treating childhood brain tumors. However, a critical step in advancing immunotherapy for these patients is to uncover targets that can be effectively translated into therapeutic interventions. Methods In this study, our team performed a transcriptomic analysis across pediatric brain tumor types to identify potential targets for immunotherapy. Additionally, we assessed components that may impact patient response to immunotherapy, including the expression of genes essential for antigen processing and presentation, inhibitory ligands and receptors, interferon signature, and overall predicted T cell infiltration. Results We observed distinct expression patterns across tumor types. These included elevated expression of antigen genes and antigen processing machinery in some tumor types while other tumors had elevated inhibitory checkpoint receptors, known to be associated with response to checkpoint inhibitor immunotherapy. Conclusion These findings suggest that pediatric brain tumors exhibit distinct potential for specific immunotherapies. We believe our findings can guide investigators in their assessment of appropriate immunotherapy classes and targets in pediatric brain tumors.

Human erythroid progenitors express antigen presentation machinery.

Early-life immune exposures can profoundly impact lifelong health. However, functional mechanisms underlying fetal immune development remain incomplete. Erythrocytes are not typically considered active immune mediators, primarily because erythroid precursors discard their organelles as they mature, thus losing the ability to alter gene expression in response to stimuli. Erythroid progenitors and precursors circulate in human fetuses and neonates. Although there is limited evidence that erythroid precursors are immunomodulatory, our understanding of the underlying mechanisms remains inadequate. To define the immunobiological role of fetal and perinatal erythroid progenitors and precursors, we analyzed single cell RNA-sequencing data and found that transcriptomics support erythroid progenitors as putative immune mediators. Unexpectedly, we discovered that human erythroid progenitors constitutively express Major Histocompatibility Complex (MHC) class II antigen processing and presentation machinery, which are hallmarks of specialized antigen presenting immune cells. Furthermore, we demonstrate that erythroid progenitors internalize and cleave foreign proteins into peptide antigens. Unlike conventional antigen presenting cells, erythroid progenitors express atypical costimulatory molecules and immunoregulatory cytokines that direct the development of regulatory T cells, which are critical for establishing maternal-fetal tolerance. Expression of MHC II in definitive erythroid progenitors begins during the second trimester, coinciding with the appearance of mature T cells in the fetus, and is absent in primitive progenitors. Lastly, we demonstrate physical and molecular interaction potential of erythroid progenitors and T cells in the fetal liver. Our findings shed light on a unique orchestrator of fetal immunity and provide insight into the mechanisms by which erythroid cells contribute to host defense.

Zebularine potentiates anti-tumor immunity by inducing tumor immunogenicity and improving antigen processing through cGAS-STING pathway

DNA methylation is an important epigenetic mechanism involved in the anti-tumor immune response, and DNA methyltransferase inhibitors (DNMTi) have achieved impressive therapeutic outcomes in patients with certain cancer types. However, it is unclear how inhibition of DNA methylation bridges the innate and adaptive immune responses to inhibit tumor growth. Here, we report that DNMTi zebularine reconstructs tumor immunogenicity, in turn promote dendritic cell maturation, antigen-presenting cell activity, tumor cell phagocytosis by APCs, and efficient T cell priming. Further in vivo and in vitro analyses reveal that zebularine stimulates cGAS-STING-NF-κB/IFNβ signaling to enhance tumor cell immunogenicity and upregulate antigen processing and presentation machinery (AgPPM), which promotes effective CD4+ and CD8+ T cell-mediated killing of tumor cells. These findings support the use of combination regimens that include DNMTi and immunotherapy for cancer treatment.

Facts and Hopes on Cancer Immunotherapy for Small Cell Lung Cancer.

Platinum-based chemotherapy plus PD1 axis blockade is the standard of care in the first-line treatment of extensive-stage small cell lung cancer (SCLC). Despite the robust and consistent increase in long-term survival with PD1 axis inhibition, the magnitude of the benefit from immunotherapy seems lower than that for other solid tumors. Several immune evasive mechanisms have been shown to be prominently altered in human SCLC, including T-cell exclusion, downregulation of components of the MHC class I antigen processing and presentation machinery, or upregulation of macrophage inhibitory checkpoints, among others. New immunotherapies aiming to target some of these dominant immune suppressive features are being intensively evaluated preclinically and clinically in SCLC. They include strategies to enhance the efficacy and/or reverse features that promote intrinsic resistance to PD1 axis inhibition (e.g., restoring MHC class I deficiency and targeting DNA damage response) and novel immunomodulatory agents beyond T-cell checkpoint blockers (e.g., T cell-redirecting strategies, antibody-drug conjugates, or macrophage checkpoint blockers). Among them, delta-like ligand 3-targeted bispecific T-cell engagers have shown the most compelling preliminary evidence of clinical efficacy and hold promise as therapies that might contribute to further improve patient outcomes in this disease. In this study, we first provide a brief overview of key tumor microenvironment features of human SCLC. Then, we update the current clinical evidence with immune checkpoint blockade and review other emerging immunotherapy strategies that are gaining increasing attention in SCLC. We finally summarize our future perspective on immunotherapy and precision oncology for this disease.

Abstract 5364: Progesterone receptor modulates the antigen processing and presentation machinery decreasing MHC class I expression on tumors

Abstract Hormone receptor (HR) positive breast cancers, which account for approximately 75% of all breast cancers and express both estrogen receptor (ER) and progesterone receptor (PR), have been the subject of much research. As such, successful anti-estrogen/ER-based therapies have been very successful in the treatment of HR+ breast cancer. Despite this success, over 1/3 of patients will eventually progress on current ER/estrogen-targeted therapies, underscoring the need for new targeted therapies in HR+ breast cancer. Unlike ER, the role of PR in breast cancer progression has received far less attention. In a previous study, we found that a mouse mammary tumor cell line E0771, modified to express the mouse progesterone receptor (mPR) and the OVA peptide, demonstrated decreased T cell-mediated cell death when treated with progesterone compared to the control group. Progesterone-mediated protection from T-cell death was only observed in mPR+ cell lines. Flow analysis of the E0771-Ova-mPR cells also revealed a decrease in MHC Class I presentation in progesterone-treated cells compared to a vehicle control. To further explore the role of the machinery required for antigen processing and presentation of MHC Class I molecules, we conducted additional experiments using the E0771 cell line. Our results showed that treatment with progesterone led to a reduction in RNA levels of all the machinery required for antigen processing and presentation (APP). Based on these findings, we hypothesize that activation of the progesterone receptor by progesterone may decrease MHC Class I expression by regulating APP. Our results showed that treatment with progesterone resulted in a reduction in RNA levels of Tap1, Tap2, Tapbp, Nlrc5, B2m, and Psmb8, which are essential for APP. We therefore propose that activation of the progesterone receptor by progesterone may decrease MHC Class I expression by directly modulating the APP pathway. The findings of this study may have significant implications for our understanding of how breast cancer cells evade T-cell mediated cytotoxicity. Citation Format: Julio Tinoco, Lauryn Werner, Eilidh Chowanec, Harmony Saunders, Sun Xiaopeng, Jay Gertz, Justin Balko, Zachary C. Hartman, Christy Hagan. Progesterone receptor modulates the antigen processing and presentation machinery decreasing MHC class I expression on tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5364.

Phase I Trial of Viral Vector-Based Personalized Vaccination Elicits Robust Neoantigen-Specific Antitumor T-Cell Responses.

Personalized vaccines targeting multiple neoantigens (nAgs) are a promising strategy for eliciting a diversified antitumor T-cell response to overcome tumor heterogeneity. NOUS-PEV is a vector-based personalized vaccine, expressing 60 nAgs and consists of priming with a nonhuman Great Ape Adenoviral vector (GAd20) followed by boosts with Modified Vaccinia Ankara. Here, we report data of a phase Ib trial of NOUS-PEV in combination with pembrolizumab in treatment-naïve patients with metastatic melanoma (NCT04990479). The feasibility of this approach was demonstrated by producing, releasing, and administering to 6 patients 11 of 12 vaccines within 8 weeks from biopsy collection to GAd20 administration. The regimen was safe, with no treatment-related serious adverse events observed and mild vaccine-related reactions. Vaccine immunogenicity was demonstrated in all evaluable patients receiving the prime/boost regimen, with detection of robust neoantigen-specific immune responses to multiple neoantigens comprising both CD4 and CD8 T cells. Expansion and diversification of vaccine-induced T-cell receptor (TCR) clonotypes was observed in the posttreatment biopsies of patients with clinical response, providing evidence of tumor infiltration by vaccine-induced neoantigen-specific T cells. These findings indicate the ability of NOUS-PEV to amplify and broaden the repertoire of tumor-reactive T cells to empower a diverse, potent, and durable antitumor immune response. Finally, a gene signature indicative of the reduced presence of activated T cells together with very poor expression of the antigen-processing machinery genes has been identified in pretreatment biopsies as a potential biomarker of resistance to the treatment.

Upregulation of MHC I antigen processing machinery gene expression in breast cancer cells by Trichostatin A

Epigenetic alterations associated with cancer have been shown to facilitate tumorigenesis and promote metastasis. In the study of cancer metastasis, epigenetics has been revealed to play a crucial role in supporting tumour immune evasion. As a result, epigenetic drugs have been identified as potential agents to activate anti-tumour immune responses and reverse tumour immunologically tolerant states. Mounting evidence is showing aberrant expression of MHC class I antigen processing molecules in cancers and their upregulation as a potential indicator for anti-tumour immunity. In this study, we demonstrate that the epigenetic drug Trichostatin A (TSA), a histone deacetylase inhibitor, can restore MHC I antigen presentation machinery (MHC I APM) genes in human breast cancer cells (MCF-7). Treatment with TSA resulted in the upregulation of MHC I, B2M, and PSMB9 in MCF-7 monolayer cells, and MHC I, B2M, PSMB9, PSMB8, TAP1, and TAP2 in MCF-7 spheroid cells. Interestingly, treatment with TSA also increased CD274 expression in these cells and enhanced the invasion ability of the MCF-7 spheroid. This aggressive behaviour was confirmed by increased expression of metastatic-related genes, SCN5A (nNav1.5 protein) and MMP1. In summary, although the restoration of MHC I APM expression was achieved by TSA, the upregulation of metastatic genes and CD274 also enhanced the invasion ability of breast cancer cells. These findings suggest the need for careful consideration when utilizing epigenetic drugs for breast cancer therapy.

Abstract B034: Characterization of MHC-I-mediated immune evasion in triple-negative breast cancer using human transcriptomic datasets and immunocompetent mouse models

Abstract In Triple-Negative Breast Cancer (TNBC) patients, adaptive immune system activity in the tumor microenvironment is associated with improved prognosis. However, tumors can evolve to evade immune detection, likely leading to worse patient outcomes. Multi-omic data of paired primary and metastatic breast tumors demonstrate that TNBC metastases frequently lose expression of genes involved in major histocompatibility complex class I (MHC-I) antigen presentation, a key pathway for adaptive immune activation (PMID: 36585450). Therefore, understanding and addressing MHC-I-mediated immune evasion may have profound clinical benefits for TNBC patients. We hypothesize that breast tumor MHC-I loss results in a unique immune-evasive phenotype, which we are characterizing using human clinical datasets and immunocompetent mouse models of breast cancer. First, using large transcriptomic datasets (i.e., SCAN-B and TCGA), we are characterizing molecular and clinical features of MHC-I-low human breast tumors using expression of the MHC-I gene human leukocyte antigen A (HLA-A) as a continuous variable. We found HLA-A expression varies significantly between PAM50 molecular and clinical subtypes of breast cancer and has differing impacts on patient survival based on clinical subtype. In TNBCs, low HLA-A expression is associated with worse outcomes, while in HR+/HER2- tumors, low HLA-A predicts improved outcomes. We also identified genes highly correlated with HLA-A regardless of molecular subtype. This list was functionally rich, related to antigen processing machinery, adaptive immune-mediated cytotoxicity, and T cell exhaustion features. To complement human genomic data, we are empirically testing the role of MHC-I in syngeneic mouse mammary tumor models by individually knocking out murine MHC-I genes–H2-K, H2-D, and B2m–through CRISPR/Cas9-mediated genome editing. We have generated and validated knockouts (KOs) of H2-K and B2m, as well as empty vector control clones from the “immune-hot” and immune checkpoint inhibitor (ICI) responsive mammary tumor cell lines KPB25L-UV and T11-APOBEC. RNA sequencing of KPB25L-UV H2-K and B2m KO versus wildtype cell lines in vitro identified lists of differentially expressed genes, many of which are related to proteolytic and peptide antigen processing. Next, these CRISPR-modified cell lines were injected into the mammary fat pad of syngeneic immunocompetent mice, and the resulting tumors were harvested for RNA sequencing. Differentially expressed genes in KPB25L-UV H2-K KO versus wildtype tumors were related to increased MHC Class II antigen presentation and dendritic cell activity. Preliminary data suggests that mice bearing KPB25L-UV H2-K KO tumors have shortened survival compared to H2-K wildtype tumors. Experiments are ongoing to further characterize the growth dynamics, metastatic potential, single-cell transcriptomic profiles, and ICI sensitivities of these KO tumors in vivo. Our long-term goal is to identify therapeutic means to overcome this common immune-evasive phenotype to improve TNBC patient outcomes. Citation Format: Constandina E O'Connell, Brooke M Felsheim, Aranzazu Fernandez-Martinez, Kevin R Mott, Charles M Perou. Characterization of MHC-I-mediated immune evasion in triple-negative breast cancer using human transcriptomic datasets and immunocompetent mouse models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr B034.

Rescue of NLRC5 expression restores antigen processing machinery in head and neck cancer cells lacking functional STAT1 and p53

The antigen processing machinery (APM) components needed for a tumor cell to present an antigen to a T cell are expressed at low levels in solid tumors, constituting an important mechanism of immune escape. More than most other solid tumors, head and neck squamous cell carcinoma (HNSCC) cells tend to have low APM expression, rendering them insensitive to immune checkpoint blockade and most other forms of immunotherapy. In HNSCC, this APM deficiency is largely driven by high levels of EGFR and SHP2, leading to low expression and activation of STAT1; however, recent studies suggest that p53, which is often mutated in HNSCCs, may also play a role. In the current study, we aimed to investigate the extent to which STAT1 and p53 individually regulate APM component expression in HNSCC cells. We found that in cells lacking functional p53, APM expression could still be induced by interferon-gamma or DNA-damaging chemotherapy (cisplatin) as long as STAT1 expression remained intact; when both transcription factors were knocked down, APM component expression was abolished. When we bypassed these deficient pathways by rescuing the expression of NLRC5, APM expression was also restored. These results suggest that dual loss of functional STAT1 and p53 may render HNSCC cells incapable of processing and presenting antigens, but rescue of downstream NLRC5 expression may be an attractive strategy for restoring sensitivity to T cell-based immunotherapy.

Enhancing the immunogenicity of Wilms tumor 1 epitope in mesothelioma cells with immunoproteasome inhibitors.

The immunogenicity of cancer cells is influenced by several factors, including the expression of the major histocompatibility complex class I (MHC-I), antigen expression, and the repertoire of proteasome-produced epitope peptides. The malignant pleural mesothelioma cell line ACC-MEOS-4 (MESO-4) expresses high levels of MHC-I and Wilms tumor 1 (WT1) tumor antigens. Using a functional T cell reporter assay specific for the HLA-A*24:02 restricted WT1 epitope (WT1235, CMTWNQMNL), we searched for factors that augmented the immunogenicity of MESO-4, focusing on proteasomes, which have a central role in the antigen processing machinery. ONX-0914, a selective inhibitor of the immunoproteasome subunit β5i, enhanced immunogenicity dose-dependently at low concentrations without cytotoxicity. In addition, CD8+ T lymphocytes recognizing WT1 showed greater cytotoxicity against MESO-4 pre-treated with ONX-0914. MESO-4 expresses a standard proteasome (SP) and immunoproteasome (IP). Notably, IP has distinct catalytic activity from SP, favoring the generation of antigenic peptides with high affinity for MHC-I in antigen-presenting cells and cancer cells. In vitro, immunoproteasome digestion assay and mass spectrometry analysis showed that IP cleaved WT1235 internally after the hydrophobic residues. Importantly, this internal cleavage of the WT1235 epitope was mitigated by ONX-0914. These results suggest that ONX-0914 prevents the internal destructive cleavage of WT1235 by IP, thereby promoting the specific presentation of the WT1 epitope by MESO-4. In conclusion, selective IP inhibitors might offer a means to modulate cancer cell immunogenicity by directing the presentation of particular tumor epitopes.

Curcuphenols facilitate the immune driven attenuation of metastatic tumour growth

One of the primary obstacles in current cancer treatments lies in the extensive heterogeneity of genetic and epigenetic changes that occur in each arising tumour. However, an additional challenge persists, as certain types of cancer display shared immune deficiencies in the antigen processing machinery (APM). This includes the downregulation of human leukocyte antigen (HLA) class I molecules, which serve as peptide antigen receptors for T lymphocyte recognition that plays a crucial role in killing emerging tumours. Consequently, this contributes to immune escape in metastatic disease. Notably, current cell-based immunotherapies primarily focusing on T lymphocytes and the implementation of immune checkpoint inhibitor modalities have largely ignored the crucial task of reversing immune escape. This oversight may explain the limited success of these approaches becoming more effective cancer immunotherapies. Hence, there is a critical need to prioritize the discovery of new therapeutic candidates that can effectively address immune escape and synergize with evolving immunotherapy strategies. In this context, we identified curcuphenol in a cell-based screen from a library of marine extracts as a chemical entity that reverses the immune-escape phenotype of metastatic cancers. To advance these findings toward clinical efficacy, the present study describes the synthesis of analogues of naturally occurring curcuphenol with enhanced chemical properties and biological efficacy. Here we test the hypothesis that these curcuphenol analogues can evoke the power of the immune system to reduce the growth of metastatic disease in tumour bearing animals. Our findings indicate that these compounds effectively restore the expression of APM genes in metastatic tumours and inhibit the growth of highly invasive tumours in preclinical models, thereby counteracting the common immune evasion phenomenon observed in metastatic cancers. We conclude that cancer immunotherapies capable of boosting APM expression, hold great potential in maximizing the effectiveness of immune blockade inhibitors and eradicating invasive tumours.

Bioinformatics analysis of immune characteristics in tumors with alternative carcinogenesis pathways induced by human papillomaviruses

BackgroundHuman papillomaviruses (HPVs) induce a subset of head and neck squamous cell carcinomas (HNSCC) and anogenital cancers, particularly cervical cancer (CC). The major viral proteins that contribute to tumorigenesis are the E6 and E7 oncoproteins, whose expression is usually enhanced after the integration of viral DNA into the host genome. Recently, an alternative tumorigenesis pathway has been suggested in approximately half of HNSCC and CC cases associated with HPV infection. This pathway is characterized by extrachromosomal HPV persistence and increased expression of the viral E2, E4, and E5 genes. The E6, E7, E5, and E2 proteins have been shown to modify the expression of numerous cellular immune-related genes. The antitumor immune response is a critical factor in the prognosis of HPV-driven cancers, and its characterization may contribute to the prediction and personalization of the increasingly used cancer immunotherapy.MethodsWe analyzed the immune characteristics of HPV-dependent tumors and their association with carcinogenesis types. Transcriptomic HNSCC and CC datasets from The Cancer Genome Atlas were used for this analysis.ResultsClustering with immune-related genes resulted in two clusters of HPV16-positive squamous cell carcinomas in both tumor types: cluster 1 had higher activation of immune responses, including stimulation of the antigen processing and presentation pathway, which was associated with higher immune cell infiltration and better overall survival, and cluster 2 was characterized by keratinization. In CC, the distribution of tumor samples into clusters 1 and 2 did not depend on the level of E2/E5 expression, but in HNSCC, most E2/E5-high tumors were localized in cluster 1 and E2/E5-low tumors in cluster 2. Further analysis did not reveal any association between the E2/E5 levels and the expression of immune-related genes.ConclusionsOur results suggest that while the detection of immune responses associated with preserved expression of genes encoding components of antigen processing and presentation machinery in HPV-driven tumors may be markers of better prognosis and an important factor in therapy selection, the type of carcinogenesis does not seem to play a decisive role in the induction of antitumor immunity.

Abstract C083: HDAC6 inhibition increases proteasome activity and modulates the myeloma immunopeptidome to promote cytotoxic T-cell activity

Abstract The proteasome functions as an essential component of immune surveillance by generating peptides from intracellular antigens that are then presented to cytotoxic T-cells. The vast majority of defined antitumor T-cell responses involves the proteasomal processing of intracellular proteins and their presentation in the context of major histocompatibility complex (MHC) class I molecules to antigenic peptide-specific CD8+ T-cells. Such antigenic peptides generated by the proteasome are generally 8-10 amino acids in length and mirror the linear sequence of the parental protein. However, immune evasion is a hallmark of cancer cells that disrupt the antigen processing and presentation machinery. Tumors develop strategies to evade host immunity and resist current immunotherapies, while others display a low tumor mutation burden limiting the presence of tumor antigenicity. Here, we postulated that pharmacologic enhancement of proteasome catalytic activity would increase MHC class I antigen presentation, modulate the myeloma immunopeptidome, and promote CD8+ T-cells anti-myeloma activity. A cell-based, high-throughput screen revealed that the histone deacetylase 6 (HDAC6) inhibitors tubastatin-A, ACY-738 and ACY-1215 increased proteasome activity in multiple myeloma (MM) cells. HDAC6 inhibitors increased pan-MHC class-I expression in MM cells as well as a panel of cancer cell lines. Treatment of MM patient tumor cells with HDAC6 inhibitors increased the cytotoxic effect of autologous patient-derived T-cells. Mechanism-based studies demonstrated that pharmacologic blockade or genetic ablation of the HDAC6 ubiquitin-binding (BUZ) domain disrupted HDAC6 association with HR23B, a substrate-shuttle factor that delivers ubiquitinylated cargo to proteasomes. HDAC6 inhibitors or HDAC6-BUZ domain deletion reduced the association of HR23B with HDAC6 and promoted binding of HR23B to proteasomes and a consequent increase in proteasome activity. The results validate the HDAC6-BUZ domain as an attractive drug discovery target and demonstrate that proteasome activators enhance antitumor immunity. Proteasome activators represent a paradigm-shifting approach to overcome mechanisms of immune escape in cancer cells. FDA-approved drugs that increase proteasome activity and boost antigen presentation and can be repositioned as cancer immunotherapeutics to overcome the existing bottlenecks in drug development. Taken together, our results highlight the breadth and magnitude of the proteasome in governing fundamental cellular processes and the immense potential of therapeutics that exploit proteasomes to treat human disease. Citation Format: Priyanka S. Rana, James J. Ignatz-Hoover, James J. Driscoll. HDAC6 inhibition increases proteasome activity and modulates the myeloma immunopeptidome to promote cytotoxic T-cell activity [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C083.

Upregulation of MHC I Antigen Processing Machinery Gene Expression in Breast Cancer Cells by Trichostatin A

Upregulation of MHC I Antigen Processing Machinery Gene Expression in Breast Cancer Cells by Trichostatin A

Targeting Proteasomes and the MHC Class I Antigen Presentation Machinery to Treat Cancer, Infections and Age-Related Diseases

The majority of T-cell responses involve proteasome-dependent protein degradation and the downstream presentation of oligopeptide products complexed with major histocompatibility complex (MHC) class I (MHC-I) molecules to peptide-restricted CD8+ T-cells. However, evasion of host immunity is a cancer hallmark that is achieved by disruption of host antigen processing and presentation machinery (APM). Consequently, mechanisms of immune evasion promote cancer growth and survival as well as de novo and acquired resistance to immunotherapy. A multitude of cell signaling pathways modulate the APM and MHC-I-dependent antigen presentation. Pharmacologics that specifically target and modulate proteasome structure and activity represent a novel emerging strategy to improve the treatment of cancers and other diseases characterized by aberrant protein accumulation. FDA-approved pharmacologics that selectively activate proteasomes and/or immunoproteasomes can be repositioned to overcome the current bottlenecks that hinder drug development to enhance antigen presentation, modulate the immunopeptidome, and enhance the cytotoxic activity of endogenous or engineered T-cells. Strategies to enhance antigen presentation may also improve the antitumor activity of T-cell immunotherapies, checkpoint inhibitors, and cancer vaccines. Proteasomes represent actionable therapeutic targets to treat difficult-to-treat infectious processes and neurodegenerative diseases that are characterized by the unwanted accrual of insoluble, deleterious, and potentially toxic proteins. Taken together, we highlight the breadth and magnitude of the proteasome and the immense potential to amplify and unmask the immunopeptidomic landscape to improve the treatment of a spectrum of human diseases.