IRF2 loss is associated with reduced MHC I pathway transcripts in subsets of most human cancers and causes resistance to checkpoint immunotherapy in human and mouse melanomas.

BACKGROUND: In order for cancers to progress, they must evade elimination by CD8 T cells or other immune mechanisms. CD8 T cells recognize and kill tumor cells that display immunogenic tumor peptides bound to MHC I molecules. One of the ways that cancers can escape such killing is by reducing expression of MHC I molecules, and loss of MHC I is frequently observed in tumors. There are multiple different mechanisms that can underly the loss of MHC I complexes on tumor and it is currently unclear whether there are particular mechanisms that occur frequently and, if so, in what types of cancers. Also of importance to know is whether the loss of MHC I is reversible and how such loss and/or its restoration would impact responses to immunotherapy. Here, we investigate these issues for loss of IRF1 and IRF2, which are transcription factors that drive expression of MHC I pathway genes and some killing mechanisms. METHODS: Bioinformatics analyses of IRF2 and IRF2-dependent gene transcripts were performed for all human cancers in the TCGA RNAseq database. IRF2 protein-DNA-binding was analyzed in ChIPseq databases. CRISRPcas9 was used to knock out IRF1 and IRF2 genes in human and mouse melanoma cells and the resulting phenotypes were analyzed in vitro and in vivo. RESULTS: Transcriptomic analysis revealed that IRF2 expression was reduced in a substantial subset of cases in almost all types of human cancers. When this occurred there was a corresponding reduction in the expression of IRF2-regulated genes that were needed for CD8 T cell recognition. To test cause and effect for these IRF2 correlations and the consequences of IRF2 loss, we gene-edited IRF2 in a patient-derived melanoma and a mouse melanoma. The IRF2 gene-edited melanomas had reduced expression of transcripts for genes in the MHC I pathway and decreased levels of MHC I complexes on the cell surface. Levels of Caspase 7, an IRF2 target gene involved in CD8 T cell killing of tumors, were also reduced. This loss of IRF2 caused both human and mouse melanomas to become resistant to immunotherapy with a checkpoint inhibitor. Importantly, these effects were reversible. Stimulation of the IRF2-deficient melanomas with interferon induced the expression of a functionally homologous transcription factor, IRF1, which then restored the MHC I pathway and responsiveness to CPI. CONCLUSIONS: Our study shows that a subset of cases within most types of cancers downregulates IRF2 and that this can allow cancers to escape immune control. This can cause resistance to checkpoint blockade immunotherapy and is reversible with currently available biologics.

IRF2 loss is associated with reduced MHC I pathway transcripts in subsets of most human cancers and causes resistance to checkpoint immunotherapy in human and mouse melanomas.

Background: In order for cancers to progress, they must evade elimination by CD8 T cells or other immune mechanisms. CD8 T cells recognize and kill tumor cells that display immunogenic tumor peptides bound to MHC I molecules. One of the ways that cancers can escape such killing is by reducing expression of MHC I molecules, and loss of MHC I is frequently observed in tumors. There are multiple different mechanisms that can underly the loss of MHC I complexes on tumor and it is currently unclear whether there are particular mechanisms that occur frequently and, if so, in what types of cancers. Also of importance to know is whether the loss of MHC I is reversible and how such loss and/or its restoration would impact responses to immunotherapy. Here, we investigate these issues for loss of IRF1 and IRF2, which are transcription factors that drive expression of MHC I pathway genes and some killing mechanisms. Methods: Bioinformatics analyses of IRF2 and IRF2-dependent gene transcripts were performed for all human cancers in the TCGA RNAseq database. IRF2 protein-DNA-binding was analyzed in ChIPseq databases. CRISRPcas9 was used to knock out IRF1 and IRF2 genes in human and mouse melanoma cells and the resulting phenotypes were analyzed in vitro and in vivo. Results: Transcriptomic analysis revealed that IRF2 expression was reduced in a substantial subset of cases in almost all types of human cancers. When this occurred there was a corresponding reduction in the expression of IRF2-regulated genes that were needed for CD8 T cell recognition. To test cause and effect for these IRF2 correlations and the consequences of IRF2 loss, we gene-edited IRF2 in a patient-derived melanoma and a mouse melanoma. The IRF2 gene-edited melanomas had reduced expression of transcripts for genes in the MHC I pathway and decreased levels of MHC I complexes on the cell surface. Levels of Caspase 7, an IRF2 target gene involved in CD8 T cell killing of tumors, were also reduced. This loss of IRF2 caused both human and mouse melanomas to become resistant to immunotherapy with a checkpoint inhibitor. Importantly, these effects were reversible. Stimulation of the IRF2-deficient melanomas with interferon induced the expression of a functionally homologous transcription factor, IRF1, which then restored the MHC I pathway and responsiveness to CPI. Conclusions: Our study shows that a subset of cases within most types of cancers downregulates IRF2 and that this can allow cancers to escape immune control. This can cause resistance to checkpoint blockade immunotherapy and is reversible with currently available biologics.

Inhibition of Autophagy by Berbamine Hydrochloride Mitigates Tumor Immune Escape by Elevating MHC-I in Melanoma Cells.

Impaired tumor cell antigen presentation contributes significantly to immune evasion. This study identifies Berbamine hydrochloride (Ber), a compound derived from traditional Chinese medicine, as an effective inhibitor of autophagy that enhances antigen presentation in tumor cells. Ber increases MHC-I-mediated antigen presentation in melanoma cells, improving recognition and elimination by CD8+ T cells. Mutation of Atg4b, which blocks autophagy, also raises MHC-I levels on the cell surface, and further treatment with Ber under these conditions does not increase MHC-I, indicating Ber's role in blocking autophagy to enhance MHC-I expression. Additionally, Ber treatment leads to the accumulation of autophagosomes, with elevated levels of LC3-II and p62, suggesting a disrupted autophagic flux. Fluorescence staining and co-localization analyses reveal that Ber likely inhibits lysosomal acidification without hindering autophagosome-lysosome fusion. Importantly, Ber treatment suppresses melanoma growth in mice and enhances CD8+ T cell infiltration, supporting its therapeutic potential. Our findings demonstrate that Ber disturbs late-stage autophagic flux through abnormal lysosomal acidification, enhancing MHC-I-mediated antigen presentation and curtailing tumor immune escape.

Low-dose SAHA enhances CD8+ T cell-mediated antitumor immunity by boosting MHC I expression in non-small cell lung cancer.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a highly aggressive type of lung cancer with poor responses to traditional therapies such as surgery, radiotherapy, and chemotherapy. While immunotherapy has become an effective approach for treating multiple types of cancer, solid tumors frequently exhibit immune escape through various mechanisms, including downregulation of MHC I expression. However, whether the upregulation of MHC I expression can improve the immunotherapeutic effect on NSCLC remains unexplored. Suberoylanilide hydroxamic acid (SAHA) is a potent histone deacetylase (HDAC) inhibitor that has been applied clinically to treat lymphoma, but a high dose of SAHA kills tumor cells and normal cells without preference. Here, we report that low-dose SAHA enhances CD8+ T cell-mediated antitumor immunity by upregulating MHC I expression in NSCLC cells. METHODS: Flow cytometric analysis, quantitative real-time PCR and western blot were used to analyze the expression of MHC I, STAT1 and Smad2/3 in both human and mouse NSCLC cell lines after SAHA treatment. The nuclear translocation of phosphorylated STAT1 and Smad2/3 was investigated by western blot and immunofluorescence staining. The mechanisms underlying STAT1 and Smad2/3 upregulation were analyzed through database searches and chromatin immunoprecipitation-qPCR. Finally, we assessed the antitumor effect of specific CD8+ T cells with SAHA treatment in vivo and in vitro. RESULTS: We showed that low-dose SAHA upregulated the expression of MHC I in NSCLC cell lines without affecting cell viability. We also provided evidence that high levels of MHC I induced by SAHA promoted the activation, proliferation, and cytotoxicity of specific CD8+ T cells in mouse models. Mechanistically, low-dose SAHA increased the levels of H3K9ac and H3K27ac in the promoters of the STAT1, Smad2 and Smad3 genes in NSCLC cells by inhibiting HDAC activity, resulting in elevated expression levels of STAT1, Smad2 and Smad3. The nuclear translocation of phosphorylated STAT1 and Smad2/3 markedly upregulated the expression of MHC I in NSCLC cells. CONCLUSIONS: Low-dose SAHA enhances CD8+ T cell-mediated antitumor immunity by boosting MHC I expression in NSCLC cells. Thus, we revealed a key mechanism of SAHA-mediated enhanced antitumor immunity, providing insights into a novel immunotherapy strategy for NSCLC.

The Proteolytic Activity of Neutrophil-Derived Serine Proteases Bound to the Cell Surface Arming Lung Epithelial Cells for Viral Defense.

The collaboration between cellular proteases and host cells is pivotal in mounting an effective innate immune defense. Of particular interest is the synergistic interaction between cathepsin G (CatG) and neutrophil elastase (NE), which are proteases secreted by activated neutrophils, and the human alveolar basal epithelial cell line (A549) and the human lung epithelial-like cell line (H1299), because of the potential implications for viral infection. Our study aimed to investigate the binding capacity of CatG and NE on the surface of A549 and H1299 cells through preincubation with purified CatG and NE; thereby, the proteolytic activity could be detected using activity-based probes. Both CatG and NE were capable of binding to the cell surface and exhibited proteolytic activity, leading to increased cell surface levels of MHC I molecules, which is crucial for displaying the endogenous antigenic repertoire. In addition, CatG cleaved the S2' site of the SARS-CoV-2 spike protein at two specific sites (815RS816 and 817FI818) as well as NE (813SK814 and 818IE819), which potentially leads to the destruction of the fusion peptide. Additionally, furin required the presence of Ca2+ ions for the distinct cleavage site necessary to generate the fusion peptide. Overall, the findings suggest that CatG and NE can fortify target cells against viral entry, underscoring the potential significance of cell surface proteases in protecting against viral invasion.

Targeted inhibition of glycogen synthase kinase-3 using 9-ING-41 (elraglusib) enhances CD8 T-cell-reactivity against neuroblastoma cells.

The prognosis of patients with high-risk neuroblastoma remains poor, partly due to inadequate immune recognition of the tumor. Neuroblastomas display extremely low surface MHC-I, preventing recognition by cytotoxic T lymphocytes (CTLs) and contributing to an immunosuppressive tumor microenvironment. Glycogen synthase kinase-3 beta (GSK-3ß) is involved in pathways that may affect the MHC-I antigen processing and presentation pathway. We proposed that therapeutic inhibition of GSK-3ß might improve the surface display of MHC-I molecules on neuroblastoma cells, and therefore tested if targeting of GSK-3ß using the inhibitor 9-ING-41 (Elraglusib) improves MHC-I-mediated CTL recognition. We analyzed mRNA expression data of neuroblastoma tumor datasets and found that non-MYCN-amplified neuroblastomas express higher GSK-3ß levels than MYCN-amplified tumors. In non-MYCN-amplified cells SH-SY5Y, SK-N-AS and SK-N-SH 9-ING-41 treatment enhanced MHC-I surface display and the expression levels of a subset of genes involved in MHC-I antigen processing and presentation. Further, 9-ING-41 treatment triggered increased STAT1 pathway activation, upstream of antigen presentation pathways in two of the three non-MYCN-amplified cell lines. Finally, in co-culture experiments with CD8 + T cells, 9-ING-41 improved immune recognition of the neuroblastoma cells, as evidenced by augmented T-cell activation marker levels and T-cell proliferation, which was further enhanced by PD-1 immune checkpoint inhibition. Our preclinical study provides experimental support to further explore the GSK-3ß inhibitor 9-ING-41 as an immunomodulatory agent to increase tumor immune recognition in neuroblastoma.

Sex and disease regulate major histocompatibility complex class I expression in human lung epithelial cells.

Major histocompatibility complex class I (MHC I) molecules present peptides to CD8+ T-cells for immunosurveillance of infection and cancer. Recent studies indicate lineage-specific heterogeneity in MHC I expression. While respiratory diseases rank among the leading causes of mortality, studies in mice have shown that lung epithelial cells (LECs) express the lowest levels of MHC I in the lung. This study aims to answer three questions: (i) Do human LECs express low levels of MHC I? (ii) Is LEC MHC I expression modulated in chronic respiratory diseases? (iii) Which factors regulate MHC I levels in human LECs? We analyzed human LECs from parenchymal explants using single-cell RNA sequencing and immunostaining. We confirmed low constitutive MHC I expression in human LECs, with significant upregulation in chronic respiratory diseases. We observed a sexual dimorphism, with males having higher MHC I levels under steady-state conditions, likely due to differential redox balance. Our study unveils the complex interplay between MHC I expression, sex, and respiratory disease. Since MHC I upregulation contributes to the development of immunopathologies in other models, we propose that it may have a similar impact on chronic lung disease.

Nilotinib boosts the efficacy of anti-PDL1 therapy in colorectal cancer by restoring the expression of MHC-I.

BACKGROUND: Although immune checkpoint inhibitors (ICIs) have revolutionized the landscape of cancer treatment, only a minority of colorectal cancer (CRC) patients respond to them. Enhancing tumor immunogenicity by increasing major histocompatibility complex I (MHC-I) surface expression is a promising strategy to boost the antitumor efficacy of ICIs. METHODS: Dual luciferase reporter assays were performed to find drug candidates that can increase MHC-I expression. The effect of nilotinib on MHC-I expression was verified by dual luciferase reporter assays, qRT-PCR, flow cytometry and western blotting. The biological functions of nilotinib were evaluated through a series of in vitro and in vivo experiments. Using RNA-seq analysis, immunofluorescence assays, western blotting, flow cytometry, rescue experiments and microarray chip assays, the underlying molecular mechanisms were investigated. RESULTS: Nilotinib induces MHC-I expression in CRC cells, enhances CD8+ T-cell cytotoxicity and subsequently enhances the antitumor effects of anti-PDL1 in both microsatellite instability and microsatellite stable models. Mechanistically, nilotinib promotes MHC-I mRNA expression via the cGAS-STING-NF-κB pathway and reduces MHC-I degradation by suppressing PCSK9 expression in CRC cells. PCSK9 may serve as a potential therapeutic target for CRC, with nilotinib potentially targeting PCSK9 to exert anti-CRC effects. CONCLUSION: This study reveals a previously unknown role of nilotinib in antitumor immunity by inducing MHC-I expression in CRC cells. Our findings suggest that combining nilotinib with anti-PDL1 therapy may be an effective strategy for the treatment of CRC.

The Wdr5-H3K4me3 Epigenetic Axis Regulates Pancreatic Tumor Immunogenicity and Immune Suppression.

The WDR5/MLL1-H3K4me3 epigenetic axis is often activated in both tumor cells and tumor-infiltrating immune cells to drive various cellular responses in the tumor microenvironment and has been extensively studied in hematopoietic cancer, but its respective functions in tumor cells and immune cells in the context of tumor growth regulation of solid tumor is still incompletely understood. We report here that WDR5 exhibits a higher expression level in human pancreatic tumor tissues compared with adjacent normal pancreas. Moreover, WDR5 expression is negatively correlated with patients' response to chemotherapy or immunotherapy in human colon cancer and melanoma. However, WDR5 expression is positively correlated with the HLA level in human cancer cells, and H3K4me3 enrichment is observed at the promoter region of the HLA-A, HLA-B, and HLA-C genes in pancreatic cancer cells. Using mouse tumor cell lines and in vivo tumor models, we determined that WDR5 deficiency or inhibition significantly represses MHC I expression in vitro and in vivo in pancreatic tumor cells. Mechanistically, we determine that WDR5 deficiency inhibits H3K4me3 deposition at the MHC I (H2K) promoter region to repress MHC I (H2K) transcription. On the other hand, WDR5 depletion leads to the effective downregulation of immune checkpoints and immunosuppressive cytokines, including TGFß and IL6, in the pancreatic tumor microenvironments. Our data determine that WDR5 not only regulates tumor cell immunogenicity to suppress tumor growth but also activates immune suppressive pathways to promote tumor immune evasion. Selective activation of the WDR5-MHC I pathway and/or selective inhibition of the WDR5-immune checkpoint and WDR5-cytokine pathways should be considered in WDR5-based epigenetic cancer immunotherapy.

Clinico-sero-pathological profiles and risk prediction model of idiopathic inflammatory myopathy (IIM) patients with different perifascicular changes.

AIMS: To explore the clinico-sero-pathological characteristics and risk prediction model of idiopathic inflammatory myopathy (IIM) patients with different muscular perifascicular (PF) changes. METHODS: IIM patients in our center were enrolled and the clinico-sero-pathological data were retrospectively analyzed. A decision tree model was established through machine learning. RESULTS: There were 231 IIM patients enrolled, including 53 with perifascicular atrophy (PFA), 39 with perifascicular necrosis (PFN), and 26 with isolated perifascicular enhancement of MHC-I/MHC-II (PF-MHCn). Clinically, PFA patients exhibited skin rashes and dermatomyositis-specific antibodies (DM-MSAs, 74.5%) except for anti-Mi2. PFN patients showed the most severe muscle weakness, highest creatine kinase (CK), anti-Mi2 (56.8%), and anti-Jo-1 (24.3%) antibodies. PF-MHCn patients demonstrated negative MSAs (48.0%) and elevated CK. Histopathologically, MAC predominantly deposited on PF capillaries in PFA but on non-necrotic myofiber in PFN (43.4% and 36.8%, p < 0.001). MxA expression was least in PF-MHCn (36.0% vs. 83.0% vs. 63.2%, p < 0.001). The decision tree model could effectively predict different subgroups, especially PFA and PFN. CONCLUSIONS: Three types of PF change of IIMs representing distinct clinico-serological characteristics and pathomechanism. Undiscovered MSAs should be explored especially in PF-MHCn patients. The three pathological features could be accurately predicted through the decision tree model.

Monocyte/Macrophage-Mediated Transport of Dual-Drug ZIF Nanoplatforms Synergized with Programmed Cell Death Protein-1 Inhibitor Against Microsatellite-Stable Colorectal Cancer.

Microsatellite-stable colorectal cancer (MSS-CRC) exhibits resistance to programmed cell death protein-1 (PD-1) therapy. Improving the infiltration and tumor recognition of cytotoxic T-lymphocytes (CTLs) is a promising strategy, but it encounters huge challenges from drug delivery and mechanisms aspects. Here, a zeolitic imidazolate framework (ZIF) coated with apoptotic body membranes derived from MSS-CRC cells is engineered for the co-delivery of ginsenoside Rg1 (Rg1) and atractylenolide-I (Att) to MSS-CRC, named as Ab@Rg1/Att-ZIF. This system is selectively engulfed by Ly-6C+ monocytes during blood circulation and utilizes a "hitchhiking" mechanism to migrate toward the core of MSS-CRC. Ab@Rg1/Att-ZIF undergoes rapid disassembly in the tumor, released Rg1 promotes the processing and transportation of tumor antigens in dendritic cells (DCs), enhancing their maturation. Meanwhile, Att enhances the activity of the 26S proteasome complex in tumor cells, leading to increased expression of major histocompatibility complex class-I (MHC-I). These coordinated actions enhance the infiltration and recognition of CTLs in the center of MSS-CRC, significantly improving the tumor inhibition of PD-1 treatment from ≈5% to ≈69%. This innovative design, involving inflammation-guided precise drug co-delivery and a rational combination, achieves synergistic engineering of the tumor microenvironment, providing a novel strategy for successful PD-1 treatment of MSS-CRC.

Tumor antigen presentation and the associated signal transduction during carcinogenesis.

Numerous developments have been achieved in the study and treatment of cancer throughout the decades that it has been common. After decades of research, about 100 different kinds of cancer have been found, each with unique subgroups within certain organs. This has significantly expanded our understanding of the illness. A mix of genetic, environmental, and behavioral variables contribute to the complicated and diverse process of cancer formation. Mutations, or changes in the DNA sequence, are crucial to the development of cancer. These mutations have the ability to downregulate the expression and function of Major Histocompatibility Complex class I (MHC I) and MHCII receptors, as well as activate oncogenes and inactivate tumor suppressor genes. Cancer cells use this tactic to avoid being recognized by cytotoxic CD8+T lymphocytes, which causes issues with antigen presentation and processing. This review goes into great length into the PI3K pathway, changes to MHC I, and positive impacts of tsMHC-II on disease-free survival and overall survival and the involvement of dendritic cells (DCs) in different tumor microenvironments. The vital functions that the PI3K pathway and its link to the mTOR pathway are highlighted and difficulties in developing effective cancer targeted therapies and feedback systems has also been mentioned, where resistance mechanisms include RAS-mediated oncogenic changes and active PI3K signalling.

Extracellular vesicle-packaged lncRNA from cancer-associated fibroblasts promotes immune evasion by downregulating HLA-A in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterised by immune evasion that contribute to poor prognosis. Cancer-associated fibroblasts (CAFs) play a pivotal role in orchestrating the PDAC tumour microenvironment. We investigated the role of CAF-derived extracellular vesicle (EV)-packaged long non-coding RNAs (lncRNAs) in immune evasion and explored gene therapy using engineered EVs loading small interfering RNAs (siRNAs) as a potential therapeutic strategy. Our findings highlight the significance of EV-packaged lncRNA RP11-161H23.5 from CAF in promoting PDAC immune evasion by downregulating HLA-A expression, a key component of antigen presentation. Mechanistically, RP11-161H23.5 forms a complex with CNOT4, a subunit of the mRNA deadenylase CCR4-NOT complex, enhancing the degradation of HLA-A mRNA by shortening its poly(A) tail. This immune evasion mechanism compromises the anti-tumour immune response. To combat this, we propose an innovative approach utilising engineered EVs as natural and biocompatible nanocarriers for siRNA-based gene therapy and this strategy holds promise for enhancing the effectiveness of immunotherapy in PDAC. Overall, our study sheds light on the critical role of CAF-derived EV-packaged lncRNA RP11-161H23.5/CNOT4/HLA-A axis in PDAC immune evasion and presents a novel avenue for therapeutic intervention.

A fluorescence polarization assay for high-throughput screening of inhibitors against HIV-1 Nef-mediated MHC-I downregulation.

The multifunctional, HIV-1 accessory protein Nef enables infected cells to evade host immunity and thus plays a key role in viral pathogenesis. One prominent function of Nef is the downregulation of major histocompatibility complex class I (MHC-I), which disrupts antigen presentation and thereby allows the infected cells to evade immune surveillance by the cytotoxic T cells. Therapeutic inhibition of this Nef function is a promising direction of antiretroviral drug discovery as it may revitalize cytotoxic T cells to identify, and potentially clear, hidden HIV-1 infections. Guided by the crystal structure of the protein complex formed between Nef, MHC-I, and the hijacked clathrin adaptor protein complex 1, we have developed a fluorescence polarization-based assay for inhibitor screening against Nef's activity on MHC-I. The optimized assay has a good signal-to-noise ratio, substantial tolerance of dimethylsulfoxide, and excellent ability to detect competitive inhibition, indicating that it is suitable for high-throughput screening.

Immunoinformatics and Reverse Vaccinology Approach for the Identification of Potential Vaccine Candidates against Vandammella animalimors.

Vandammella animalimorsus is a Gram-negative and non-motile bacterium typically transmitted to humans through direct contact with the saliva of infected animals, primarily through biting, scratches, or licks on fractured skin. The absence of a confirmed post-exposure treatment of V. animalimorsus bacterium highlights the imperative for developing an effective vaccine. We intended to determine potential vaccine candidates and paradigm a chimeric vaccine against V. animalimorsus by accessible public data analysis of the strain by utilizing reverse vaccinology. By subtractive genomics, five outer membranes were prioritized as potential vaccine candidates out of 2590 proteins. Based on the instability index and transmembrane helices, a multidrug transporter protein with locus ID A0A2A2AHJ4 was designated as a potential candidate for vaccine construct. Sixteen immunodominant epitopes were retrieved by utilizing the Immune Epitope Database. The epitope encodes the strong binding affinity, nonallergenic properties, non-toxicity, high antigenicity scores, and high solubility revealing the more appropriate vaccine construct. By utilizing appropriate linkers and adjuvants alongside a suitable adjuvant molecule, the epitopes were integrated into a chimeric vaccine to enhance immunogenicity, successfully eliciting both adaptive and innate immune responses. Moreover, the promising physicochemical features, the binding confirmation of the vaccine to the major innate immune receptor TLR-4, and molecular dynamics simulations of the designed vaccine have revealed the promising potential of the selected candidate. The integration of computational methods and omics data has demonstrated significant advantages in discovering novel vaccine targets and mitigating vaccine failure rates during clinical trials in recent years.

DHODH inhibition enhances the efficacy of immune checkpoint blockade by increasing cancer cell antigen presentation.

Pyrimidine nucleotide biosynthesis is a druggable metabolic dependency of cancer cells, and chemotherapy agents targeting pyrimidine metabolism are the backbone of treatment for many cancers. Dihydroorotate dehydrogenase (DHODH) is an essential enzyme in the de novo pyrimidine biosynthesis pathway that can be targeted by clinically approved inhibitors. However, despite robust preclinical anticancer efficacy, DHODH inhibitors have shown limited single-agent activity in phase 1 and 2 clinical trials. Therefore, novel combination therapy strategies are necessary to realize the potential of these drugs. To search for therapeutic vulnerabilities induced by DHODH inhibition, we examined gene expression changes in cancer cells treated with the potent and selective DHODH inhibitor brequinar (BQ). This revealed that BQ treatment causes upregulation of antigen presentation pathway genes and cell surface MHC class I expression. Mechanistic studies showed that this effect is (1) strictly dependent on pyrimidine nucleotide depletion, (2) independent of canonical antigen presentation pathway transcriptional regulators, and (3) mediated by RNA polymerase II elongation control by positive transcription elongation factor B (P-TEFb). Furthermore, BQ showed impressive single-agent efficacy in the immunocompetent B16F10 melanoma model, and combination treatment with BQ and dual immune checkpoint blockade (anti-CTLA-4 plus anti-PD-1) significantly prolonged mouse survival compared to either therapy alone. Our results have important implications for the clinical development of DHODH inhibitors and provide a rationale for combination therapy with BQ and immune checkpoint blockade.

Enhancing immunotherapy efficacy against MHC-I deficient triple-negative breast cancer using LCL161-loaded macrophage membrane-decorated nanoparticles.

Current cytotoxic T lymphocyte (CTL) activating immunotherapy requires a major histocompatibility complex I (MHC-I)-mediated presentation of tumor-associated antigens, which malfunctions in around half of patients with triple-negative breast cancer (TNBC). Here, we create a LCL161-loaded macrophage membrane decorated nanoparticle (LMN) for immunotherapy of MHC-I-deficient TNBC. SIRPα on the macrophage membrane helps LMNs recognize CD47-expressing cancer cells for targeted delivery of LCL161, which induces the release of high mobility group protein 1 and proinflammatory cytokines from cancer cells. The released cytokines and high mobility group protein 1 activate antitumor immunity by increasing the intratumoral density of the phagocytic macrophage subtype by 15 times and elevating the intratumoral concentration of CTL lymphotoxin by 4.6 folds. LMNs also block CD47-mediated phagocytosis suppression. LMNs inhibit the growth of MHC-I-deficient TNBC tumors, as well as those resistant to combined therapy of anti-PDL1 antibody and albumin-bound paclitaxel, and prolong the survival of animals, during which process CTLs also play important roles. This macrophage membrane-decorated nanoparticle presents a generalizable platform for increasing macrophage-mediated antitumor immunity for effective immunotherapy of MHC-I-deficient cancers.

A monoclonal antibody that recognizes a unique 13-residue epitope in the cytoplasmic tail of HLA-E.

The Class I MHC molecule (MHC-I) HLA-E presents peptides that are derived from the signal sequences, either those of other MHC-I products, or of viral type I membrane glycoproteins. Monoclonal antibodies with proven specificity for HLA-E, and with no cross-reactions with other MHC-I products, have yet to be described. To obtain anti-HLA-E-specific antibodies suitable for a range of applications, we generated monoclonal antibodies against a unique feature of HLA-E: its cytoplasmic tail. We created an immunogen by performing an enzymatically catalyzed transpeptidation reaction to obtain a fusion of the cytoplasmic tail of HLA-E with a nanobody that recognizes murine Class II MHC (MHC-II) products. We obtained a mouse monoclonal antibody that recognizes a 13-residue stretch in the HLA-E cytoplasmic tail. We cloned the genes that encode this antibody in expression vectors to place an LPETG sortase recognition motif at the C-terminus of the heavy and light chains. This arrangement allows the site-specific installation of fluorophores or biotin at these C-termini. The resulting immunoglobulin preparations, labeled with 4 equivalents of a fluorescent or biotinylated payload of choice, can then be used for direct immunofluorescence or detection of the tag by fluorescence or by streptavidin-based methods. We also show that the 13-residue sequence can serve as an epitope tag, independent of the site of its placement within a protein's sequence. The antibody can be used diagnostically to stain for HLA-E on patient tumor samples, it can be used as an antibody-epitope tag for extracellular proteins, and it enables research into the unique role of the cytoplasmic tail of HLA-E.

PACSIN1 promotes immunosuppression in gastric cancer by degrading MHC-I.

Gastric cancer (GC) is a common gastrointestinal system malignancy. PACSIN1 functions as an oncogene in various cancers. This study aims to investigate the potential of PACSIN1 as a target in GC treatment. Gene expression is determined by RT-qPCR, immunofluorescence staining, and immunohistochemistry assay. FISH is performed to determine the colocalization of PACSIN1 and the major histocompatibility complex (MHC-I). Cytokine release and cell functions are analyzed by flow cytometry. In vivo assays are also conducted. Histological analysis is performed using H&E staining. The results show that PACSIN1 is overexpressed in GC patients, especially in those with immunologically-cold tumors. A high level of PACSIN1 is associated with poor prognosis. PACSIN1 deficiency inhibits autophagy but increases antigen presentation in GC cells. Moreover, PACSIN1 deficiency inhibits the lysosomal fusion and selective autophagy of MHC-I, increases CD8 + T-cell infiltration, and suppresses tumor growth and liver metastasis in vivo. Additionally, PACSIN1 knockout enhances the chemosensitivity of cells to immune checkpoint blockade. In summary, PACSIN1 mediates lysosomal fusion and selective autophagy of MHC-I and suppresses antigen presentation and CD8 + T-cell infiltration, thus inhibiting antitumor immunity in GC.

Insights into PCSK9-LDLR Regulation and Trafficking via the Differential Functions of MHC-I Proteins HFE and HLA-C.

PCSK9 is implicated in familial hypercholesterolemia via targeting the cell surface PCSK9-LDLR complex toward lysosomal degradation. The M2 repeat in the PCSK9's C-terminal domain is essential for its extracellular function, potentially through its interaction with an unidentified "protein X". The M2 repeat was recently shown to bind an R-x-E motif in MHC-class-I proteins (implicated in the immune system), like HLA-C, and causing their lysosomal degradation. These findings suggested a new role of PCSK9 in the immune system and that HLA-like proteins could be "protein X" candidates. However, the participation of each member of the MHC-I protein family in this process and their regulation of PCSK9's function have yet to be determined. Herein, we compared the implication of MHC-I-like proteins such as HFE (involved in iron homeostasis) and HLA-C on the extracellular function of PCSK9. Our data revealed that the M2 domain regulates the intracellular sorting of the PCSK9-LDLR complex to lysosomes, and that HFE is a new target of PCSK9 that inhibits its activity on the LDLR, whereas HLA-C enhances its function. This work suggests the potential modulation of PCSK9's functions through interactions of HFE and HLA-C.