Antigen Processing Machinery Components Research Articles

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.

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.

Correlation of the tumor escape phenotype with loss of PRELP expression in melanoma

BackgroundDespite immunotherapies having revolutionized the treatment of advanced cutaneous melanoma, effective and durable responses were only reported in a few patients. A better understanding of the interaction of melanoma cells with the microenvironment, including extracellular matrix (ECM) components, might provide novel therapeutic options. Although the ECM has been linked to several hallmarks of cancer, little information is available regarding the expression and function of the ECM protein purine-arginine-rich and leucine-rich protein (PRELP) in cancer, including melanoma.MethodsThe structural integrity, expression and function of PRELP, its correlation with the expression of immune modulatory molecules, immune cell infiltration and clinical parameters were determined using standard methods and/or bioinformatics.ResultsBioinformatics analysis revealed a heterogeneous, but statistically significant reduced PRELP expression in available datasets of skin cutaneous melanoma when compared to adjacent normal tissues, which was associated with reduced patients’ survival, low expression levels of components of the MHC class I antigen processing machinery (APM) and interferon (IFN)-γ signal transduction pathway, but increased expression of the transforming growth factor (TGF)-β isoform 1 (TFGB1) and TGF-β receptor 1 (TGFBR1). In addition, a high frequency of intra-tumoral T cells directly correlated with the expression of MHC class I and PRELP as well as the T cell attractant CCL5 in melanoma lesions. Marginal to low PRELP expression levels were found in the 47/49 human melanoma cell lines analysis. Transfection of PRELP into melanoma cell lines restored MHC class I surface expression due to transcriptional upregulation of major MHC class I APM and IFN-γ pathway components. In addition, PRELP overexpression is accompanied by high CCL5 secretion levels in cell supernatant, an impaired TGF-β signaling as well as a reduced cell proliferation, migration and invasion of melanoma cells.ConclusionsOur findings suggest that PRELP induces the expression of MHC class I and CCL5 in melanoma, which might be involved in an enhanced T cell recruitment and immunogenicity associated with an improved patients’ outcome. Therefore, PRELP might serve as a marker for predicting disease progression and its recovery could revert the tumorigenic phenotype, which represents a novel therapeutic option for melanoma.

Effects of hypoxia on antigen presentation and T cell-based immune recognition of HPV16-transformed cells.

Attempts to develop a therapeutic vaccine against human papillomavirus (HPV)-induced malignancies have mostly not been clinically successful to date. One reason may be the hypoxic microenvironment present in most tumors, including cervical cancer. Hypoxia dysregulates the levels of human leukocyte antigen (HLA) class I molecules in different tumor entities, impacts the function of cytotoxic T cells, and leads to decreased protein levels of the oncoproteins E6 and E7 in HPV-transformed cells. Therefore, we investigated the effect of hypoxia on the presentation of HPV16 E6- and E7-derived epitopes in cervical cancer cells and its effect on epitope-specific T cell cytotoxicity. Hypoxia induced downregulation of E7 protein levels in all analyzed cell lines, as assessed by Western blotting. However, contrary to previous reports, no perturbation of antigen processing and presentation machinery (APM) components and HLA-A2 surface expression upon hypoxia treatment was detected by mass spectrometry and flow cytometry, respectively. Cytotoxicity assays performed in hypoxic conditions showed differential effects on the specific killing of HPV16-positive cervical cancer cells by epitope-specific CD8+ Tcell lines in a donor- and peptide-specific manner. Effects of hypoxia on the expression of PD-L1 were ruled out by flow cytometry analysis. Altogether, our results under hypoxia show a decreased expression of E6 and E7, but an intact APM, and epitope- and donor-dependent effects on T cell cytotoxicity towards HPV16-positive target cells. This suggests that successful immunotherapies can be developed for hypoxic HPV-induced cervical cancer, with careful choice of target epitopes, and ideally in combination with hypoxia-alleviating measures.

Biglycan as a potential regulator of tumorgenicity and immunogenicity in K-RAS-transformed cells

ABSTRACT The extracellular matrix component biglycan (BGN) plays an essential role in various physiological and pathophysiological processes. A deficient BGN expression associated with reduced immunogenicity was found in HER-2/neu-overexpressing cells. To determine whether BGN is suppressed by oncogene-driven regulatory networks, the expression and function of BGN was analyzed in murine and human BGNlow/BGNhigh K-RASG12V-transformed model systems as well as in different patients’ datasets of colorectal carcinoma (CRC) lesions. K-RAS-mutated CRC tissues expressed low BGN mRNA and protein levels when compared to normal colon epithelial cells, which was associated with a reduced patients’ survival. Transfection of BGN in murine and human BGNlow K-RAS-expressing cells resulted in a reduced growth and migration of BGNhigh vs BGNlow K-RAS cells. In addition, increased MHC class I surface antigens as a consequence of an enhanced antigen processing machinery component expression was found upon restoration of BGN, which was confirmed by RNA-sequencing of BGNlow vs. BGNhigh K-RAS models. Furthermore, a reduced tumor formation of BGNhigh versus BGNlow K-RAS-transformed fibroblasts associated with an enhanced MHC class I expression and an increased frequency of tumor-infiltrating lymphocytes in tumor lesions was found. Our data provide for the first time an inverse link between BGN and K-RAS expression in murine and human K-RAS-overexpressing models and CRC lesions associated with altered growth properties, reduced immunogenicity and worse patients’ outcome. Therefore, reversion of BGN might be a novel therapeutic option for K-RAS-associated malignancies.

HLA AND CANCER

This review provides updated information on HLA class I and II antigens in cancer. The expression of HLA antigens in normal and tumor tissues, the physiological organization of the components of HLA antigen-processing machinery, the expression patterns of HLA antigens associated with the molecular and regulatory defects identified to date, as well as their functional and clinical significance, are described. This review summarizes clinical and experimental data on the complexity of immune escape mechanisms used by tumour cells to avoid T and natural killer cell responses. The variety of class I HLA phenotypes that can be produced by tumor cells during this process is presented. We also discuss here the potential capacity of metastatic lesions to recover MHC/HLA class I expression after immunotherapy, which depends on the reversible/ soft or irreversible/hard nature of the molecular mechanism responsible for the altered HLA class I phenotypes, and which determines the progression or regression of metastatic lesions in response to treatment. HLA сlass II genes play key roles in connecting innate and adaptive immunity in tumor rejection and when the escape route via HLA-I is already established. Antigens сlass II HLA expression in tumor cells and gives tumor cells the ability to present antigens, becoming less aggressive, and improves prognosis. Malignant tumors, as a genetic disease, are caused by structural alterations of the genome which can give rise to the expression of tumor-associated antigens in the form of either structurally altered molecules or of overexpressed normal molecules. Tumor associated antigens recognized by the immune system and induce a T-cell-mediated immune response. Outgrowing cancers use different strategies to evade destruction by the immune system. Immune evasion mechanisms affecting the expression and/or function of HLA-antigens are of special interest to tumor immunologists, since these molecules play a crucial role in the interaction of malignant cells with immune cells. This review describes the potential role of immunity control points in immunosuppression and therapeutic strategies for restoring the cytotoxicity of immune cells.

910 Overexpression of miR-155–5p can upregulate antigen processing and presentation pathway via targeting tapasin

BackgroundDysregulation of major histocompatibility complex (MHC) class I antigen processing and presentation machinery (APM) components in the tumor as one main molecular mechanism of immune escape leading to deactivation of T cell immune surveillance could be due to post-transcriptional regulation via immune-modulatory microRNAs (miRNA). It is now well established from a variety of studies that several miRNAs could effectively modulate the expression of some MHC class I APM components in tumors. Tapasin is an important APM molecule involved in the association of MHC class I with transporter associated with antigen processing (TAP) and peptide loading. Since so far no detailed investigation of the posttranscriptional regulation of tapasin exists, the aim of this study is to identify and functionally characterize miRNAs targeting tapasin in melanoma.MethodsUsing miRNA trapping by RNA in vitro affinity purification (miTRAP) and in silico as well as small RNA sequencing, miRNAs will be identified, which bind to the 3’untranslated region (3’ UTR) of tapasin. Dual-luciferase assays will be performed to determine to bind of the miRNA. In silico analysis was performed to predict the effect of miRNAs on the survival of melanoma patients in correlation to tapasin. RT-qPCR, Western blot, flow cytometry, and other functional assays were performed after transfecting miRNA mimics in three melanoma cell lines.ResultsUsing the combination strategy of miTRAP and RNA seq we identified miR-155-5p to bind to the 3’UTR of tapasin, which was further confirmed by in silico analysis and dual-luciferase reporter assay. Transfection of miR-155-5p mimics demonstrated that miR-155-5p upregulate tapasin protein level, which was accompanied by an upregulation of the MHC class I (HLA-ABC) surface expression. Simultaneously, in several different types of cancer, including melanoma, the expression of miR-155-5p is significantly positively correlated with the patient‘s survival and HLA-A protein.ConclusionsOur data revealed for the first time a positive role of miR-155-5p in the posttranscriptional regulation of tapasin in melanoma and provide further insights into the miR-155-5p-mediated induction of HLA-ABC surface expression. This might lead to a better T cell response, avoidance tumor cell escape, improvement of patients‘ survival and thus might be a potential therapeutic target.AcknowledgementsThe work was supported by a grant from the DKH (BS).

Distinct Molecular Mechanisms of Altered HLA Class II Expression in Malignant Melanoma.

Simple SummaryThere exists limited knowledge about the underlying molecular processes controlling the expression of HLA class II APM components and their prognostic significance in melanoma. Therefore, this study analyzed the basal and regulated expression of HLA class II antigens and components in melanoma cell lines and patients’ lesions in conjunction to T-cell infiltration. The heterogeneous constitutive HLA class II APM expression was caused by distinct molecular mechanisms and was partially linked to immune cell infiltration and clinical parameters. These results contribute not only to a better understanding of the regulation of HLA class II expression in melanoma, but might have an impact on the design of novel (immuno)therapies for the treatment of this disease.Background: The human leukocyte antigen (HLA) class II molecules are constitutively expressed in some melanoma, but the underlying molecular mechanisms have not yet been characterized. Methods: The expression of HLA class II antigen processing machinery (APM) components was determined in melanoma samples by qPCR, Western blot, flow cytometry and immunohistochemistry. Immunohistochemical and TCGA datasets were used for correlation of HLA class II expression to tumor grading, T-cell infiltration and patients’ survival. Results: The heterogeneous HLA class II expression in melanoma samples allowed us to characterize four distinct phenotypes. Phenotype I totally lacks constitutive HLA class II surface expression, which is inducible by interferon-gamma (IFN-γ); phenotype II expresses low basal surface HLA class II that is further upregulated by IFN-γ; phenotype III lacks constitutive and IFN-γ controlled HLA class II expression, but could be induced by epigenetic drugs; and in phenotype IV, lack of HLA class II expression is not recovered by any drug tested. High levels of HLA class II APM component expression were associated with an increased intra-tumoral CD4+ T-cell density and increased patients’ survival. Conclusions: The heterogeneous basal expression of HLA class II antigens and/or APM components in melanoma cells is caused by distinct molecular mechanisms and has clinical relevance.

Defective HLA Class I Expression and Patterns of Lymphocyte Infiltration in Chordoma Tumors.

There are no effective systemic therapies for chordoma. The recent successes of immunotherapeutic strategies in other cancers have resulted in a resurgence of interest in using immunotherapy in chordoma. These approaches rely on a functional interaction between the host's immune system and the expression of tumor peptides via the human leukocyte antigen (HLA) Class I antigen. It is not known whether chordoma cells express the HLA Class I antigen. (1) Do chordoma tumors exhibit defects in HLA Class I antigen expression? (2) What is the pattern of lymphocyte infiltration in chordoma tumors? Patients with chordoma treated at Massachusetts General Hospital between 1989 and 2009 were identified with permission from the institutional review board. Of the 75 patients who were identified, 24 human chordoma tumors were selected from 24 distinct patients based on tissue availability. Histology slides from these 24 formalin-fixed paraffin-embedded chordoma tissue samples were deparaffinized using xylene and ethanol and underwent heat-induced antigen retrieval in a citrate buffer. Samples were incubated with monoclonal antibodies directed against HLA Class I antigen processing machinery components. Antibody binding was detected via immunohistochemical staining. Staining intensity (negative, weakly positive, strongly positive) was assessed semiquantitatively and the percentage of chordoma cells stained for HLA Class I antigen subunits was assessed quantitatively. Hematoxylin and eosin-stained histology slides from the same 24 chordoma samples were assessed qualitatively for the presence of tumor-infiltrating lymphocytes and histologic location of these lymphocytes. Immunohistochemical staining with monoclonal antibodies directed against CD4 and CD8 was performed in a quantitative manner to identify the lymphocyte subtype present in chordoma tumors. All results were scored independently by two investigators and were confirmed by a senior bone and soft tissue pathologist. Seven of 24 chordoma samples exhibited no staining by the anti-HLA-A heavy chain monoclonal antibody HC-A2, two had weak staining intensity, and eight had a heterogeneous staining pattern, with fewer than 60% of chordoma cells exhibiting positive staining results. Four of 24 samples tested were not stained by the anti-HLA-B/C heavy chain monoclonal antibody HC-10, five had weak staining intensity, and 11 displayed a heterogeneous staining pattern. For the anti-β-2-microglobulin monoclonal antibody NAMB-1, staining was detected in all samples, but 11 had weak staining intensity and four displayed a heterogeneous staining pattern. Twenty-one of 24 samples tested had decreased expression in at least one subunit of HLA Class I antigens. No tumors were negative for all three subunits. Lymphocytic infiltration was found in 21 of 24 samples. Lymphocytes were primarily found in the fibrous septae between chordoma lobules but also within the tumor lobules and within the fibrous septae and tumor lobules. Twenty-one of 24 tumors had CD4+ T cells and 11 had CD8+ T cells. In chordoma tissue samples, HLA Class I antigen defects commonly were present, suggesting a mechanism for escape from host immunosurveillance. Additionally, nearly half of the tested samples had cytotoxic CD8+ T cells present in chordoma tumors, suggesting that the host may be capable of mounting an immune response against chordoma tumors. The resulting selective pressure imposed on chordoma tumors may lead to the outgrowth of chordoma cell subpopulations that can evade the host's immune system. These findings have implications in the design of immunotherapeutic strategies for chordoma treatment. T cell recognition of tumor cells requires HLA Class I antigen expression on the targeted tumor cells. Defects in HLA Class I expression may play a role in the clinical course of chordoma and may account for the limited or lack of efficacy of T cell-based immunity triggered by vaccines and/or checkpoint inhibitors.

Polymorphisms of Antigen-Presenting Machinery Genes in Non-Small Cell Lung Cancer: Different Impact on Disease Risk and Clinical Parameters in Smokers and Never-Smokers.

Lung cancer is strongly associated with cigarette smoking; nevertheless some never-smokers develop cancer. Immune eradication of cancer cells is dependent on polymorphisms of HLA class I molecules and antigen-processing machinery (APM) components. We have already published highly significant associations of single nucleotide polymorphisms (SNPs) of the ERAP1 gene with non-small cell lung cancer (NSCLC) in Chinese, but not in Polish populations. However, the smoking status of participants was not known in the previous study. Here, we compared the distribution of APM polymorphic variants in larger cohorts of Polish patients with NSCLC and controls, stratified according to their smoking status. We found significant but opposite associations in never-smokers and in smokers of all tested SNPs (rs26653, rs2287987, rs30187, and rs27044) but one (rs26618) in ERAP1. No significant associations were seen in other genes. Haplotype analysis indicated that the distribution of many ERAP1/2 haplotypes is opposite, depending on smoking status. Additionally, haplotypic combination of low activity ERAP1 and the lack of an active form of ERAP2 seems to favor the disease in never-smokers. We also revealed interesting associations of some APM polymorphisms with: age at diagnosis (ERAP1 rs26653), disease stage (ERAP1 rs27044, PSMB9 rs17587), overall survival (ERAP1 rs30187), and response to chemotherapy (ERAP1 rs27044). The results presented here may suggest the important role for ERAP1 in the anti-cancer response, which is different in smokers versus never-smokers, depending to some extent on the presence of ERAP2, and affecting NSCLC clinical course.

Tumor Microenvironment, HLA Class I and APM Expression in HPV-Negative Oral Squamous Cell Carcinoma.

Simple SummaryOral squamous cell carcinoma has developed different strategies to escape from T-cell-mediated immune surveillance, which is mediated by changes in the composition of cellular and soluble components of the tumor microenvironment as well as an impaired expression of molecules of the antigen processing machinery leading to a downregulation of HLA class I surface antigens. In depth characterization of these escape mechanisms might help to develop strategies to overcome this tolerance. In this study, human papilloma virus negative oral squamous cell carcinoma lesions were analyzed regarding the protein expression of major components of the HLA class I antigen processing/presentation pathway in correlation to the intra-tumoral immune cell composition, IFN-γ signaling and clinical parameters, which was further confirmed by bioinformatics analyses of datasets obtained from The Cancer Genome Atlas. This novel knowledge could be used for optimizing the design of immunotherapeutic approaches of this disease.Progression of oral squamous cell carcinoma (OSCC) has been associated with an escape of tumor cells from the host immune surveillance due to an increased knowledge of its underlying molecular mechanisms and its modulation by the tumor microenvironment and immune cell repertoire. In this study, the expression of HLA class I (HLA-I) antigens and of components of the antigen processing machinery (APM) was analyzed in 160 pathologically classified human papilloma virus (HPV)-negative OSCC lesions and correlated to the intra-tumoral immune cell response, IFN-γ signaling and to the patient’s outcome. A heterogeneous but predominantly lower constitutive protein expression of HLA-I APM components was found in OSCC sections when compared to non-neoplastic cells. Tumoral HLA-I APM component expression was further categorized into the three major phenotypes HLA-Ihigh/APMhigh, HLA-Ilow/APMlow and HLA-Idiscordant high/low/APMhigh. In the HLA-Ihigh/APMhigh group, the highest frequency of intra-tumoral CD8+ T cells and lowest number of CD8+ T cells close to FoxP3+ cells were found. Patients within this group presented the most unfavorable survival, which was significantly evident in stage T2 tumors. Despite a correlation with the number of intra-tumoral CD8+ T cells, tumoral JAK1 expression as a surrogate marker for IFN-γ signaling was not associated with HLA-I/APM expression. Thus, the presented findings strongly indicate the presence of additional factors involved in the immunomodulatory process of HPV-negative OSCC with a possible tumor-burden-dependent complex network of immune escape mechanisms beyond HLA-I/APM components and T cell infiltration in this tumor entity.

The HDAC Inhibitor Domatinostat Promotes Cell-Cycle Arrest, Induces Apoptosis, and Increases Immunogenicity of Merkel Cell Carcinoma Cells

Merkel cell carcinoma (MCC) is a rare, highly aggressive skin cancer for which immune modulation by immune checkpoint inhibitors shows remarkable response rates. However, primary or secondary resistance to immunotherapy prevents benefits in a significant proportion of patients. For MCC, one immune escape mechanism is insufficient for recognition by T cells owing to the downregulation of major histocompatibility complex I surface expression. Histone deacetylase inhibitors have been demonstrated to epigenetically reverse the low major histocompatibility complex I expression caused by the downregulation of the antigen-processing machinery. Domatinostat, an orally available small-molecule inhibitor targeting histone deacetylase class I, is currently in clinical evaluation to overcome resistance to immunotherapy. In this study, we present preclinical data on domatinostat's efficacy and mode of action in MCC. Single-cell RNA sequencing revealed a distinct gene expression signature of antigen processing and presentation, cell-cycle arrest, and execution phase of apoptosis on treatment. Accordingly, functional assays showed that domatinostat induced G2M arrest and apoptosis. In the surviving cells, antigen-processing machinery component gene transcription and translation were upregulated, consequently resulting in increased major histocompatibility complex I surface expression. Altogether, domatinostat not only exerts direct antitumoral effects but also restores HLA class I surface expression on MCC cells, therefore, restoring surviving MCC cells' susceptibility to recognition and elimination by cognate cytotoxic T cells.

Abstract B11: Antagonist of cIAP1/2 and XIAP induces immunogenic cell death and alters antigen-processing machinery in preclinical head and neck cancer models

Abstract Background/Rationale: The Cancer Genome Atlas (TCGA) analysis of head and neck squamous cell carcinomas (HNSCCs) has revealed common alterations in the Tumor Necrosis Factor (TNF) Receptor signaling pathway, with 30% overexpressing genes encoding for Fas-Associated Death Domain (FADD) and/or cellular Inhibitor of Apoptosis Proteins 1/2 (cIAP1/2). We have previously shown that ASTX660, an antagonist of cIAP1/2 and XIAP, sensitizes murine oral cancer (MOC) cells to TNFα and also demonstrates synergistic antitumor activity when used in combination with radiation therapy (XRT) and PD-1 immune checkpoint blockade, leading to tumor growth delay or eradication in a syngeneic mouse model (Xiao et al., OncoImmunology 2018). These combination treatments also enhanced the number and function of CD8+ T cells and dendritic cells. However, the immune effects of IAP antagonists at the tumor cell level are still unknown. We hypothesized that ASTX660 enhances immune-based killing of tumor cells through induction of immunogenic cell death (ICD) and upregulation of antigen-processing machinery (APM) components. Methods/Results: We performed ICD vaccination experiments in two syngeneic mouse models of HNSCC. We vaccinated mice with MOC1 cells killed by XRT, mitoxantrone (MTX), ASTX660 + TNFα or the combination of ASTX660 + TNFα + XRT in the left flank. One week later we rechallenged with live MOC1 cells in the right flank and monitored subsequent tumor formation/growth. Positive controls XRT and MTX, known ICD inducers, resulted in tumor rejection rates of 40% and 20%, respectively. Cells treated with ASTX660 + TNFα demonstrated growth of tumors at the vaccination site on the left flank, but 90% rejection when rechallenged with viable cells on the right. Combination vaccination group ASTX660 + TNFα + XRT showed 100% tumor rejection with no vaccination site growth. Similar results were obtained with the MEER (HPV-positive) mouse model. To test our hypothesis that ASTX660 upregulates APM components, we analyzed expression of intracellular APM components by flow cytometry in human HNSCC cell lines (UMSCC-46, -11B, and -74A) treated with interferon gamma (positive control), ASTX660 alone, TNFα alone, or ASTX660 + TNFα. Changes in APM components were variable among cell lines treated with ASTX660 + TNFα, though HLA-A,B,C was consistently upregulated. Conclusions/Future Directions: Our results demonstrate that ASTX660 induces ICD and upregulates APM components, providing a mechanism contributing to synergism between IAP antagonists with XRT as an effective treatment modality for HNSCC. Furthermore, with a significantly more favorable drug toxicity profile than cisplatin, combination treatment with ASTX660 + XRT may be a potential replacement for traditional chemoradiation in HNSCC. Citation Format: Wenda Ye, Sreenivasulu Gunti, Carter Van Waes, Nicole C. Schmitt. Antagonist of cIAP1/2 and XIAP induces immunogenic cell death and alters antigen-processing machinery in preclinical head and neck cancer models [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr B11.

ASTX660, an antagonist of cIAP1/2 and XIAP, increases antigen processing machinery and can enhance radiation-induced immunogenic cell death in preclinical models of head and neck cancer

ABSTRACT Inhibitor of apoptosis protein (IAP) antagonists have shown activity in preclinical models of head and neck squamous cell carcinoma (HNSCC), and work across several cancer types has demonstrated diverse immune stimulatory effects including enhancement of T cell, NK cell, and dendritic cell function. However, tumor-cell-intrinsic mechanisms for this immune upregulation have been largely unexplored. In this study, we show that ASTX660, an antagonist of cIAP1/2 and XIAP, induces expression of immunogenic cell death (ICD) markers in sensitive HNSCC cell lines in vitro. Experiments in syngeneic mouse models of HNSCC showed that ASTX660 can also enhance radiation-induced ICD in vivo. On a functional level, ASTX660 also enhanced killing of multiple murine cell lines by cytotoxic tumor-infiltrating lymphocytes, and when combined with XRT, stimulated clonal expansion of antigen-specific T lymphocytes and expression of MHC class I on the surface of tumor cells. Flow cytometry experiments in several human HNSCC cell lines showed that MHC class I (HLA-A,B,C) was reliably upregulated in response to ASTX660 + TNFα, while increases in other antigen processing machinery (APM) components were variable among different cell lines. These findings suggest that ASTX660 may enhance anti-tumor immunity both by promoting ICD and by enhancing antigen processing and presentation.

MHC class I dysfunction of glioma stem cells escapes from CTL-mediated immune response via activation of Wnt/β-catenin signaling pathway.

Glioma stem cells (GSCs) decrease T cells cognition and evade systemic immunosurveillance via downregulations or defects of major histocompatibility complex class I (MHC-I) molecule and antigen-processing machinery (APM) components. Improvement of tumor surface antigens of GSCs may be effective strategy to trigger an adaptive immune response and activate cytotoxic T cells (CTLs) to eliminate glioma. In this study, our data indicated that downregulations of MHC-I and APM components expressions were associated with Wnt pathway activation in GSCs. Histone deacetylases (HDAC) inhibition improved MHC-I and APM components expressions, which could be partly reverted by Wnt pathway activation. Blocking CTLs-mediated killing decreased the anti-tumor effect of tumor lysate vaccine. The enhancement of T cells immune response resulting from HDAC inhibition was dependent on CTLs cognition on tumor antigens presented by upregulated MHC-I molecule in GSCs. These data suggest that suppression of stemness pathway may be effective for GSCs-based immunotherapy against immune-escaped tumors.

HLA Class I Antigen Processing Machinery Defects in Cancer Cells-Frequency, Functional Significance, and Clinical Relevance with Special Emphasis on Their Role in T Cell-Based Immunotherapy of Malignant Disease.

MHC class I antigen abnormalities have been shown to be one of the major immune escape mechanisms murine and human cancer cells utilize to avoid recognition and destruction by host immune system. This mechanism has clinical relevance, since it is associated with poor prognosis and/or reduced patients' survival in many types of malignant diseases. The recent impressive clinical responses to T cell-based immunotherapies triggered by checkpoint inhibitors have rekindled tumor immunologists and clinical oncologists' interest in the analysis of the human leukocyte antigen (HLA) class I antigen processing machinery (APM) expression and function in malignant cells. Abnormalities in the expression, regulation and/or function of components of this machinery have been associated with the development of resistances to T cell-based immunotherapies. In this review, following the description of the human leukocyte antigen (HLA) class I APM organization and function, the information related to the frequency of defects in HLA class I APM component expression in various types of cancer and the underlying molecular mechanisms is summarized. Then the impact of these defects on clinical response to T cell-based immunotherapies and strategies to revert this immune escape process are discussed.

Abstract 569: Expression and modulation by IFN of HLA class I APM components in melanoma: Potential biomarkers of clinical response to checkpoint inhibitors

Abstract Purpose: The goals of this study are i) to analyze the expression of human leukocyte antigen class I (HLA-I) antigen processing machinery (APM) components in surgically removed melanoma tumors, ii) to assess their modulation by Interferons (IFNs) and iii) to determine their value as predictive biomarkers of the clinical response to immunotherapy with checkpoint inhibitors (CI). Methods: The expression levels of HLA class I APM components [HLA-I (ABC) chains, beta(2)microglobulin (β2m), Calnexin, Calreticulin, ERp57, Tapasin, TAP1-2, LMP2, LMP7, LMP10] were evaluated by staining with mAbs and flow cytometry in melanoma cells isolated from surgical biopsies from 16 patients with advanced melanoma. In addition the modulation of these molecules by IFN-γ or IFN-α2 (IFNs) was evaluated. Results: The frequency of cells expressing HLA-I/β2m was >90% in 15/16 Mel tumors. In 1 case extracellular HLA-I/β2m molecules were not detected. The expression levels of extracellular HLA class I APM molecules were quite heterogeneous. Based on Mean Fluorescence Intensity (MFI) of HLA-I/β2m, Mel segregated into 2 groups with high (MFI >200, n=8) and low (MFI<200, n=8) expression levels. In 11 cases (5 from the high and 6 from the low expression groups) we could explore the modulatory activity of IFNs. In vitro exposure to IFNs globally enhanced the expression levels of HLA-I/β2m, allowing the transition from the low to the highexpression group in 1 case. The distribution of expression defects in the intracellular APM components resulted intensely heterogeneous. Highly defective (<25%) expressions were detected notably for LMP2 (12/16), TAP1 (13/16), TAP2 (10/16) and Tapasin (14/16). In 10 cases we could explore the beneficial modulatory activity of IFNs on defective APM components. IFNs could restore the expression of LMP2 (4/10), TAP1 (6/10), and TAP2 (7/8), but not that of Tapasin. Seven patients were treated with CI (either anti-PD1 or anti-CTLA4). Four of them had clinical responses (PR or CR); in 3 of them Mel expressed high levels of HLA-I/β2m. Conversely, Mel from all 3 patients that did not respond to CI expressed low HLA-I/β2m levels which could not be upregulated by IFNs. Highly defective expression of APM components, not responsive to IFN restoration, was detected in all 3 Mel from non-responders. Conclusions: We report a wide heterogeneous distribution of HLA-I/β2m expression levels and deficits of APM components in Melanoma, partially restorable by IFNs. Our data support the possibility that defects in HLA expression, not responsive to IFN restoration, may predict low responsiveness to CI. We provide rationale to incorporate the evaluation of HLA-I/β2m and APM in clinical immunotherapy studies with CI. Citation Format: Lidia Giraudo, Loretta Gammaitoni, Bortolot Valentina, Ilenia Iaia, Giulia Cattaneo, Paolo Becco, Susanna Gallo, Alessandro Zaccagna, Alberto Pisacane, Luca Crotto, Soldano Ferrone, Massimo Aglietta, Fabrizio Carnevale Schianca, Dario Sangiolo. Expression and modulation by IFN of HLA class I APM components in melanoma: Potential biomarkers of clinical response to checkpoint inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 569.

Abstract 2247: Platinoid-induced MHCI antigen presentation enables checkpoint inhibitor responsiveness

Abstract Many cancers are refractory to immune checkpoint therapy (ICT), but the causes of ICT resistance remain unknown. Despite low mutational burden, hepatocellular carcinoma (HCC) responds to PD-1 blockade, but prostate cancer (PCa) does not. RNA-seq analysis revealed that, unlike HCC, PCa cells poorly express MHC-I molecules and components of the MHC-I antigen processing and presentation machinery. We found that treatment of PCa cells and other tumor types with platin-based drugs, especially low dose Oxaliplatin, led to a pronounced upregulation of MHC-I molecules and their antigen generating and loading machinery. Most importantly, Oxaliplatin synergistically potentiated the effect of exogenous IFNγ; a conclusion based on two cumulative observations. First, ATAC-seq analyses revealed that Oxaliplatin activates MHC machinery through different pathways compared to well-known IFNγ signaling. Second, Oxaliplatin upregulates IFNγR2 expression in cancer cells. Induction of IFNγR2, which was needed for Oxaliplatin-potentiation of ICT-induced tumor rejection in vivo, depended on NF-κB signaling. Induction of IFNγR2, which was needed for Oxaliplatin-potentiation of ICT-induced tumor rejection in vivo, depended on NF-κB signaling. Moreover, we found that Oxaliplatin activates histone acetyltransferases (HATs) which alter chromatin structure of genes encoding MHC-I complex components, thereby enabling recruitment of a diverse array of activating transcription factors. Our data indicates that combining ICT with an appropriate low dose platinoid chemotherapeutic agent is useful in reinvigorating the immune response to tumors that have escaped immune destruction due to MHC-I downregulation and do not respond to single agent therapy. Citation Format: Ingmar Niels Bastian, Weihua Li, Qui Phung, Kathleen Fisch, Jennie Lill, Michael Karin, Shabnam Shalapour. Platinoid-induced MHCI antigen presentation enables checkpoint inhibitor responsiveness [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2247.

Molecular and Functional Profiles of Exosomes From HPV(+) and HPV(-) Head and Neck Cancer Cell Lines.

Exosomes produced by tumor cells have been shown to reprogram functions of human immune cells. Molecular cargos of exosomes isolated from supernatants of HPV(+) and HPV(−) head and neck cancer (HNC) cell lines or from HNC patients' plasma were compared. The exosome protein profiles resembled those of respective parent tumor cells. Only HPV(+) exosomes carried E6/E7, p16, and survivin. HPV(−) exosomes were negative for cyclin D1 and carried low p53 levels. Immunomodulatory molecules (TGF-β, FasL, OX40, OX40L, and HSP70) were carried by HPV(+) and HPV(−) exosomes. These exosomes co-incubated with human T cells induced apoptosis and suppressed T cell activation and proliferation. HPV(−) exosomes suppressed DC maturation and expression of antigen processing machinery (APM) components. In contrast, HPV(+) exosomes promoted DC maturation and did not suppress expression of APM components in mature DCs. While DCs readily internalized exosomes, T lymphocytes resisted their uptake during the initial 12 h co-culture. Thus, HPV(+) exosomes capable of sustaining DC functions may play a key role in promoting anti-tumor immune responses thereby improving outcome in patients with HPV(+) cancers.

Defective HLA class I antigen processing machinery in cancer.

Malignant transformation of cells is frequently associated with defective HLA class I antigen processing machinery (APM) component expression. This abnormality may have functional relevance, since it may have a negative impact on tumor cell recognition by cognate T cells. Furthermore, HLA class I APM abnormalities appear to have clinical significance, since they are associated with poor prognosis in several malignant diseases and may play a role in the resistance to immune checkpoint inhibitor-based immunotherapy. In this paper, we have reviewed the literature describing abnormalities in HLA class I APM component expression in many types of cancer. These abnormalities have been reported in all types of cancer analyzed with a frequency ranging between a minimum of 35.8% in renal cancer and a maximum of 87.9% in thyroid cancer for HLA class I heavy chains. In addition, we have described the molecular mechanisms underlying defects in HLA class I APM component expression and function by malignant cells. Lastly, we have discussed the clinical significance of HLA class I APM component abnormalities in malignant tumors.