Programmed Death Ligand 1 Expression In Cancer Research Articles

USP2 promotes tumor immune evasion via deubiquitination and stabilization of PD-L1.

The abnormal upregulation of programmed death ligand-1 (PD-L1) on tumor cells impedes T-cell mediated cytotoxicity through PD-1 engagement, and further exploring the mechanisms regulation of PD-L1 in cancers may enhance the clinical efficacy of PD-L1 blockade. Here, using single-guide RNAs (sgRNAs) screening system, we identify ubiquitin-specific processing protease 2 (USP2) as a novel regulator of PD-L1 stabilization for tumor immune evasion. USP2 directly interacts with and increases PD-L1 abundance in colorectal and prostate cancer cells. Our results show that Thr288, Arg292 and Asp293 at USP2 control its binding to PD-L1 through deconjugating the K48-linked polyubiquitination at lysine 270 of PD-L1. Depletion of USP2 causes endoplasmic reticulum (ER)-associated degradation of PD-L1, thus attenuates PD-L1/PD-1 interaction and sensitizes cancer cells to T cell-mediated killing. Meanwhile, USP2 ablation-induced PD-L1 clearance enhances antitumor immunity in mice via increasing CD8+ T cells infiltration and reducing immunosuppressive infiltration of myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), whereas PD-L1 overexpression reverses the tumor growth suppression by USP2 silencing. USP2-depletion combination with anti-PD-1 also exhibits a synergistic anti-tumor effect. Furthermore, analysis of clinical tissue samples indicates that USP2 is positively associated with PD-L1 expression in cancer. Collectively, our data reveal a crucial roleof USP2 for controlling PD-L1 stabilization in tumor cells, and highlight USP2 as a potential therapeutic target for cancer immunotherapy.

99mTc]-labelled anti-Programmed Death-Ligand 1 single-domain antibody SPECT/CT: a novel imaging biomarker for myocardial PD-L1 expression

BackgroundMyocardial programmed death-ligand 1 (PD-L1) expression is implicated in immune checkpoint inhibitor (ICI)-associated myocarditis. Measurement of myocardial PD-L1 expression may have potential use as a mechanistic and predictive biomarker. The aim of this study was to determine non-invasive assessment of myocardial PD-L1 expression using [99mTc]-labelled anti-PD-L1 single-domain antibody (NM-01) SPECT/CT.MethodsThoracic [99mTc]NM-01 SPECT/CT was performed in lung cancer patients (n = 10) at baseline and 9-weeks following anti-programmed cell death protein 1 (PD-1) therapy. Baseline and 9-week left ventricular and right ventricular to blood pool ratios (LVmax:BP) and (RVmax:BP) were measured. LVmax was compared to background skeletal muscle (musclemax). Intra-rater reliability was determined by intraclass correlation coefficient (ICC) and Bland–Altman analysis.ResultsMean LVmax:BP values were 2.76 ± 0.67 at baseline vs 2.55 ± 0.77 at 9 weeks (p = 0.42). Mean RVmax:BP was 1.82 ± 0.32 at baseline vs 1.76 ± 0.45 at 9 weeks (p = 0.67). Myocardial PD-L1 expression was at least threefold greater than skeletal muscle at baseline for the LV (LVmax to musclemax 3.71 ± 0.77 vs 0.98 ± 0.20 (p < 0.001)) and at least twofold for the RV (LVmax to musclemax 2.49 ± 0.63 vs 0.98 ± 0.20 (p < 0.001)). There was excellent intra-rater reliability for LVmax:BP with ICC 0.99 (95% confidence interval 0.94–0.99, p < 0.001), mean bias -0.05 ± 0.14 (95% limits of agreement -0.32 to 0.21). There were no major adverse cardiovascular events or myocarditis during follow-up.ConclusionThis study is the first to report PD-L1 expression of the heart that can be quantified non-invasively without invasive myocardial biopsy, with high reliability and specificity. This technique can be applied to investigate myocardial PD-L1 expression in ICI-associated myocarditis and cardiomyopathies.Clinical trial registration PD-L1 Expression in Cancer (PECan) study (NCT04436406). https://clinicaltrials.gov/ct2/show/NCT04436406 June 18th, 2020.

CMTM6, a potential immunotherapy target.

The CKLF-like MARVEL transmembrane domain-containing protein 6 (CMTM6), which binds to the programmed death ligand 1 (PD-L1) and stabilizes the expression of PD-L1 on the cell surface, has been recently discovered as a novel regulator of PD-L1 expression in cancer. PD-L1 is an immune checkpoint inhibitory molecule that can mediate the immune escape of tumor cells in various tumors and has been studied intensively in recent years. In 2017, two articles simultaneously reported that CMTM6 can stabilize the expression of PD-L1 on the plasma membrane and prevent PD-L1 from being degraded by lysosomes; therefore, CMTM6 may play an important role in tumor cell immune escape and immunosuppression. At present, there are few studies on the relationship between the expression of CMTM6 and PD-L1 in different tumors and diseases. These studies together suggested that CMTM6 may be a potential novel immunotherapy target. In this review, we briefly describe the latest research progresses of CMTM6 in various cancers and other diseases.

MiR-105-5p regulates PD-L1 expression and tumor immunogenicity in gastric cancer.

Cancer immunotherapies targeting the interaction between Programmed death 1 (PD-1) and Programmed death ligand 1 (PD-L1) have recently been approved for the treatment of multiple cancer types, including gastric cancer. However, not all patients respond to these therapies, while some eventually acquire resistance. A partial predictive biomarker for positive response to PD-1/PD-L1 therapy is PD-L1 expression, which has been shown to be under strict post-transcriptional control in cancer. By fractionating the PD-L1 3' untranslated region (3'UTR) into multiple overlapping fragments, we identified a small 100-nucleotide-long cis-acting region as being necessary and sufficient for post-transcriptional repression of PD-L1 expression in gastric cancer. In parallel, we performed a correlation analysis between PD-L1 expression and all host miRNAs in stomach cancer patient samples. A single miRNA, miR-105-5p, was predicted to bind to the identified cis-acting 3'UTR region and to negatively correlate with PD-L1 expression. Overexpression of miR-105-5p in gastric cancer cell lines resulted in decreased expression of PD-L1, both at the total protein and surface expression levels, and induced CD8+ T cell activation in co-culture assays. Finally, we show that expression of miR-105-5p in gastric cancer is partly controlled by DNA methylation of a cancer- and germline-specific promoter of its host gene, GABRA3. Dysregulation of miR-105-5p is observed in many cancer types and this study shows the importance of this miRNA in controlling the immunogenicity of cancer cells, thus highlighting it as a potential biomarker for PD-1/PD-L1 therapy and target for combinatorial immunotherapy.

Screening and characterization of aptamers for recombinant human programmed death-1 and recombinant extracellular domain of human programmed death ligand-1.

Programmed death ligand-1 (PD-L1) is expressed on tumor cells and macrophages. The detection of PD-L1 expression in cancer and the treatment by targeting the PD-L1/programmed death-1 (PD-1) are of great clinical significance. This work aims to screen the aptamers with high affinity and specificity for recombinant human PD-1 (rhPD-1)/recombinant human PD-L1 extracellular domain (rhPD-L1). In this study, we have expressed, purified, prepared, and identified rhPD-1 and rhPD-L1. The rhPD-L1/rhPD-1 aptamers with high affinity and specificity were obtained by systematic evolution of ligands by exponential enrichment technique. Ten aptamers sequences to rhPD-L1 and 10 aptamers sequences to rhPD-1 were obtained by cloning and sequencing. The affinity and specificity of candidate aptamers were analyzed by gold nanoparticles-based colorimetric assay, dot blot assay, and electrophoretic mobility shift assay. The aptamers named A6 were picked out as the optimal aptamers that recognize PD-1, specifically with the Kd value of 47.84 ± 24.78 nM. The aptamers named B10 were picked out as the optimal aptamers that recognize PD-L1, specifically with the Kd value of 59.72 ± 15.87 nM. The study lays a foundation for the development of detection methods and therapeutic drugs targeting PD-L1/PD-1.

Immuno-PET imaging of 68Ga-labeled nanobody Nb109 for dynamic monitoring the PD-L1 expression in cancers.

The checkpoint blockade immunotherapy has become a potent treatment strategy for cancers, and programmed death ligand-1 (PD-L1) is a prominent checkpoint ligand that is highly expressed in some cancers. The identification of immune checkpoint marker PD-L1 is critical for improving the success of immunotherapy. Accordingly, the binding specificity and dynamic monitoring property of a non-blocking nanobody tracer 68Ga-NOTA-Nb109 to PD-L1 were assessed in this study. The endogenous expression level of PD-L1 in several cancer cells was measured by flow cytometry, Western blot, and cellular uptake assay. Sensitivity and specificity of 68Ga-NOTA-Nb109 in monitoring the expression of PD-L1 in vivo were evaluated by PET imaging of different tumor-bearing models (U87, high PD-L1 expression; HCT 116, medium PD-L1 expression; and NCI-H1299, low PD-L1 expression). In vivo PET imaging results agreed well with those detected in vitro. In addition, PET imaging of PD-L1 expression in U87 and NCI-H1299 xenografts using 18F-FDG was also performed for comparison. The maximum tumor-to-muscle uptake ratio of 68Ga-NOTA-Nb109 was more than twofold that of 18F-FDG in U87 xenograft. The change of PD-L1 expression in NCI-H1299 cells and xenografts induced by cisplatin (CDDP) was sensitively monitored by 68Ga-NOTA-Nb109. This study demonstrated the feasibility of tracer 68Ga-NOTA-Nb109 for specifically targeting endogenous PD-L1 and dynamic monitoring the change of PD-L1 expression, and could guide the immunotherapy and immunochemotherapy for refractory cancers.

Enhanced histone H3 acetylation of the PD-L1 promoter via the COP1/c-Jun/HDAC3 axis is required for PD-L1 expression in drug-resistant cancer cells

BackgroundDrug resistance is a major obstacle to treating cancers because it desensitizes cancer cells to chemotherapy. Recently, attention has been focused on changes in the tumor immune landscape after the acquisition of drug resistance. Programmed death-ligand-1 (PD-L1) is an immune suppressor that inhibits T cell-based immunity. Evidence has shown that acquired chemoresistance is associated with increased PD-L1 expression in cancer cells. However, the underlying mechanism is still largely unknown.MethodsPD-L1 expression in three drug-resistant A549/CDDP, MCF7/ADR and HepG2/ADR cell lines was detected by qRT-PCR, western blotting and flow cytometry, and a T cell proliferation assay was performed to test its functional significance. Then, the potential roles of JNK/c-Jun, histone H3 acetylation, histone deacetylase 3 (HDAC3) and the E3 ligase COP1 in the PD-L1 increase were explored through ChIP assays and gain- and loss-of-function gene studies. Furthermore, murine xenograft tumor models were used to verify the role of JNK/c-Jun and HDAC3 in PD-L1 expression in A549/CDDP cells in vivo. Finally, the correlations of PD-L1, c-Jun and HDAC3 expression in clinical cisplatin-sensitive and cisplatin-resistant non-small cell lung cancer (NSCLC) tissues were analyzed by immunohistochemistry and Pearson’s correlation coefficient.ResultsPD-L1 expression was significantly increased in A549/CDDP, MCF7/ADR and HepG2/ADR cells and was attributed mainly to enhanced JNK/c-Jun signaling activation. Mechanistically, decreased COP1 increased c-Jun accumulation, which subsequently inhibited HDAC3 expression and thereby enhanced histone H3 acetylation of the PD-L1 promoter. Furthermore, PD-L1 expression could be inhibited by JNK/c-Jun inhibition or HDAC3 overexpression in vivo, which could largely reverse inhibited CD3+ T cell proliferation in vitro. PD-L1 expression was significantly increased in the cisplatin-resistant clinical NSCLC samples and positively correlated with c-Jun expression but negatively correlated with HDAC3 expression.ConclusionsEnhanced histone H3 acetylation of the PD-L1 promoter via the COP1/c-Jun/HDAC3 axis was crucial for the PD-L1 increase in drug-resistant cancer cells. Our study reveals a novel regulatory network for the PD-L1 increase in drug-resistant cancer cells and that combined PD-L1-targeting strategies could improve T cell-based immunity in drug-resistant cancers.

Reactive versus Constitutive: Reconcile the Controversial Results about the Prognostic Value of PD-L1 Expression in cancer.

The prognostic value of programmed death-ligand 1 (PD-L1) has been controversial in recent studies. PD-L1 is known to play a major role in suppressing the immune response, yet increasing studies have reported that PD-L1 expression has a favorable prognostic value for cancer patients. This raises the concern about how to understand PD-L1 expression: merely an immune inhibitory signal, or more likely a reactive process to T-cell response that indicates cytotoxic T lymphocyte (CTL) level in a tumor? To solve this dilemma, an integrative investigation is required. We compared the PD-L1 expression between tumor cells and immune cells, and characterized the inter- and intra-tumor correlation between CTL and PD-L1 expression. The prognostic values between PD-L1 and CTL is compared across 15 solid cancers and 11 independent cohorts of ovarian cancer. PD-L1 and PD-L1-adjusted CTL are analyzed in immunotherapy dataset receiving nivolumab. We observed unexpected high concordance between the prognostic value of PD-L1 and CTL across different cancers and cohorts. We found primarily reactive rather than constitutive PD-L1 expression in most tumors. We revealed that PD-L1-adjusted CTL level, rather than the expression of PD-L1, effectively predicts the responders to immune checkpoint inhibitors. This study highlights the importance of PD-L1 expression, as primarily a signature of reacting efficiency of pre-existing anti-tumor immunity, in balancing the tumor microenvironment. Importantly, it suggests that the reactive efficiency of PD-L1 is more useful to predict the response to immunotherapy.

Radiopharmacology and molecular imaging of PD-L1 expression in cancer

Immunotherapy [(specifically, antibodies blocking programmed death ligand-1 (PD-L1)] is a valuable option in cancer treatment because it leads to durable tumour regression and improves survival in several cancers. Patients with PD-L1 expressing tumours benefit from this therapy, but currently it can only be determined through biopsy, which may be inconclusive or impossible due to lesion location, associated risks, intratumoral and interlesional heterogeneity. Therefore, radio-immune-imaging with a specific radiopharmaceutical is ideally placed to play an important role when performing real-time, in vivo, whole-body and non-invasive PD-L1 expression mapping. To describe and summarise published scientific data on imaging PD-L1 expression using radiopharmaceuticals and discuss future directions in this research field. A summary review of the literature was done through PubMed to search papers that described and included radiopharmaceuticals to image PD-L1 expression. Only English papers published until April 2018 that detailed laboratorial and animal procedures were selected. Eleven pre-clinical papers published between 2015 and 2018 were included. Four studies used anti-PD-L1 radiopharmaceuticals labelled with Indium-111, 4 with Copper-64, 2 with Fluoride-18 and 1 with both Copper-64 and Gallium-68. All of them had identical protocols and showed similar radiopharmaceutical biodistribution. They reported successful anti-PD-L1 labelling, with high tumour–background ratio (mainly when spleen uptake was saturated with unlabelled/cold antibody). All reported radiopharmaceuticals had high sensitivity and specificity to identify tumours with PD-L1 expression in animal model. Clinical experiments appear to be now justifiable.

MicroRNA-155 induction via TNF-α and IFN-γ suppresses expression of programmed death ligand-1 (PD-L1) in human primary cells

Programmed death ligand-1 (PD-L1) is a critical regulator of T cell function contributing to peripheral immune tolerance. Although it has been shown that posttranscriptional regulatory mechanisms control PD-L1 expression in cancer, it remains unknown whether such regulatory loops operate also in non-transformed cells. Here we studied PD-L1 expression in human dermal lymphatic endothelial cells (HDLECs), which play key roles in immunity and cancer. Treatment of HDLECs with the pro-inflammatory cytokines IFN-γ and TNF-α synergistically up-regulated PD-L1 expression. IFN-γ and TNF-α also affected expression of several microRNAs (miRNAs) that have the potential to suppress PD-L1 expression. The most highly up-regulated miRNA following IFN-γ and TNF-α treatment in HDLECs was miR-155, which has a central role in the immune system and cancer. Induction of miR-155 was driven by TNF-α, the effect of which was significantly enhanced by IFN-γ. The PD-L1 3′-UTR contains two functional miR-155-binding sites. Endogenous miR-155 controlled the kinetics and maximal levels of PD-L1 induction upon IFN-γ and TNF-α treatments. We obtained similar findings in dermal fibroblasts, demonstrating that the IFN-γ/TNF-α/miR-155/PD-L1 pathway is not restricted to HDLECs. These results reveal miR-155 as a critical component of an inflammation-induced regulatory loop controlling PD-L1 expression in primary cells.