Network pharmacology study of Citrus reticulata and Pinellia ternata in the treatment of non-small cell lung cancer

It has been recognized that Citrus reticulata and Pinellia ternata have a good therapeutic effect on NSCLC. However, the potential mechanism of C. reticulata and P. ternata in the treatment of NSCLC based on network pharmacology analysis is not clear. The “Drug-Component-Target-Disease” network was constructed by Cytoscape, and the protein interaction (PPI) network was constructed by STRING. Our study indicated that 18 active ingredients of C. reticulata and P. Ternata were screened from the TCMSP database, and 56 target genes of C. reticulata and P. Ternata for the treatment of NSCLC were identified, and we constructed the “Drug-Component-Target-Disease” network. In this study, we screened 56 PPI core genes to establish a PPI network. We concluded that the network pharmacology mechanism of the effect of C. reticulata and P. Ternata on NSCLC may be closely related to the protein expressed by TP53, ESR1, FOS, NCOA3 and MAPK8, and these may play the therapeutic roles by regulating the IL-17 signaling pathway, antigen processing and presentation, microRNAs in cancer and endocrine resistance.

TP53 Mutation Infers a Poor Prognosis and Is Correlated to Immunocytes Infiltration in Breast Cancer

Background: This study aimed to investigate the TP53 mutation, its potential immune features, its prognostic value, and its impact on immune infiltration in patients with breast cancer (BC).Methods: We downloaded the somatic mutation data and clinicopathologic features of BC patients from the TCGA GDC database, UCSC Xena platform, and International Cancer Genome Consortium (ICGC) database. The association between the TP53 mutation, clinicopathology features, and overall survival (OS) in BC patients was analyzed. We evaluated the potential role of the TP53 mutation in the immune therapy response, including the tumor mutation burden (TMB), microsatellite instability (MSI), and tumor immune dysfunction and exclusion (TIDE). Moreover, ESTIMATE was employed to assess the ImmuneScore and StromalScore in BC patients. We also explored immunocyte infiltration related to the TP53 mutation and its potential mechanism. Immunohistochemistry (IHC) was performed to validate the association between the expression of CXCL1, CXCL10, and CCL20 and TP53 status.Results: We found that the TP53 mutation was significantly associated with the shorter OS (p = 0.038) and was also an independent predictive factor of OS for BC patients (p < 0.001). Compared to that in the wild type group, the TP53-mutant group showed a higher TMB value (P< 0.001), MSI value (p = 0.077), and TIDE value (p < 0.001) with respect to BC patient immunotherapy. In addition, the ImmuneScore and StromalScore were both significantly increased in the TP53-mutant group (ImmuneScore: p < 0.001; StromalScore: p = 0.003). The results of CIBERSORT suggested that the TP53 mutation significantly promoted the infiltration of Tregs, T helper cells, and M0-type macrophages. KEGG and GSEA enrichment results suggested that the IL-17 signaling pathway and antigen processing and presentation pathways were significantly enriched in the TP53-mutant group. Importantly, based on IHC results of immune-related hub-genes, the chemokines CXCL1, CXCL10, and CCL20 were significantly upregulated in the TP53-mutant group in BC patients.Conclusion: These results indicate that a TP53 mutation might serve as a biomarker for BC prognosis and is related to immunocyte infiltration in the tumor microenvironment.

Dual Targeting of EZH2 and HDAC with Tazemetostat and Belinostat Promotes Immunogenicity in GC-DLBCL

Epigenetic remodeling is essential for the proper differentiation and function of germinal center (GC) B cells. Competing actions of histone acetyltransferases (HATs), such as CREBBP and EP300, and inhibitory histone deacetylases (HDACs) and methyltransferases (HMTs), such as EZH2, modulate the epigenomic program of GC B cells and control key activating and immunogenic processes, such as BCR and CD40 signaling, antigen processing and presentation, and cell cycle regulation. In line with the critical importance of epigenetic regulation of GC B cell function, inactivating mutations in CREBBPor EP300and gain-of-function mutations in EZH2 have been identified in 39% and 21% of GC B-cell like diffuse large B-cell lymphoma (GC-DLBCL), respectively. Strategies to target epigenetic dysfunction in GC-DLBCL have largely focused on indirectly restoring the activity of HATs through the use of histone deacetylase (HDAC) inhibitors, such as belinostat, and by directly inhibiting EZH2 with tazemetostat. Despite showing great promise in preclinical studies, single agent therapies with HDAC or EZH2 inhibitors have shown only modest efficacy in the clinic. Recently, our group demonstrated synergistic effects of dual therapy with EZH2 and HDAC inhibitors in inducing lethality of GC-DLBCL lines. As combination therapies begin to be tested in clinical trials, whether dual inhibition of EZH2 and HDAC is able to restore immunogenic features of GC-DLBCL and lead to enhanced T cell-mediated killing in vivo remains unknown. Here, we utilize transcriptomic and flow cytometric methods to assess whether epigenetic remodeling with tazemetostat (EZH2 inhibitor) and belinostat (HDAC inhibitor) can alter the immunogenicity of GC-DLBCL. We find that combination treatment with both tazemetostat and belinostat promotes an antigen processing and presentation program in GC-DLBCL lines and results in significant increases in MHC-I and MHC-II surface expression. This effect was only modestly appreciated with single agent treatment and required at least 4-7 days post-treatment to become apparent. Importantly, the increased MHC expression in response to combination therapy was not dependent on EZH2 or CREBBPmutation status, suggesting broad applications of dual therapy in patients with diverse mutation burdens. Furthermore, we found that combination treatment also altered surface expression of costimulatory molecules, such as CD80 and CD86, suggesting that tazemetostat and belinostat can modulate interactions with T cells in a multifaceted manner. These findings thus uncover that dual targeting of EZH2 and HDAC with tazemetostat and belinostat promotes antigen presentation pathways in GC-DLBCL and suggests that dual therapies could restore immunogenicity in GC-DLBCL and enhance immune-mediated killing of tumor cells. This work was funded, in part, by the ASH HONORS Award Summer Program. Figure 1 Figure 1. Disclosures Amengual: Seagen: Consultancy; Appia Pharmaceuticals: Research Funding; Daiichi Sankyo, Inc: Consultancy; Epizyme, Inc.: Speakers Bureau.

Analysis of the Expression and Prognosis for Leukocyte Immunoglobulin-Like Receptor Subfamily B in Human Liver Cancer

Background: Leukocyte immunoglobulin-like receptor subfamily B (LILRB), including 5 subtypes, is a group of inhibitory receptors in immune system. The LILRB family is known to be involved in the tumor progression of various cancer types, especially liver cancer. However, the expression patterns and prognostic values of LILRB family members in liver cancer tissues remain unclear.Methods: We used the Oncomine database, GEPIA database, Kaplan–Meier Plotter, Timer, TISIDB and cBioPortal to assess the expression and prognostic value of the LILRB family in liver cancer patients. We also verified the expression of the LILRB family in tumor tissues and tumor-free liver tissues at the protein level by using immunohistochemistry. The STRING website was used to explore the interaction between the LILRB family and their related genes. The DAVID database was used to perform the GO and KEGG analyses. Flow cytometry was used to assess the infiltrated NK cells in liver cancer tissues.Results: Our study revealed that the mRNA expression of LILRB1, LILRB2, LILRB3 and LILRB5 was downregulated, while compared with normal tissues, the mRNA expression of LILRB4 was upregulated in liver cancer tissues. Survival analysis revealed that LILRB2 and LILRB5 mRNA expression levels were significantly associated with OS and DSS and that the mRNA expression of all LILRB family members was significantly correlated with RFS and PFS. Next, we further found that the mRNA expression of all LILRB family members was significantly associated with the infiltration of B cells, CD8+ T cells, CD4+ T cells, macrophages, and neutrophil dendritic cells in liver cancer. Finally, GO and KEGG analyses found that the LILRB family and its related genes were involved in antigen processing and presentation and natural killer cell-mediated cytotoxicity pathways.Conclusions: Our study suggested that LILRB family expression was associated with the prognosis of liver cancer patients and infiltrated immune cells. The LILRB family might be involved in antigen processing and presentation and natural killer cell-mediated cytotoxicity pathways.

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

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

914 Loss of LKB1 is associated with resistance to IFN-gamma and T cell killing in non-small cell lung cancer

BackgroundKRAS-mutant non-small cell lung cancers (NSCLC) have exhibited unique response patterns to immunotherapy based on their co-occurring mutations. Patients harboring KRAS & STK11/LKB1 co-mutations (KL) have experienced shorter progression-free and overall survival compared to those with only KRAS mutations (K). Despite their limited responses, KL tumors exhibit a tumor mutational burden comparable to their K counterparts, suggesting the presence of additional mechanisms impairing antigen-specific responses. Accordingly, here we investigated the role of the MHC I antigen processing and presentation pathway in KL tumors.MethodsTCGA lung adenocarcinoma (LUAD) data were investigated for changes in expression of HLA molecules and chaperones involved in antigen processing and presentation. In mice, we performed single cell RNA sequencing of resected LKR13 K and KL tumors to evaluate changes in the tumor microenvironment and intrinsic differences in tumor antigen processing machinery. In vitro experiments were performed using the ovalbumin antigen to evaluate changes in antigen-specific T cell responses.ResultsExpression of HLA-A (p<0.0001), -B (p<0.0001), -C (p<0.0001), and beta2-microglobulin (B2M, p<0.0002) was downregulated in KL tumors from TCGA, as were expression of the TAP1 (p<0.001) and TAP2 (p<0.001) transporter associated with antigen processing subunits. LKR13 KL tumors exhibited similar patterns with lower H2-k1 (p<0.0001), H2-d1 (p<0.0001), B2m (p<0.0001), Tap1 (p<0.0001) and Tap2 (p<0.0001). As a result, LKR13 KL were resistant to recognition (p<0.005) and killing (56.9% K versus 7.8% KL) by OT-I T cells. Decreased expression of IFN-gamma-regulated genes such as PSMB8 (p<0.001), PSMB9 (p<0.0001), PSMB10 (p<001), CIITA (p<0.0001), NLRC5 (p<0.0001), IFNGR1 (p<0.0001), and IFNGR2 (p<0.0001) was also noted in KL tumors. Accordingly, KL tumors were unresponsive to exogenous IFN-gamma stimulation, maintaining repression of surface H2-Kb and resistance to T cell recognition (p<0.05) and killing (12.8% K versus 4% KL). Expression of T cell chemokines and receptors CXCR3 (p<0.0001), CXCL9 (p<0.0001), and CXCL10 (p<0.0001) was also repressed, potentially contributing to the lack of T cell infiltration in KL tumors.ConclusionsKRAS-mutant tumors harboring STK11/LKB1 alterations have an immunosuppressed phenotype and resistance to PD-1/PD-L1 inhibitors. Our findings provide evidence that these alterations are associated with markedly reduced antigen presentation and resistance to T cell killing, responsiveness to IFN-gamma stimulation, and impaired production of T cell chemokines, providing mechanistic insights into this immunosuppressed phenotype that could help guide the development of new therapeutic strategies for enhancing anti-tumor immunity.

Proteomic analysis of necroptotic extracellular vesicles

Necroptosis is a regulated and inflammatory form of cell death. We, and others, have previously reported that necroptotic cells release extracellular vesicles (EVs). We have found that necroptotic EVs are loaded with proteins, including the phosphorylated form of the key necroptosis-executing factor, mixed lineage kinase domain-like kinase (MLKL). However, neither the exact protein composition, nor the impact, of necroptotic EVs have been delineated. To characterize their content, EVs from necroptotic and untreated U937 cells were isolated and analyzed by mass spectrometry-based proteomics. A total of 3337 proteins were identified, sharing a high degree of similarity with exosome proteome databases, and clearly distinguishing necroptotic and control EVs. A total of 352 proteins were significantly upregulated in the necroptotic EVs. Among these were MLKL and caspase-8, as validated by immunoblot. Components of the ESCRTIII machinery and inflammatory signaling were also upregulated in the necroptotic EVs, as well as currently unreported components of vesicle formation and transport, and necroptotic signaling pathways. Moreover, we found that necroptotic EVs can be phagocytosed by macrophages to modulate cytokine and chemokine secretion. Finally, we uncovered that necroptotic EVs contain tumor neoantigens, and are enriched with components of antigen processing and presentation. In summary, our study reveals a new layer of regulation during the early stage of necroptosis, mediated by the secretion of specific EVs that influences the microenvironment and may instigate innate and adaptive immune responses. This study sheds light on new potential players in necroptotic signaling and its related EVs, and uncovers the functional tasks accomplished by the cargo of these necroptotic EVs.

The Mannose Receptor: From Endocytic Receptor and Biomarker to Regulator of (Meta)Inflammation

The mannose receptor is a member of the C-type lectin (CLEC) family, which can bind and internalize a variety of endogenous and pathogen-associated ligands. Because of these properties, its role in endocytosis as well as antigen processing and presentation has been studied intensively. Recently, it became clear that the mannose receptor can directly influence the activation of various immune cells. Cell-bound mannose receptor expressed by antigen-presenting cells was indeed shown to drive activated T cells towards a tolerogenic phenotype. On the other hand, serum concentrations of a soluble form of the mannose receptor have been reported to be increased in patients suffering from a variety of inflammatory diseases and to correlate with severity of disease. Interestingly, we recently demonstrated that the soluble mannose receptor directly promotes macrophage proinflammatory activation and trigger metaflammation. In this review, we highlight the role of the mannose receptor and other CLECs in regulating the activation of immune cells and in shaping inflammatory responses.