Programmed Death-ligand 1 Protein Research Articles

Pan-Cancer Proteomics Analysis Reveals Wiskott-Aldrich Syndrome Protein as a Potential Regulator of Programmed Death-Ligand 1.

The programmed death-ligand 1 (PD-L1) is a key mediator of immunosuppression in the tumor microenvironment. The expression of PD-L1 in cancer cells is useful for the clinical determination of an immune checkpoint blockade (ICB). However, the regulatory mechanism of the PD-L1 abundance remains incompletely understood. Here, we integrated the proteomics of 52 patients with solid tumors and examined immune cell infiltration to reveal PD-L1-related regulatory modules. Wiskott-Aldrich syndrome protein (WASP) was identified as a potential regulator of PD-L1 transcription. In two independent cohorts containing 164 cancer patients, WASP expression was significantly associated with PD-L1. High WASP expression contributed to immunosuppressive cell composition, including cells positive for immune checkpoints (PD1, CTLA4, TIGIT, and TIM3), FoxP3+ Treg cells, and CD163+ tumor-associated macrophages. Overexpression of WASP increased, whereas knockdown of WASP decreased the protein level of PD-L1 in cancer cells without alteration of PD-L1 protein stability. The WASP-mediated cell migration and invasion were markedly attenuated by the silence of PD-L1. Collectively, our data suggest that WASP is a potential regulator of PD-L1 and the WASP/PD-L1 axis is responsible for cell migration and an immunosuppressive microenvironment.

Acesulfame potassium upregulates PD-L1 in HCC cells by attenuating autophagic degradation

Artificial sweeteners, which contain no or few calories, have been widely used in various foods and beverages, and are regarded as safe alternatives to sugar by the Food and Drug Administration. While several studies suggest that artificial sweeteners are not related to cancer development, some research has reported their potential association with the risk of cancers, including hepatocellular carcinoma (HCC). Here, we investigated whether acesulfame potassium (Ace K), a commonly used artificial sweetener, induces immune evasion of HCC cells by upregulating programmed death ligand-1 (PD-L1). Ace K elevated the protein levels of PD-L1 in HCC cells without increasing its mRNA levels. The upregulation of PD-L1 protein levels in HCC cells by Ace K was induced by attenuated autophagic degradation of PD-L1, which was mediated by the Ace K-stimulated ERK1/2-mTORC1 signaling pathway. Ace K-induced upregulation of PD-L1 attenuated T cell-mediated death of HCC cells, thereby promoting immune evasion of HCC cells. In summary, the present study suggests that Ace K promotes HCC progression by upregulating the PD-L1 protein level.

Lanosterol synthase deficiency promotes tumor progression by orchestrating PDL1-dependent tumor immunosuppressive microenvironment.

Lipid metabolic reprogramming is closely related to tumor progression with the mechanism not fully elucidated. Here, we report the immune-regulated role of lanosterol synthase (LSS), an essential enzyme in cholesterol synthesis. Database analysis and clinical sample experiments suggest that LSS was lowly expressed in colon and breast cancer tissues, which indicates poor prognosis. The biological activity of tumor cell lines and tumor progression in NOD scid gamma (NSG) mice were not affected after LSS knockdown, whereas LSS deficiency obviously aggravated tumor burden in fully immunized mice. Flow cytometry analysis showed that LSS knockdown significantly promoted the formation of tumor immunosuppressive microenvironment, characterized by the increase in M2 macrophages and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs), as well as the decrease in anti-tumoral T lymphocytes. With the inhibition of myeloid infiltration or loss function of T lymphocytes, the propulsive effect of LSS knockdown on tumor progression disappeared. Mechanistically, LSS knockdown increased programmed death ligand 1 (PDL1) protein stability by 2,3-oxidosqualene (OS) binding to PDL1 protein. Anti-PDL1 therapy abolished LSS deficiency-induced immunosuppressive microenvironment and cancer progression. In conclusion, our results show that LSS deficiency promotes tumor progression by establishing an OS-PDL1 axis-dependent immunosuppressive microenvironment, indicative of LSS or OS as a potential hallmark of response to immune checkpoint blockade.

Computational analysis of PD-L1 dimerization mechanism induced by small molecules and potential dynamical properties

The design of small molecule inhibitors that target the programmed death ligand-1 (PD-L1) is a forefront issue in immune checkpoint blocking therapy. Small-molecule inhibitors have been shown to exert therapeutic effects by inducing dimerization of the PD-L1 protein, however, the specific mechanisms underlying this dimerization process remain largely unexplored. Furthermore, there is a notable lack of comparative studies examining the binding modes of structurally diverse inhibitors. In view of the research gaps, this work employed molecular dynamics simulations to meticulously examine the interactions between two distinct types of inhibitors and PD-L1 in both monomeric and dimeric forms, and predicted the dimerization mechanism. The results revealed that inhibitors initially bind to a PD-L1 monomer, subsequently attracting another monomer to form a dimer. Notably, symmetric inhibitors observed superior binding efficiency compared to other inhibitors. Key residues, including Ile54, Tyr56, Met115 and Tyr123 played a leading role in binding. Structurally, symmetric inhibitors were capable of thoroughly engaging the binding pocket, promoting a more symmetrical formation of PD-L1 dimers. Furthermore, symmetric inhibitors formed more extensive hydrophobic interactions with protein residues. The insights garnered from this research are expected to significantly contribute to the rational design and optimization of small molecule inhibitors targeting PD-L1.

PD-L1 mRNA and protein expression in canine mammary carcinomas: Correlation with histopathological grade and molecular markers.

Programmed death ligand 1 (PD-L1) is an immune checkpoint molecule that plays a crucial role in regulating antitumor immune responses. Canine mammary carcinomas (CMCs) are common tumors of dogs. Despite extensive studies on the heterogeneity of CMCs, there is still a lack of effective precision therapies for the treatment of CMCs. In this study, we aimed to investigate the correlation between PD-L1 mRNA and protein expression in CMCs and explore its association with histopathological grade and molecular markers, including the estrogen receptor, epidermal growth factor receptor 2, and cytokeratin 5/6 (CK5/6). Formalin-fixed paraffin-embedded samples were evaluated for PD-L1 mRNA expression using RNA in situ hybridization and PD-L1 protein expression using immunohistochemistry. We observed no substantial correlation between PD-L1 mRNA and protein expression in CMCs; however, PD-L1 mRNA levels were significantly higher in grade 3 than in grade 1 tumors (P = .001). In addition, we observed a positive correlation between PD-L1 protein expression and CK5/6 expression in CMCs (P = .032). These findings suggest that PD-L1 expression in CMCs is heterogeneous and may be regulated post-transcriptionally. Further studies are needed to explore the prognostic and therapeutic implications of PD-L1 expression in different molecular subtypes of CMCs and their potential as predictive biomarkers for immunotherapy.

Exosomal circ_0032704 confers sorafenib resistance to hepatocellular carcinoma and contributes to cancer malignant progression by modulating the miR-514a-3p/PD-L1 pathway.

This study aims to explore the role of circ_0032704 in sorafenib-resistant hepatocellular carcinoma (HCC). The expression of circ_0032704, miR-514a-3p, and programmed death-ligand 1 (PD-L1) mRNA was detected by quantitative real-time PCR (qPCR). The expression of multidrug resistant-related proteins, migration/invasion-related proteins, exosome-related proteins, and PD-L1 protein was detected by western blot. Cell viability was detected by CCK-8 assay. Cell proliferation, migration, and invasion were assessed by EdU assay, wound healing assay, and transwell assay. The binding between miR-514a-3p and circ_0032704 or PD-L1 was verified by RIP assay, pull-down assay, and dual-luciferase reporter assay. Cell- or serum-derived exosomes were isolated and identified by TEM and NTA. Xenograft models were established to determine the effect of circ_0032704 on drug resistance invivo. Circ_0032704 was overexpressed in sorafenib-resistant HCC tissues and cells. Circ_0032704 knockdown reduced sorafenib resistance in HCC cells and inhibited cell proliferation, migration, and invasion of sorafenib-resistant HCC cells, while these effects were reversed by PD-L1 overexpression. We found that circ_0032704 positively regulated PD-L1 expression via targeting miR-514a-3p. Exosomes with circ_0032704 inhibition reduced sorafenib resistance in HCC cells and inhibited cell proliferation, migration, and invasion of sorafenib-resistant HCC cells. Exosomes with circ_0032704 inhibition also inhibited tumor growth invivo. The expression of circ_0032704 in exosomes was stable and possessed diagnostic value. Circ_0032704 enhanced sorafenib resistance in HCC and promoted the malignant development of sorafenib-resistant HCC. Circ_0032704 could be transported by exosomes, and exosomal circ_0032704 had diagnostic value.

Mitochondrial Disruption Nanosystem Simultaneously Depressed Programmed Death Ligand-1 and Transforming Growth Factor-β to Overcome Photodynamic Immunotherapy Resistance.

Currently, limited photosensitizers possess the capacity to reverse tumor hypoxia and reduce programmed death ligand-1 (PD-L1) and transforming growth factor-β (TGF-β) expression simultaneously, hindering the perfect photodynamic therapy (PDT) effect due to acquired immune resistance and the tumor hypoxic microenvironment. To tackle these challenges, in this research, we demonstrated that mitochondrial energy metabolism depression can be utilized as an innovative and efficient approach for reducing the expression of PD-L1 and TGF-β simultaneously, which may offer a design strategy for a more ideal PDT nanosystem. Through proteomic analysis of 5637 cells, we revealed that tamoxifen (TMX) can incredibly regulate PD-L1 expression in tumor cells. Then, to selectively deliver clinically used mitochondrial energy metabolism depressant TMX to solid tumors as well as design an ideal PDT nanosystem, we synthesized MHI-TMX@ALB by combining a mitochondria-targeted heptamethine cyanine PDT-dye MHI with TMX through self-assembly with albumin (ALB). Interestingly enough, the MHI-TMX@ALB nanoparticle demonstrated effective reversion of tumor hypoxia and inhibition of PD-L1 protein expression at a lower dosage (7.5 times to TMX), which then enhanced the efficacy of photodynamic immunotherapy via enhancing T-cell infiltration. Apart from this, by leveraging the heptamethine dye's targeting capacity toward tumors and TMX's role in suppressing TGF-β, MHI-TMX@ALB also more effectively mitigated 4T1 tumor lung metastasis development. All in all, the MHI-TMX@ALB nanoparticle could be used as a multifunctional economical PD-L1 and TGF-β codepression immune-regulating strategy, broadening the potential clinical applications for a more ideal PDT nanosystem.

IKZF4/NONO-RAB11FIP3 axis promotes immune evasion in gastric cancer via facilitating PD-L1 endosome recycling

As an immune checkpoint protein expressed by diverse cancer cells, programmed death ligand 1 (PD-L1) facilitates immune evasion by interacting with programmed cell death-1 (PD-1) on T cells. Despite the clinical benefits observed in various cancer types, strategies targeting PD-1/PD-L1 have demonstrated limited efficacy in gastric cancer (GC). Furthermore, the regulation of PD-L1, especially at post-translational modification levels, remains largely unknown. Therefore, it is crucial to elucidate the mechanisms governing PD-L1 expression to enhance anti-tumor immunity. In this study, we have identified that IKAROS family zinc finger 4 (IKZF4) and Non-POU domain-containing octamer-binding (NONO) synergistically regulate and enhance the expression of RAB11 family-interacting protein 3 (RAB11FIP3) in GC. The IKZF4/NONO-RAB11FIP3 axis facilitates the endosomal recycling of PD-L1, particularly on the cell membrane of GC cells. Moreover, overexpression of RAB11FIP3 mitigates the hypo-expression of PD-L1 protein resulting from IKZF4 or NONO deletion. Functionally, the silencing of RAB11FIP3 or IKZF4 promotes T cell proliferation, and enhances T-cell cytotoxicity towards GC cells in vitro, which further inhibits tumor immune evasion in mice via increasing the infiltration of CD8+ T cells into the tumor microenvironment (TME) to suppress GC progression. Our study suggests that the IKZF4/NONO-RAB11FIP3 axis promotes immune evasion by facilitating PD-L1 endosome recycling, thus presenting a potential therapeutic target for GC treatment.

Fluorescence Super-Resolution Imaging Chip for Gene Silencing Exosomes.

Tumor cell-derived extracellular vesicles and their cargo of bioactive substances have gradually been recognized as novel biomarkers for cancer diagnosis. Meanwhile, the PD-L1 (Programmed Death-Ligand 1) protein, as an immune checkpoint molecule, is highly expressed on certain tumor cells and holds significant potential in immune therapy. In comparison to PD-L1 monoclonal antibodies, the inhibitory effect of PD-L1 siRNA (small interfering RNA) is more advantageous. In this article, we introduced a microfluidic chip integrating cell cultivation and exosome detection modules, which were intended for the investigation of the gene silencing effect of PD-L1 siRNA. Basically, cells were first cultured with PD-L1 siRNA in the chip. Then, the secreted exosomes were detected via super-resolution imaging, to validate the inhibitory effect of siRNA on PD-L1 expression. To be specific, a "sandwich" immunological structure was employed to detect exosomes secreted from HeLa cells. Immunofluorescence staining and DNA-PAINT (DNA Point Accumulation for Imaging in Nanoscale Topography) techniques were utilized to quantitatively analyze the PD-L1 proteins on HeLa exosomes, which enabled precise structural and content analysis of the exosomes. Compared with other existing PD-L1 detection methods, the advantages of our work include, first, the integration of microfluidic chips greatly simplifying the cell culture, gene silencing, and PD-L1 detection procedures. Second, the utilization of DNA-PAINT can provide an ultra-high spatial resolution, which is beneficial for exosomes due to their small sizes. Third, qPAINT could allow quantitative detection of PD-L1 with better precision. Hence, the combination of the microfluidic chip with DNA-PAINT could provide a more powerful integrated platform for the study of PD-L1-related tumor immunotherapy.

Extra villous trophoblast-derived PDL1 can ameliorate macrophage inflammation and promote immune adaptation associated with preeclampsia

IntroductionSevere preeclampsia (sPE) is a systemic syndrome that may originate from chronic inflammation. Maintaining maternal-fetal hemostasis by the co-inhibitory molecule programmed death ligand 1 (PDL1) can be favorable for ameliorating inflammation from immune cells. Apart from programmed death 1 (PD1) expression, decidual macrophages (dMs) produce inflammatory cytokines, in response to cells which express PDL1. However, strong evidence is lacking regarding whether the PDL1/PD1 interaction between trophoblasts and decidual macrophages affects inflammation during sPE development. MethodsTo determine whether the trophoblast-macrophage crosstalk via the PDL1/PD1 axis modulates the inflammatory response in sPE-like conditions, at first, maternal-fetal tissues from sPE and normal patients were collected, and the PDL1/PD1 distribution was analyzed by Western blot, immunohistochemistry/ immunofluorescence and flow cytometry. Next, a coculture system was established and flow cytometry was used to identify how PDL1 was involved in macrophage-related inflammation under hypoxic stress. Transcriptional analysis was performed to clarify the inflammation-associated pathway induced by the PDL1/PD1 interaction. Finally, the Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME) mouse model was used to examine the effect of PDL1 on macrophage-related inflammation by measuring PE-like symptoms. ResultsIn maternal-fetal tissue from sPE patients, placental extravillous trophoblasts (EVTs) and dMs had a surprisingly increase of PDL1 and PD1 expression, respectively, accompanied by a higher percentage of CD68 +CD86 + dMs. In vitro experiments showed that trophoblast-derived PDL1 under hypoxia interacted with PD1 on CD14 +CD80 +macrophages, leading to suppression of inflammation through the TNFα-p38/NFκB pathway. Accordingly, the PE-like mouse model showed a reversal of PE-like symptoms and a reduced F4/80 + CD86 + macrophage percentage in the uterus in response to recombinant PDL1 protein administration, indicating the protective effect of PDL1. DiscussionOur results initially explained an immunological adaptation of trophoblasts under placental hypoxia, although this protection was insufficient. Our findings suggest the possible capacity of modulating PDL1 expression as a potential therapeutic strategy to target the inflammatory response in sPE.

Plasma cell-free RNA PD-L1 or tissue PD-L1 protein expression and outcomes with first-line immunotherapy in metastatic non-small cell lung cancer

BackgroundTissue programmed death ligand-1 (PD-L1) protein expression is associated with immune checkpoint inhibitor (ICI) treatment benefit in metastatic non-small cell lung cancer (NSCLC). However, tissue PD-L1 protein testing is limited by sampling tumor heterogeneity and fraught with tissue acquisition difficulties. A liquid biopsy-based PD-L1 assay could overcome these limitations of tissue PD-L1 testing. MethodsAn observational cohort of patients with metastatic NSCLC treated with first-line ICI-based therapies were retrospectively assessed for a pre-planned endpoint of median and 3-year landmark overall survival (OS) based upon plasma cell-free RNA (cfRNA) PD-L1 expression by a commercial exosome-free real-time qPCR assay or tissue PD-L1 protein expression (Dako 22C3) performed in CLIA/CAP accredited laboratories. Results53 contemporaneous patients in 3 patient cohorts were compared with a median follow-up 34 months. 16 patients were cfRNA plasma PD-L1 positive, including 6 (37 %) tissue PD-L1 negative or tissue quantity not sufficient for testing; 16 were plasma PD-L1 negative but tissue PD-L1 positive; 21 were both plasma and tissue PD-L1 negative. OS was identical whether positive plasma cfRNA PD-L1 expression or positive tissue PD-L1 protein expression (median OS 15 months; 3-year landmark OS 30 %; hazard ratio (HR) 0.97; 95 % CI, 0.44–2.10; p-value = 0.93). Within the positive plasma PD-L1 cohort there was no differing OS whether tissue PD-L1 positive, negative, or unknown (median OS 15 months; 3-year landmark OS 30 %; HR 1.15; 95 % CI, 0.322–4.05; p-value = 0.81). Positive plasma cfRNA expression was associated with a numerically longer median and higher 3-year OS compared to patients lacking PD-L1 expression (median 15 months versus 8 months; 3-year landmark OS 30 % versus 15 %; HR 0.57, 95 % CI 0.26–1.20; p-value = 0.11). ConclusionsIn this retrospective study of real-world metastatic NSCLC patients, plasma cfRNA PD-L1 expression was similarly predictive of ICI benefit as tissue PD-L1 protein expression.

Label Free Detection of Programmed Death Ligand 1 Protein Biomarker by Quartz Tuning Fork-Based Biosensor

Programmed death-ligand 1 (PD-L1) is expressed on the membrane of many types of cancer cells such as breast and lung cancer cells. PD-L1 helps the cancerous cells to escape the surveillance of the immune system. PD-L1 also exists in a soluble form, reaching detectable levels in cancer patient’s serum. The available detection techniques for PD-L1 are time and cost demanding, requiring advance instrumentations and efforts. To overcome the challenges of the current detection techniques, functionalized quartz tuning forks are employed as biosensors for the real-time detection of PD-L1. In this study, the QTF gold coated biosensors have been functionalized by Anti PD-L1 monoclonal antibodies as a probe layer. The functionalized QTF biosensors were tested against different concentrations of PD-L1 protein ranging from 0.05 up to 500 ng ml−1, with an incubation time of 15 min. It has been observed that the QTF resonance frequency shifted correlatively with the PD-L1 concentration. Testing of other proteins has not shown significant responses indicating the suitability of the probe layer’s selectivity for PD-L1. This result is expected to open the way for a fast and early yet simple approach for the possible discovery of cancer cells in initial stages, and cancer treatment monitoring.

Abstract LB_C21: Exogenous N-acetylgalactosamine-4-sulfatase (ARSB)-mediated tumor suppression involves effects on chondroitin 4-sulfate binding with SHP-2 and galectin-3, MAPK signaling, and transcriptional events, including epigenetic regulation of PD-L1

Abstract In experiments in melanoma, prostate, colon, and mammary cells and tissues, the enzyme N-acetylgalactosamine-4-sulfatase (Arylsulfatase B: ARSB), which removes the 4-sulfate group at the non-reducing end of chondroitin 4-sulfate (C4S), acts as a tumor suppressor. Following treatment with recombinant human (rh) ARSB in previous experiments and in recent experiments in the B16F10 mouse model of malignant melanoma, specific molecular pathways have been identified that retard tumor progression. These pathways involve more or less binding with chondroitin 4-sulfate when ARSB activity is modified. Galectin-3 binding with C4S increases following exogenous ARSB, leading to lower nuclear galectin-3 and reduced expression of chondroitin sulfate proteoglycan (CSPG)4 and of carbohydrate sulfotransferase (CHST)15 in melanoma cells and tissues. Nuclear Sp1 also declines, inhibiting transcription of both CSPG4, also known as the melanoma-associated chondroitin sulfate proteoglycan (MCSP), and of CHST15, which is required for 6-sulfation of N-acetylgalactosamine 4-sulfate. These proteins are associated with more aggressive tumors, and decline in their expression is consistent with the tumor suppressive function of ARSB. Inverse to the effect on galectin-3, SHP2, the ubiquitous non-receptor tyrosine phosphatase (PTPN11), binds less to C4S following treatment with exogenous ARSB. The resulting activation of SHP2 impacts on MAPK signaling, reducing phospho-ERK1/2 and phospho(T180/Y182)-p38-MAPK. These effects lead to decline in expression of the matrix metalloproteinases MMP2 and MMP9 and thereby restrain invasiveness and metastasis. Treatment of cultured melanoma cells with siRNA for ARSB leads to opposite effects to those of exogenous ARSB, including epigenetic effects modifying Programmed Death-Ligand 1 (PD-L1) expression. Treatment with rhARSB significantly reduces PD-L1 protein and gene expression, in contrast to increases when ARSB is silenced. The effects of rhARSB on PD-L1 expression are attributable to declines in phospho-JNK and nuclear c-Jun when free galectin-3 is less and to associated increases in histone deacetylase (HDAC) 3 expression and activity. Consistent with these effects, the ARSB-silencing induced increase in PD-L1 is inhibited by peptide inhibitors of the JNK-c-Jun interaction and by HDAC3 inhibitor. ARSB silencing reduces PD-L1 promoter acetylation, as shown by chromatin immunoprecipitation with anti-acetylhistone H3 antibody. The impact of rhARSB on signaling pathways, transcriptional events, and epigenetic mechanisms demonstrates how a critical sulfation and the resulting modification of chondroitin 4-sulfate can influence vital cell processes and can suppress tumor development. Effects on subcutaneous and pulmonary melanomas demonstrate the impact of exogenous ARSB in melanoma and suggest future prospects as a therapeutic agent. Recombinant human ARSB is an approved agent for the treatment of Mucopolysaccharidosis VI and is used safely and effectively for this congenital deficiency of ARSB. Citation Format: Joanne K. Tobacman, Insug O-Sullivan, Sumit Bhattacharyya. Exogenous N-acetylgalactosamine-4-sulfatase (ARSB)-mediated tumor suppression involves effects on chondroitin 4-sulfate binding with SHP-2 and galectin-3, MAPK signaling, and transcriptional events, including epigenetic regulation of PD-L1 [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_C21.

Chromosome 9p Duplication Promotes T-Cell Exhaustion and Enhances Stem Cell Clonogenic Potential in JAK2-Mutant Myeloproliferative Neoplasms

Myeloproliferative Neoplasms (MPNs) are a diverse group of clonal hematopoietic disorders originating from a single hematopoietic stem cell, causing an excessive production of mature blood cells. Classic forms of MPNs include polycythemia vera, essential thrombocythemia, and myelofibrosis. The most common mutation is a gain-of-function point mutation in the JAK2 gene, known as JAK2V617F. This mutation leads to constant activation of the JAK-STAT signaling pathway, resulting in the overgrowth of MPN cells. Furthermore, the variant allele frequency (VAF) of JAK2V617F has a significant impact on the severity and phenotype of the disease. Alongside many described genetic mutations, cytogenetic abnormalities are commonly observed in MPNs, particularly involving chromosome 9. As the JAK2 gene is located on the short arm of this chromosome, we hypothesized that chromosome 9 copy number abnormalities might be a disease modifier in JAK2V617F-mutant MPN patients. To characterize the biological effects of chromosome 9 copy number abnormalities on JAK2-mutated MPN cells, we analyzed circulating CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as monocytes and granulocytes from 32 MPN patients. Through Next Generation Sequencing, we categorized the patients into three main groups based on JAK2 mutation and copy number status: patients with two copies of the JAK2 gene with either heterozygous (VAF between 20% and 60%; n=10) or homozygous (VAF > 60% n=10) JAK2V617F mutation, or patients carrying mutant JAK2 and gene amplification (n=12). In-depth analysis of JAK2-amplified patients revealed that the amplification involved the entire chromosome 9p, thus including other gene loci like CD274, which encodes programmed death-ligand 1 (PD-L1). Further investigation of the order-of-events and clonal hierarchies through droplet digital PCR on CD34+ cell-derived colonies showed that most colonies of 9p-duplicated patients had three copies of JAK2, with 2 out of 3 alleles harboring the JAK2 mutation and that point mutations are frequently the initial pathogenic event in clonal evolution, followed by amplification of the JAK2-mutated allele. Functionally, CD34+ cells from +9p patients displayed high clonogenicity and gave rise to a greater number of primitive colonies (Colony Forming Unit-Granulocyte, Erythrocyte, Monocyte, Megakaryocyte, CFU-GEMM). As JAK2 hyperactivation had been previously reported to lead to increased PD-L1 expression, we further explored the functional significance of CD274 amplification in +9p patients. Our analysis showed increased PD-L1 messenger RNA and protein levels in +9p patient CD14+ monocytes compared to those from patients carrying only two copies of chromosome 9. Moreover, immunofluorescence analysis demonstrated significant re-localization of PD-L1 to the cytoplasmic membrane in monocytes from +9p patients, but not in JAK2V617F-homozygous patients (panel A). Increased levels of PD-L1, an immune checkpoint known to curb T cell activation, led us to analyze the T cell compartment, which resulted enriched in CD3+/CD8+/CD57-/PD-1+ exhausted T-cells in +9p patients compared to other MPN patients and healthy donors (panel B). In conclusion, our comprehensive characterization of the molecular interplay between JAK2V617F and chromosome 9 alterations, along with their immunological implications due to PD-L1 hyperactivation, fills a critical knowledge gap and provides valuable insights into the disease progression of 9p-MPNs. Further analysis is ongoing to explore the associations between 9p duplication and hematological parameters in 9p-MPN patients for a better understanding of the clinical implications of this genetic abnormality in MPNs.

Genomic Landscape of Pulmonary Sarcomatoid Carcinoma.

Pulmonary sarcomatoid carcinoma (PSC) is a rare aggressive subtype of non-small cell lung cancer (NSCLC) with limited therapeutic strategies. We attempted to elucidate the evolutionary trajectories of PSC using multiregional and longitudinal tumor samples. A total of 31 patients were enrolled in this study and 11 longitudinal samples were available from them. Using whole exome sequencing data, we analyzed the mutational signatures in both carcinomatous and sarcomatous areas in primary tumors of the 31 patients and longitudinal samples obtained from 11 patients. Furthermore, digital droplet polymerase chain reaction (ddPCR), and programmed death-ligand 1 (PD-L1) immunohistochemistry using the Ventana SP263 assay were performed. TP53 was identified as the most frequently altered gene in the primary (74%) and metastatic (73%) samples. MET exon 14 skipping mutations, confirmed by ddPCR, and TP53 mutations were mutually exclusive; whereas, MET exon 14 skipping mutations frequently co-occurred with MDM2 amplification. Metastatic tumors showed dissimilar genetic profiles from either primary component. During metastasis, the signatures of APOBEC decreased in metastatic lesions compared with that in primary lesions. PSC showed higher MET and KEAP1 mutations and stronger PD-L1 protein expression compared with that recorded in other NSCLCs. Decreased APOBEC signatures and subclonal diversity were detected during malignant progression in PSC. Frequent MET mutations and strong PD-L1 expression distinguished PSC from other NSCLCs. The aggressiveness and therapeutic difficulties of PSC were possibly attributable to profound intratumoral and intertumoral genetic diversity. Next-generation sequencing could suggest the appropriate treatment strategy for PSC.

TP53/mTORC1-mediated bidirectional regulation of PD-L1 modulates immune evasion in hepatocellular carcinoma

BackgroundImmunotherapy has facilitated great breakthroughs in the treatment of hepatocellular carcinoma (HCC). However, the efficacy and response rate of immunotherapy are limited and vary among different patients with HCC. TP53...

Astragaloside IV Attenuates Programmed Death-Ligand 1-Mediated Immunosuppression during Liver Cancer Development via the miR-135b-5p/CNDP1 Axis.

Astragaloside IV (AS-IV) is a pivotal contributor to anti-tumour effects and has garnered extensive attention in research. Tumour cell immune suppression is closely related to the increase in Programmed Death-Ligand 1 (PD-L1). Hepatocellular carcinoma (HCC) is a malignant tumour originating from hepatic epithelial tissue, and the role of AS-IV in regulating PD-L1 in anti-HCC activity remains unclear. Various concentrations of AS-IV were administered to both human liver immortalised cells (THEL2) and HCC (Huh-7 and SMMC-7721), and cell growth was assessed using the CCK-8 assay. HCC levels and cell apoptosis were examined using flow cytometry. Mice were orally administered AS-IV at different concentrations to study its effects on HCC in vivo. Immunohistochemistry was employed to evaluate PD-L1 levels. Western blotting was employed to determine PD-L1 and CNDP1 protein levels. We carried out a qRT-PCR to quantify the levels of miR-135b-3p and CNDP1. Finally, a dual-luciferase reporter assay was employed to validate the direct interaction between miR-135b-3p and the 3'UTR of CNDP1. AS-IV exhibited a dose-dependent inhibition of proliferation in Huh-7 and SMMC-7721 while inhibiting PD-L1 expression induced by interferon-γ (IFN-γ), thus attenuating PD-L1-mediated immune suppression. MiR-135b-5p showed significant amplification in HCC tissues and cells. AS-IV mitigated PD-L1-mediated immune suppression through miR-135b-5p. MiR-135b-5p targeted CNDP1, and AS-IV mitigated PD-L1-induced immunosuppression by modulating the miR-135b-5p/CNDP1 pathway. AS-IV decreases cell surface PD-L1 levels and alleviates PD-L1-associated immune suppression via the miR-135b-5p/CNDP1 pathway. AS-IV may be a novel component for treating HCC.

The Crosstalk between the EGFR and IFN-γ Pathways and Synergistic Roles in Survival Prediction and Immune Escape in Gliomas.

Glioma is the most common primary malignant brain tumor. The poor prognosis of gliomas, especially glioblastoma (GBM), is associated with their unique molecular landscape and tumor microenvironment (TME) features. The epidermal growth factor receptor (EGFR) gene is one of the frequently altered loci in gliomas, leading to the activation of the EGFR signaling pathway and thus, promoting the genesis of gliomas. Whether there exist factors within the TME that can lead to EGFR activation in the context of gliomas is currently unexplored. In total, 702 samples from The Cancer Genome Atlas (TCGA) and 325 samples from The Chinese Glioma Genome Atlas (CGGA) were enrolled in this study. Gene signatures related to EGFR signaling and interferon-γ (IFN-γ) response were established via the LASSO-COX algorithm. Gene Set Enrichment Analysis (GSEA) and Gene Ontology (GO) analysis were applied for function exploration. Kaplan-Meier (KM) curves and single sample GSEA (ssGSEA) of immune cell subpopulations were performed to analyze the prognosis and TME characteristics of different subgroups. Moreover, Western blotting (WB) and flow cytometry (FCM) demonstrated the correlation between IFN-γ and EGFR signaling activation and the subsequent induction of programmed death ligand 1 (PD-L1) expression. An EGFR signaling-related risk score was established, and a higher score was correlated with poorer prognosis and a more malignant phenotype in gliomas. Biological function analysis revealed that a higher EGFR-related score was significantly associated with various cytokine response pathways, especially IFN-γ. Long-term (7 days) exposure to IFN-γ (400 ng/mL) induced the activation of EGFR signaling in the u87 cell line. Next, an IFN-γ response-related risk score was established; the combination of these two scores could be used to further reclassify gliomas into subtypes with different clinical features and TME features. Double high-risk samples tended to have a poorer prognosis and more immunosuppressive TME. Additionally, FCM discovered that the activation of EGFR signaling via EGF (100 ng/mL) could trigger PD-L1 protein expression. This research indicates that IFN-γ, an inflammatory cytokine, can activate the EGFR pathway. The combination of EGFR signaling and IFN-γ response pathway can establish a more precise classification of gliomas.

Soluble PD-L1 as a diagnostic and prognostic biomarker in resectable gastric cancer patients.

In this study, we compared programmed death-ligand 1 (PD-L1) expression in primary tissue samples and its soluble form (sPD-L1) concentration in matched preoperative plasma samples from gastric cancer patients to understand the relationship between tissue and plasma PD-L1 expression and to determine its diagnostic and prognostic value. PD-L1 expression in tissue was assessed by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA), and sPD-L1 concentration in plasma was quantified by ELISA. The levels of the CD274 gene, which encodes for PD-L1 protein, were examined as part of bulk tissue RNA-sequencing analyses. Additionally, we evaluated the association between sPD-L1 levels and various laboratory parameters, disease characteristics, and patient outcomes. GC patients had significantly higher levels of sPD-L1 in their plasma (71.69pg/mL) compared to healthy controls (35.34pg/mL) (p < 0.0001). Moreover, sPD-L1 levels were significantly correlated with tissue PD-L1 protein, CD274 mRNA expression, larger tumor size, advanced tumor stage, and lymph node metastasis. Elevated sPD-L1 levels (> 103.5ng/mL) were associated with poor overall survival (HR = 2.16, 95%CI 1.15-4.08, p = 0.017). Furthermore, intratumoral neutrophil and dendritic cell levels were directly correlated with plasma sPD-L1 concentration in the GC patients. sPD-L1 was readily measurable in GC patients, and its level was associated with GC tissue PD-L1 expression, greater inflammatory cell infiltration, disease progression, and survival. Thus, sPD-L1 may be a useful minimally invasive diagnostic and prognostic biomarker in GC patients.

Transcriptional factor BRD4 promotes the stemness of esophageal cancer by activating the nuclear PD-L1/RelB axis.

Esophageal cancer (EC) is a prevalent malignancy associated with therapeutic resistance and poor prognosis. This study investigates the role of programmed death-ligand 1 (PD-L1) in esophageal cancer stem cell (ECSC) formation. ECSCs were enriched and characterized using various assays. We found that both PD-L1 and bromodomain-containing protein 4 (BRD4) were upregulated in ECSCs, promoting their stemness. Inhibiting BRD4 suppressed ECSC markers expression and sphere formation. Furthermore, BRD4 inhibitors downregulated membrane and nuclear PD-L1 levels, with knockdown of PD-L1 inhibiting ECSC formation. PD-L1 degraders also affected PD-L1 and its downstream effector RelB expression. Moreover, inhibiting RelB influenced sphere formation through interleukin-6 expression. This study reveals the critical role of the BRD4/nuclear PD-L1/RelB axis in ECSC formation, highlighting nuclear PD-L1 as a potential immunotherapeutic target for refractory EC.