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Abstract Background Checkpoint immunotherapy unleashes tumor control by T cells, but it is undermined in non-immunogenic tumors, e.g. with low MHC class I expression and low neoantigen burden, such as neuroblastoma (NB). Endoplasmic reticulum aminopeptidase 1 (ERAP1) is an enzyme that trims peptides before loading on MHC class I molecules. Inhibition of ERAP1 results in the generation of new antigens able of inducing potent anti-tumor immune responses. Here, we identify a novel non-toxic combinatorial strategy based on genetic inhibition of ERAP1 and administration of the HDAC inhibitor (HDACi) entinostat that increase the immunogenicity of NB, making it responsive to PD-1 therapy. Methods CRISPR/Cas9-mediated gene editing was used to knockout (KO) the ERAP1 gene in 9464D NB cells derived from spontaneous tumors of TH-MYCN transgenic mice. The expression of MHC class I and PD-L1 was evaluated by flow cytometry (FC). The immunopeptidome of these cells was studied by mass spectrometry. Cocultures of splenocytes derived from 9464D bearing mice and tumor cells allowed the assessment of the effect of ERAP1 inhibition on the secretion of inflammatory cytokines and activation and migration of immune cells towards ERAP1 KO cells by FC. Tumor cell killing was evaluated by Caspase 3/7 assay and flow cytometry analysis. The effect of ERAP1 inhibition on the immune content of tumors was analyzed by FC, immunohistochemistry and multiple immunofluorescence. Results We found that inhibition of ERAP1 makes 9464D cells more susceptible to immune cell-mediated killing by increasing both the recall and activation of CD4+ and CD8+ T cells and NK cells. Treatment with entinostat induces the expression of MHC class I and PD-L1 molecules in 9464D both in vitro and in vivo. This results in pronounced changes in the immunopeptidome induced by ERAP1 inhibition, but also restrains the growth of ERAP1 KO tumors in vivo by remodelling the tumor-infiltrating T-cell compartment. Interestingly, the absence of ERAP1 in combination with entinostat and PD-1 blockade overcomes resistance to PD-1 immunotherapy and increases host survival. Conclusions These findings demonstrate that ERAP1 inhibition combined with HDACi entinostat treatment and PD-1 blockade remodels the immune landscape of a non-immunogenic tumor such as NB, making it responsive to checkpoint immunotherapy.

Neuroblastoma (NB) is a childhood tumor that originates in the peripheral sympathetic nervous system and is responsible for 15% of cancer-related deaths in the pediatric population. Despite intensive multimodal treatment, many patients with high-risk NB relapse and develop a therapy-resistant tumor. One of the phenomena related to therapeutic resistance is intratumor heterogeneity resulting from the adaptation of tumor cells in response to different selective environmental pressures. The transcriptional and epigenetic profiling of NB tissue has recently revealed the existence of two distinct cellular identities in the NB, termed adrenergic (ADRN) and mesenchymal (MES), which can spontaneously interconvert through epigenetic regulation. This phenomenon, known as tumor plasticity, has a major impact on cancer pathogenesis. The aim of this review is to describe the peculiarities of these two cell states, and how their plasticity affects the response to current therapeutic treatments, with special focus on the immunogenic potential of MES cells. Furthermore, we will discuss the opportunity to combine immunotherapy with chemotherapy to counteract NB phenotypic interconversion.

Abstract Background Poor infiltration of functioning T cells renders tumors unresponsive to checkpoint-blocking immunotherapies. Here, we identified a combinatorial in situ immunomodulation strategy based on the administration of selected immunogenic drugs and immunotherapy to sensitize poorly T-cell-infiltrated neuroblastoma (NB) to the host antitumor immune response. Methods 975A2 and 9464D NB cell lines derived from spontaneous tumors of TH-MYCN transgenic mice were employed to study drug combinations able of enhancing the antitumor immune response using in vivo and ex vivo approaches. Migration of immune cells towards drug-treated murine-derived organotypic tumor spheroids (MDOTS) were assessed by microfluidic devices. Activation status of immune cells co-cultured with drug-treated MDOTS was evaluated by flow cytometry analysis. The effect of drug treatment on the immune content of subcutaneous or orthotopic tumors was comprehensively analyzed by flow-cytometry, immunohistochemistry and multiplex immunofluorescence. The chemokine array assay was used to detect soluble factors released into the tumor microenvironment. Patient-derived organotypic tumor spheroids (PDOTS) were generated from human NB specimens. Migration and activation status of autologous immune cells to drug-treated PDOTS were performed. Results We found that treatment with low-doses of mitoxantrone (MTX) recalled immune cells and promoted CD8+ T and NK cell activation in MDOTS when combined with TGFβ and PD-1 blockade. This combined immunotherapy strategy curbed NB growth resulting in the enrichment of a variety of both lymphoid and myeloid immune cells, especially intratumoral dendritic cells (DC) and IFNγ- and granzyme B-expressing CD8+ T cells and NK cells. A concomitant production of inflammatory chemokines involved in remodelling the tumor immune landscape was also detected. Interestingly, this treatment induced immune cell recruitment against PDOTS and activation of CD8+ T cells and NK cells. Conclusions Combined treatment with low-dose of MTX and anti-TGFβ treatment with PD-1 blockade improves antitumor immunity by remodelling the tumor immune landscape and overcoming the immunosuppressive microenvironment of aggressive NB.

Despite the significant clinical advances with the use of immune checkpoint inhibitors (ICIs) in a wide range of cancer patients, response rates to the therapy are variable and do not always result in long-term tumor regression. The development of ICI-resistant disease is one of the pressing issue in clinical oncology, and the identification of new targets and combination therapies is a crucial point to improve response rates and duration. Antigen processing and presentation (APP) pathway is a key element for an efficient response to ICI therapy. Indeed, malignancies that do not express tumor antigens are typically poor infiltrated by T cells and unresponsive to ICIs. Therefore, improving tumor immunogenicity potentially increases the success rate of ICI therapy. In this review, we provide an overview of the key elements of the APP machinery that can be exploited to enhance tumor immunogenicity and increase the efficacy of ICI-based immunotherapy.

The endoplasmic reticulum aminopeptidase ERAP1 regulates innate and adaptive immune responses by trimming peptides for presentation by major histocompatibility complex (MHC) class I molecules. Previously, we have shown that genetic or pharmacological inhibition of ERAP1 on murine and human tumor cell lines perturbs the engagement of NK cell inhibitory receptors Ly49C/I and Killer-cell Immunoglobulin-like receptors (KIRs), respectively, by their specific ligands (MHC class I molecules), thus leading to NK cell killing. However, the effect of ERAP1 inhibition in tumor cells was highly variable, suggesting that its efficacy may depend on several factors, including MHC class I typing. To identify MHC class I alleles and KIRs that are more sensitive to ERAP1 depletion, we stably silenced ERAP1 expression in human HLA class I-negative B lymphoblastoid cell line 721.221 (referred to as 221) transfected with a panel of KIR ligands (i.e. HLA-B*51:01, -Cw3, -Cw4 and -Cw7), or HLA-A2 which does not bind any KIR, and tested their ability to induce NK cell degranulation and cytotoxicity. No change in HLA class I surface expression was detected in all 221 transfectant cells after ERAP1 depletion. In contrast, CD107a expression levels were significantly increased on NK cells stimulated with 221-B*51:01 cells lacking ERAP1, particularly in the KIR3DL1-positive NK cell subset. Consistently, genetic or pharmacological inhibition of ERAP1 impaired the recognition of HLA-B*51:01 by the YTS NK cell overexpressing KIR3DL1*001, suggesting that ERAP1 inhibition renders HLA-B*51:01 molecules less eligible for binding to KIR3DL1. Overall, these results identify HLA-B*51:01/KIR3DL1 as one of the most susceptible combinations for ERAP1 inhibition, suggesting that individuals carrying HLA-B*51:01-like antigens may be candidates for immunotherapy based on pharmacological inhibition of ERAP1.

Patients with coronavirus disease 2019 (COVID-19) have a wide variety of clinical outcomes ranging from asymptomatic to severe respiratory syndrome that can progress to life-threatening lung lesions. The identification of prognostic factors can help to improve the risk stratification of patients by promptly defining for each the most effective therapy to resolve the disease. The etiological agent causing COVID-19 is a new coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that enters cells via the ACE2 receptor. SARS-CoV-2 infection causes a reduction in ACE2 levels, leading to an imbalance in the renin-angiotensin system (RAS), and consequently, in blood pressure and systemic vascular resistance. ERAP1 and ERAP2 are two RAS regulators and key components of MHC class I antigen processing. Their polymorphisms have been associated with autoimmune and inflammatory conditions, hypertension, and cancer. Based on their involvement in the RAS, we believe that the dysfunctional status of ERAP1 and ERAP2 enzymes may exacerbate the effect of SARS-CoV-2 infection, aggravating the symptomatology and clinical outcome of the disease. In this review, we discuss this hypothesis.

Human cytomegalovirus (HCMV) is a β-herpesvirus that causes serious problems in people with a compromised immune system, whereas it coexists asymptomatically within the host with a healthy immune system. Like other viruses, HCMV has adopted multiples strategies to manipulate the host’s immune responses. Among them, expression of viral microRNAs (miRNAs) is one of the most intriguing. HCMV miR-UL112-5p and miR-US4-1 have been found to contribute to immune evasion by targeting the endoplasmic reticulum aminopeptidase 1 (ERAP1), a highly polymorphic key component of antigen processing. The current incomplete picture on the interplay between viral miRNAs and host immunity implies the need to better characterize the host genetic determinants. Naturally occurring single nucleotide polymorphisms (SNPs) within the miRNA binding sites of target genes may affect miRNA–target interactions. In this review, we focus on the relevance of 3′ untranslated region (3′UTR) ERAP1 SNPs within miRNA binding sites in modulating miRNA–mRNA interactions and the possible consequent individual susceptibility to HCMV infection. Moreover, we performed an in silico analysis using different bioinformatic algorithms to predict ERAP1 variants with a putative powerful biological function. This evidence provides a basis to deepen the knowledge on how 3′UTR ERAP1 variants may alter the mechanism of action of HCMV miRNAs, in order to develop targeted antiviral therapies.