Immune Response-related Genes Research Articles

Spinster Homologue 2 Expression Correlates With Improved Patient Survival in Hepatocellular Carcinoma Despite Association With Lymph-Angiogenesis.

Spinster homologue 2 (SPNS2) is a transporter of sphingosine-1-phosphate (S1P), a bioactive lipid linked to cancer progression. We studied the link between SPNS2 gene expression, tumor aggressiveness, and outcomes in patients with hepatocellular carcinoma (HCC). Gene expression in patients with HCC was analyzed from the Cancer Genome Atlas (TCGA) (n = 350) and GSE76427 (n = 115) as a validation cohort, as well as liver tissue cohort GSE6764 (n = 75). High-SPNS2 HCC was significantly associated with high level of lymph-angiogenesis-related factors. SPNS2 expression was significantly higher in normal liver and early HCC versus advanced HCC (P < 0.02). High SPNS2 levels enriched immune response-related gene sets; inflammatory, interferon (IFN)-α, IFN-γ responses, and tumor necrosis factor (TNF)-α, interleukin (IL)-6/Janus kinase/signal transducer and activator of transcription (JAK/STAT3) signaling, complement and allograft rejection, but did not significantly infiltrate specific immune cells nor cytolytic activity score. High-SPNS2 HCC enriched tumor aggravating pathway gene sets such as KRAS (Kirsten rat sarcoma virus) signaling, but inversely correlated with Nottingham histological grade, MKI67 (marker of proliferation Ki-67) expression, and cell proliferation-related gene sets. Further, high-SPNS2 HCC had significantly high infiltration of stromal cells, showing that low-SPNS2 HCC is highly proliferative. Finally, high-SPNS2 HCC was associated with better disease-free, disease-specific, and overall survival (P = 0.031, 0.046, and 0.040, respectively). Although SPNS2 expression correlated with lymph-angiogenesis and other cancer-promoting pathways, it also enriched immune response. SPNS2 levels were higher in normal liver compared to HCC, and inversely correlated with cancer cell proliferation and better survival. SPNS2 expression may be beneficial in HCC patients despite detrimental in-vitro effects.

Low- and high-grade glioma-associated vascular cells differentially regulate tumor growth.

A key feature distinguishing high-grade glioma (HG) from low-grade glioma (LG) is the extensive neovascularization and endothelial hyperproliferation. Prior work has shown that tumor-associated vasculature from HG is molecularly and functionally distinct from normal brain vasculature and expresses higher levels of pro-tumorigenic factors that promote glioma growth and progression. However, it remains unclear whether vessels from LG also express pro-tumorigenic factors, and to what extent they functionally contribute to glioma growth. Here, we profile the transcriptomes of glioma-associated vascular cells (GVC) from IDH-mutant (mIDH) LG and IDH-wildtype (wIDH) HG and show that they exhibit significant molecular and functional differences. LG-GVC show enrichment of extracellular matrix-related gene sets and sensitivity to anti-angiogenic drugs, whereas HG-GVC display an increase in immune response-related gene sets and anti-angiogenic resistance. Strikingly, conditioned media from LG-GVC inhibits the growth of wIDH glioblastoma cells, whereas HG-GVC promotes growth. In vivo co-transplantation of LG-GVC with tumor cells reduces growth, whereas HG-GVC enhances tumor growth in orthotopic xenografts. We identify ASPORIN (ASPN), a small leucine-rich repeat proteoglycan, highly enriched in LG-GVC as a growth suppressor of wIDH glioblastoma cells in vitro and in vivo. Together, these findings indicate that GVC from LG and HG are molecularly and functionally distinct and differentially regulate tumor growth. Implications: This study demonstrated that vascular cells from IDH-mutant LG and IDH-wildtype HG exhibit distinct molecular signatures and have differential effects on tumor growth via regulation of ASPN-TGFβ1-GPM6A signaling.

Abstract 3925: Cancer-associated fibroblast-derived Dickkopf-1 impairs anti-tumor immunity in breast cancer by suppressing NK cell-mediated cytotoxicity

Abstract Despite early diagnosis, 10-20% of breast cancer patients experience metastatic disease within 5-10 years. While it is established that breast cancer cells have the capacity to evade immune responses, the mechanisms driving this evasion remain elusive. Therefore, finding ways to enhance anti-tumor immunity is crucial to improve patient outcomes. Dickkopf-1 (DKK1) is a Wnt inhibitor whose levels correlate with poor prognosis and decreased immune infiltration in various cancers. In this study, we used mice orthotopically injected with the PyMT, EO771, and 4T1 breast cancer lines. In all models, we found elevated DKK1 serum levels, and its neutralization with an αDKK1 Ab resulted in a significant reduction in primary tumor growth but also in metastatic dissemination. To identify the source of DKK1, we examined its expression in the tumor. DKK1 was detected in stromal cells with a fibroblast-like morphology but not in cancer cells or immune infiltrates. Co-staining confirmed colocalization between DKK1 and the CAF marker αSMA. To address the role of CAF-derived DKK1, we specifically deleted DKK1 in CAFs by generating FSP1CreDkk1fl/fl and αSMACreERT2Dkk1fl/fl mice. Both models showed a significant reduction in primary tumor growth while maintaining comparable serum DKK1 levels, indicating locally produced DKK1 supports tumor growth. To understand how DKK1 promotes tumor progression, we conducted bulk RNAseq of tumor cells isolated from IgG or αDKK1 treated mice. While no changes related to cell viability or cell cycle were observed, immune response-related genes were upregulated in αDKK1-treated cells. In support of this, αDKK1 did not reduce tumor growth in NSG immune-compromised mice. To pinpoint the immune cell targeted by DKK1, we depleted T, NK cells, or macrophages in WT tumor-bearing mice treated with αDKK1. Remarkably, only NK cell depletion negated αDKK1 anti-tumor effects. In vitro assays further demonstrated that rDKK1 or the presence of CAFs reduced NK cell cytotoxicity against tumor cells while αDKK1 restored it. To assess whether DKK1 levels correlated with tumor progression and immune suppression in breast cancer patients, we analyzed DKK1 serum levels and circulating NK cells at the diagnosis of metastatic bone disease and 15-18 months after standard care. DKK1 levels were significantly elevated in patients with progressive bone disease compared to those with stable disease. Importantly, NK cells from the progressive disease cohort had reduced perforin and granzyme B expression at the follow-up visit compared to baseline, indicating reduced cytotoxicity. Furthermore, rDKK1 reduced perforin in NK cells from healthy donors. In sum, our data show DKK1 is an important regulator of breast cancer progression, and we report for the first time DKK1 directly impairs NK cell cytotoxicity, creating an immune suppressive environment resistant to treatment. Citation Format: Seunghyun Lee, Biancamaria Ricci, Roberta Faccio. Cancer-associated fibroblast-derived Dickkopf-1 impairs anti-tumor immunity in breast cancer by suppressing NK cell-mediated cytotoxicity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3925.

Effects of Combined Treatment with Sodium Dichloroacetate and Sodium Valproate on the Genes in Inflammation- and Immune-Related Pathways in T Lymphocytes from Patients with SARS-CoV-2 Infection with Pneumonia: Sex-Related Differences.

The study presents data on the anti-inflammatory effects of a combination of sodium dichloroacetate and sodium valproate (DCA-VPA) on the expression of inflammation- and immune response-related genes in T lymphocytes of SARS-CoV-2 patients. The study aimed to assess the effects of DCA-VPA on the genes of cytokine activity, chemokine-mediated signaling, neutrophil chemotaxis, lymphocyte chemotaxis, T-cell chemotaxis, and regulation of T-cell proliferation pathways. The study included 21 patients with SARS-CoV-2 infection and pneumonia: 9 male patients with a mean age of 68.44 ± 15.32 years and 12 female patients with a mean age of 65.42 ± 15.74 years. They were hospitalized between December 2022 and March 2023. At the time of testing, over 90% of sequences analyzed in Lithuania were found to be of the omicron variant of SARS-CoV-2. The T lymphocytes from patients were treated with 5 mmol DCA and 2 mmol VPA for 24 h in vitro. The effect of the DCA-VPA treatment on gene expression in T lymphocytes was analyzed via gene sequencing. The study shows that DCA-VPA has significant anti-inflammatory effects and apparent sex-related differences. The effect is more potent in T cells from male patients with SARS-CoV-2 infection and pneumonia than in females.

MeIS: DNA methylation-based immune response signatures for thyroid nodule diagnostics.

Accurately distinguishing between benign thyroid nodules (BTNs) and papillary thyroid cancers (PTCs) with current conventional methods poses a significant challenge. We identify DNA methylation markers of immune response-related genes for distinguishing BTNs and PTCs. In this study, we analyzed a public reduced representative bisulfite sequencing (RRBS) dataset and revealed distinct methylation patterns associated with immune signals in PTCs and BTNs. Based on these findings, we developed a diagnostic classifier named as the Methylation-based Immune Response Signature (MeIS), which was composed of fifteen DNA methylation markers associated with immune response-related genes. We validated the MeIS's performance in two independent cohorts: ZS's retrospective cohort (50 PTC and 18 BTN surgery-leftover samples) and ZS's preoperative cohort (31 PTC and 30 BTN fine-needle aspiration (FNA) samples). The MeIS classifier demonstrated significant clinical promise, achieving AUCs of 0.96, 0.98, 0.89 and 0.90 in the training set, validation set, ZS's retrospective cohort, and ZS's preoperative cohort, respectively. For the cytologically indeterminate thyroid nodules, in the ZS's retrospective cohort, MeIS exhibited a sensitivity of 91% and a specificity of 82%; in the ZS's preoperative cohort, MeIS achieved a sensitivity of 84% and a specificity of 74%. Additionally, combining MeIS and BRAFV600E detection improved the detecting performance of cytologically indeterminate thyroid nodules, yielding sensitivities of 98% and 87%, and specificities of 82% and 74% in the ZS's retrospective cohort and ZS's preoperative cohort, respectively. The fifteen markers we identified can be employed to improve the diagnostic of cytologically indeterminate thyroid nodules.

Longitudinal analysis of influenza vaccination implicates regulation of RIG-I signaling by DNA methylation

Influenza virus infection alters the promoter DNA methylation of key immune response-related genes, including type-1 interferons and proinflammatory cytokines. However, less is known about the effect of the influenza vaccine on the epigenome. We utilized a targeted DNA methylation approach to study the longitudinal effects (day 0 pre-vaccination and day 28 post-vaccination) on influenza vaccination responses in peripheral blood mononuclear cells. We found that baseline, pre-vaccination methylation profiles are associated with pre-existing, protective serological immunity. Additionally, we identified 481 sites that were differentially methylated between baseline and day 28 post-vaccination. These were enriched for genes involved in the regulation of the RIG-I signaling pathway, an important regulator of viral responses. Our results suggest that DNA methylation changes to components of the RIG-I pathway are associated with vaccine effectiveness. Therefore, immunization strategies that target this pathway may improve serological responses to influenza vaccination.

The Identification Distinct Antiviral Factors Regulated Influenza Pandemic H1N1 Infection.

Influenza pandemic with H1N1 (H1N1pdms) causes severe lung damage and "cytokine storm," leading to higher mortality and global health emergencies in humans and animals. Explaining host antiviral molecular mechanisms in response to H1N1pdms is important for the development of novel therapies. In this study, we organised and analysed multimicroarray data for mouse lungs infected with different H1N1pdm and nonpandemic H1N1 strains. We found that H1N1pdms infection resulted in a large proportion of differentially expressed genes (DEGs) in the infected lungs compared with normal lungs, and the number of DEGs increased markedly with the time of infection. In addition, we found that different H1N1pdm strains induced similarly innate immune responses and the identified DEGs during H1N1pdms infection were functionally concentrated in defence response to virus, cytokine-mediated signalling pathway, regulation of innate immune response, and response to interferon. Moreover, comparing with nonpandemic H1N1, we identified ten distinct DEGs (AREG, CXCL13, GATM, GPR171, IFI35, IFI47, IFIT3, ORM1, RETNLA, and UBD), which were enriched in immune response and cell surface receptor signalling pathway as well as interacted with immune response-related dysregulated genes during H1N1pdms. Our discoveries will provide comprehensive insights into host responding to pandemic with influenza H1N1 and find broad-spectrum effective treatment.

Evaluation of a live attenuated vaccine against Edwardsiella piscicida by 16S rRNA sequencing

A complex and dynamic community of microorganisms is attached to the mucosal surface of the fish gut. Gut microbes play important roles in fish metabolism and immunity. Edwardsiella piscicida infection of turbot causes Edwardsiellosis, and vaccination is an effective method to prevent and control aquaculture diseases. Here, intraperitoneal vaccination by E. piscicida 10Δ, a live attenuated vaccine candidate, significantly upregulated the expression of immune response-related genes in the turbot spleen and enhanced the serum antibody titers of IgM. 16S rRNA amplicon sequencing revealed that the vaccination of E. piscicida 10Δ slightly reduced the relative abundance of intestinal microbiota in turbot. Meanwhile, E. piscicida 10Δ potentially conferred the intestinal microbiota metabolic capacity associated with carbohydrate metabolism, while hypothetically impaired their metabolic capacity related to fatty acid degradation and amino acid metabolism. Collectively, E. piscicida 10Δ can effectively stimulate the host immune responses and has no apparent influence on the relative abundance of intestinal microbiota, which can thus be considered as a potentially safe live attenuated vaccine.

Exercise Reverses Immune-Related Genes in the Hippocampus of Multiple Sclerosis Patients.

Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory demyelinating lesions in the white matter of the central nervous system. Studies have shown that exercise is beneficial for multiple sclerosis (MS). However, the molecular basis is largely unknown. We integrated multiple blood and hippocampus transcriptome data from subjects with physical activity or MS. Transcription change associations between physical activity and MS were analyzed with bioinformatic methods including GSEA (Gene Set Enrichment Analysis) and GO (Gene Ontology) analysis. We find that exercise can specifically reverse immune-related genes in the hippocampus of MS patients, while this effect is not observable in blood. Moreover, many of these reversed genes encode immune-related receptors. Interestingly, higher levels of physical activity have more pronounced effects on the reversal of MS-related transcripts. The immune-response related genes or pathways in the hippocampus may be the targets of exercise in alleviating MS conditions, which may offer new therapeutic clues for MS.

Effect of Lincomycin and Butyrate Glycerides Supplementation on Performance, Blood Biochemical Constituents, Immune Response and Nutrient Absorption Related Gene Expression in Broilers

Abstract Over 35 days feeding trial, the effects of lincomycin (LM) and butyrate glycerides (BG) supplementation on the growth performance, blood biochemical changes, immune response, and nutrient absorption related gene expression in broiler chickens were evaluated. A total of 480 one- day-old broiler chicks (Ross 308) were allotted into 6 groups (8 replicates/group with 10 birds/replicate). Experimental groups were arranged as follows; control group (A) fed the basal diet (BD); B, C, and D groups were fed the BD supplemented with 350, 500 and 1000 g BG/ton of feed respectively. While group E was fed the BD with 350 + 500 + 1000 g BG/ton of feed during starter, grower, and finisher periods, respectively, and finally group F which received lincomycin (LM) antibiotic (100 g/ton) in overall periods. Broilers that received BG in group E (birds supplemented with BG at 350 + 500 + 1000 g/ton feed) and LM (F) supplements both gained more body weight and weight growth (P&lt;0.05). Lower feed intake and improved fcr were observed in all the experimental groups that received either BG or lm compared to control birds (P&lt;0.05). Lower serum total cholesterol, triglycerides, and glutamic oxaloacetic transaminase (GOT) (P&lt;0.05) were recorded in the lm or BG supplemented birds compared to the control. Higher immune response represented by enhanced antibody titer against H9N1, and newcastle disease (ND) and lower hepatic malondialdehyde (MDA) was obtained in groups D, E and F (P&lt;0.05) compared to the other groups. Higher gene expression of GSH-Px, Glut 2, PepT1 and FAS was obtained in groups D, E and F (P&lt;0.05) compared to the control. Supplemental BG or LM increased the unsaturated fatty acids (oleic, linoleic and linolenic acids) while lowered the palmitic acid contents in breast muscle (P&lt;0.05) compared to control. In conclusion, supplementing broilers’ diets with BG or lincomycin improved their growth performance, immune response and muscle fatty acid profile as well as modulatory role in lipid metabolism. Supplementing starter, grower, and finisher broiler diets with BG at 350 + 500 + 1000 g/ton each may be a useful substitute for lincomycin.

Repeated intratracheal instillation of whole-cigarette smoke condensate to assess lung damage in a rat model.

Cigarette smoke induces an inflammatory response in the lungs by recruiting inflammatory cells, leading to lung diseases such as lung cancer, chronic obstructive pulmonary disease, and pulmonary fibrosis. Existing inhalation exposure methods for assessing the adverse effects of cigarette smoke require expensive equipment and are labor-intensive. Therefore, we attempted to develop a novel method to assess these adverse effects using intratracheal instillation (ITI) of whole cigarette smoke condensate (WCSC). The WCSC (0, 5, 10, or 20 mg/mL) was administered by ITI once daily for 6 or 12 days using an automatic video instillator. Repeated WCSC ITI increased the lung weight, and monocyte chemoattractant protein-1 (MCP-1), neutrophil, and lymphocyte levels within bronchoalveolar lavage fluid compared to the control. In the histopathological analysis of the lung tissue, a mild inflammatory response was observed in the 6 and 12 days 20 mg/mL WCSC exposure groups. The genome-wide RNA-seq expression patterns revealed that inflammatory and immune response-related genes, such as the chemokine signaling pathway, Th1/Th2 cell differentiation, and cytokine-cytokine receptor interaction, were employed following WCSC exposure. In addition, MCP-1 was time-dependent and increased in the 10 mg/mL exposure group compared to the control group. These results suggested that the WCSC might induce the potential pulmonary inflammatory response. Furthermore, we proposed that ITI may be a rapid and effective method of evaluating the adverse effects of WCSC within a short exposure period (less than 2 weeks), and it can be used to evaluate cigarette inhalation toxicity studies as an alternative method.

Vitamin D3 activates the innate immune response and xenophagy against Nocardia seriolae through the VD receptor in liver of largemouth bass (Micropterus salmoides)

Nocardia seriolae is a pathogenic bacterium that commonly infects largemouth bass (Micropterus salmoides), causing chronic wasting disease characterized by multiple nodules. The mechanism by which Vitamin D3 (VD3) can resist Nocardia seriolae (N. seriolae)infection is unknown, although VD3 has been found to regulate innate immune response and xenophagy in mammals. Therefore, we investigated the effects of VD3 on the immune response and xenophagy of largemouth bass after N. seriolae challenge. The results demonstrate that increasing dietary VD3 intake can alleviate focal necrosis of liver cells, increase mRNA and protein levels of innate immune response-related genes, such as cGAS, STING1, TBK1, IRF3, and IFNα, decrease the content of liver autophagosomes, and increase the content of liver autophagolysosomes. Additionally, VD3 can increase mRNA expression and protein levels of genes related to liver autophagosome-lysosome fusion, including ATG16L1, ATPV0Ca, ATPV0Cb, ATPV1D, LAMP2, STX17, and VAMP8, as well as VDR. These findings suggest that VD3 contributes to the resistance of largemouth bass to N. seriolae infection by activating innate immune response and xenophagy through VDR-mediated immunomodulation.

Effects of dietary lipid levels on lipid accumulation and health status of adult Onychostomamacrolepis

The effects of different dietary lipid levels on lipid accumulation, inflammatory response, serum bio-chemical index and histological features of intestine and hepatopancreas of O.macrolepis was experimentally evaluated in an eight-weeks study. Fish (initial weight 50.11 ± 2.86 g) were fed with five iso-nitrogenous diets (around 390 g/kg protein) varying with lipid level (5%, 7%, 9%, 11%, 13%, being LL5, LL7, LL9, LL11, and LL13 respectively) in triplicates. Results showed that the content of crude lipid in carcass and hepatopancreas were not affected by dietary lipid levels (P > 0.05). Serum ALT, TP, HDL-c and MDA, etc., were not significantly affected by diets (P > 0.05), while serum total antioxidant capacity in LL9 and LL11 groups were significantly higher than the other groups (P < 0.05). Histological features of hepatopancreas and intestine showed no significant difference among the five diets (P > 0.05), while the height of intestine villus showed the higher trend in LL9 compared with other groups. The relative expression of lipid metabolism related genes (ppar α, cpt-1α, fas, and hsl) and immune response related genes (tlr 22, nrf 2, tnfα, and il-γ) in the hepatopancreas of fish fed diets differing in their crude lipid levels were not significantly different (P > 0.05). The results suggested that a proper dietary lipid level of 9%–11% could maintain higher antioxidant and health status of adult O.macrolepis.

Optic Nerve Head Gene Transcription Sequelae to a Single Elevated IOP Exposure Provides Insights Into Known Responses to Chronically Elevated IOP.

To clarify the optic nerve head (ONH) gene expression responses associated with a single, axon-damaging exposure to elevated IOP in relation to the composite cellular events previously identified in models of chronically elevated IOP. Anesthetized rats were exposed unilaterally to an 8-hour pulse-train controlled elevation of IOP (PT-CEI) at 60mm Hg, while others received normotensive CEI at 20mm Hg. ONH RNA was harvested at 0hours and 1, 2, 3, 7, and 10 days after either CEI and from naïve animals. RNA sequencing was performed to analyze ONH gene expression. DAVID Bioinformatics tools were used to identify significant functional annotation clusters. Gene function was compared between PT-CEI and two models of chronic ocular hypertension from the literature. The number of significantly changed genes peaked immediately (n = 1354) after PT-CEI (0hours). This was followed by a lull (<4 genes per time point) at 1 and 2days after PT-CEI. Gene activity increased again at 3 days (136 genes) and persisted at 7 (78 genes) and 10 (339 genes) days. Significant gene functional categories included an immediate upregulation of Defense Response at 0hours, followed by upregulation in Cell Cycle, a reduction in Axonal-related genes at 3 to 10 days, and upregulation of Immune Response-related genes at 10 days following PT-CEI. The most commonly upregulated gene expression across our PT-CEI study and two chronic models of ocular hypertension were cell cycle related. The PT-CEI model places in sequence ONH gene expression responses previously reported in models with chronically elevated IOP and may provide insights into their role in optic nerve damage.

Fructo-oligosaccharide-mediated alteration in claudin expression in small intestinal absorptive Caco-2 cells is positively associated with the induction of inflammatory genes and the glucan receptor gene CLEC7A

ObjectivesIndigestible carbohydrates may strengthen tight junctions (TJs) independently of intestinal bacteria. This study investigated whether indigestible carbohydrates (i.e., fructo-oligosaccharides [FOS]) promote TJs directly to intestinal absorptive Caco-2 cells and examined the association between the expression of genes constructing TJs and other genes using mRNA microarray analysis. MethodsCaco-2 cells at 1.0 × 105/mL were seeded in a type I collagen plate and cultured in high-glucose Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS); the cells reached confluence at 7 d after seeding. Ten days after the cells reached confluency, they were cultured for 24 h in 10% FCS-containing DMEM medium supplemented with 0%, 5%, or 10% FOS. We performed mRNA microarray to identify the genes whose expression was altered by FOS. Subsequently, quantitative reverse transcription polymerase chain reaction was performed for these altered genes, including CLEC7A encoding the glucan receptor, and for the claudin (CLDN) family genes. The expression of CLDN2, CLDN4, and CLEC7A proteins was assessed using western blot analysis. ResultsFOS decreased the mRNA and protein expression of CLDN2, which weakens TJs, and increased those of CLDN4, which strengthens TJs, in Caco-2 cells. FOS treatment (10%) reduced the mRNA expression of antioxidative genes and induced the expression of immune response–related genes, including CLEC7A, CCL2, and ITGA2. Furthermore, the expression of CLEC7A protein was enhanced by FOS. ConclusionsInduction of TJ-strengthening CLDN4 and reduction of TJ-weakening CLDN2 by FOS treatment in small intestinal absorptive Caco-2 cells is positively associated with the induction of inflammatory genes, including CLEC7A.

Integrated transcriptomics reveals the brain and blood biomarkers in Alzheimer's disease.

The systematic molecular associations between the peripheral blood cells and brain in Alzheimer's disease (AD) remains unclear, which hinders our understanding of AD pathological mechanisms and the exploration of new diagnostic biomarkers. Here, we performed an integrated analysis of the brain and peripheral blood cells transcriptomics to establish peripheral biomarkers of AD. By employing multiple statistical analyses plus machine learning, we identified and validated multiple regulated central and peripheral network in patients with AD. By bioinformatics analysis, a total of 243 genes were differentially expressed in the central and peripheral systems, mainly enriched in three modules: immune response, glucose metabolism and lysosome. In addition, lysosome related gene ATP6V1E1 and immune response related genes (IL2RG, OSM, EVI2B TNFRSF1A, CXCR4, STAT5A) were significantly correlated with Aβ or Tau pathology. Finally, receiver operating characteristic (ROC) analysis revealed that ATP6V1E1 showed high-diagnostic potential for AD. Taken together, our data identified the main pathological pathways in AD progression, particularly the systemic dysregulation of the immune response, and provided peripheral biomarkers for AD diagnosis.

Abstract B031: Investigation of the ubiquitin ligase RNF31 as a mediator of immune evasion in advanced prostate cancer

Abstract Background: Metastatic Castration-Resistance Prostate cancer (mCRPC) is a disease state associated with poor survival. Current FDA-approved therapies are non-curative and can only extend mCRPC patient survival by a few months. Therefore, there is an unmet need to improve therapies for patients with advanced disease. Immunotherapy is an effective treatment strategy for a number of cancers but showed limited success in mCRPC patients. Prostate cancer cells exhibit reduced surface expression of major histocompatibility complex (MHC) molecules as an evasive mechanism from cytotoxic CD8+ T-cells. Natural killer (NK) cells are another class of immune cells with anti-tumor characteristics and can target cancer cells independently of MHC expression. Increased NK cell infiltration and activity in prostate cancer has been associated with favorable patient outcomes, but cancer cells have evolved resistance mechanisms against NK cell targeting. We hypothesize that there are druggable pathways that can be exploited to enhance anti-tumor activities of NK cells and improve efficacy of immunotherapies. Methods: To investigate these pathways, we established and characterized an in vitro co-culture system between prostate cancer and NK cells. We conducted the first targeted pool CRISPR screen identifying genes that, when knockout (KO) in prostate cancer cells, enhance killing by NK cells. We validated the screen by individually KO the gene hits. To explore the potential resistance mechanisms conferred by these hits, we performed RNA seq in control and KO cells. Results: Our screen nominated several known ligands of NK cells including CD58 and PVRL2, as well as RNF31 as the top novel target in promoting NK-mediated kill of cancer cells. RNF31 is upregulated in prostate cancer and is part of a druggable linear ubiquitin chain assembly complex (LUBAC). Single sgRNA validation experiments confirmed that RNF31 KO significantly sensitized LNCaP cells to NK92-mediated depletion. Importantly, RNF31 KO did not affect cancer cell proliferation, suggesting that the observed effects are due to the actions of NK cells. We observed that immune response related genes were upregulated in the RNF31 KO cells compare to the wildtype cells. Furthermore, pharmacologic inhibiting RNF31 via LUBAC inhibitor Hopin-8 sensitizes LNCaP cell to NK cell mediated kill. Conclusions: We anticipate that results from this study will yield insights into a novel tumor-intrinsic mechanism of immune evasion in prostate cancer and evaluate the therapeutic potential of targeting this pathway. We hope this work will help identify new therapeutic options or combination treatments that can improve the efficacy of immunotherapy in prostate cancer and significantly extend the lives of patients with advanced prostate cancer. Acknowledgements: This study was supported by the DOD Early Investigator Research Award to TL. Citation Format: Tianyi Liu, Haolong Li, Tony Junjie Hua, Hyunjin Shin, Tanushree Shenoy, Martin Sjoestroem, Arian Lundberg, Luke Gilbert, David Quigley, Alan Ashworth, Lewis Lanier, Felix Feng. Investigation of the ubiquitin ligase RNF31 as a mediator of immune evasion in advanced prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr B031.

Association of a high E2F targets score with survival in hepatocellular carcinoma patients.

4106 Background: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality. Multiple staging systems have all been proposed to date; however, the optimal tool for stratification of prognosis is still not universally accepted. E2F targets are the essential component of cell proliferation, thus we hypothesized that a score to quantify E2F activity is expected to reflect the aggressiveness and prognosis of HCC. Methods: Total of 655 HCC patients from TCGA (The Cancer Genome Atlas) -HCC ( n=358), GSE6764 ( n=75), GSE76427 ( n = 115), and GSE89377 ( n=107) cohorts were analyzed. The score was generated using MSigDb Hallmark E2F Targets gene set using Gene Set Variation Analysis. Results: The E2F targets scores of TGCA-HCC patients showed a bimodal distribution, thus high vs. low score groups were divided by median. As expected, high E2F targets enriched all of the Hallmark cell proliferation-related gene sets; E2F targets, G2M checkpoint, MYC targets v1 and v2, and Mitotic spindle. It also enriched gene sets known to aggravate cancer, such as Glycolysis, mTORC1 signaling, DNA repair, and Unfolded protein response. E2F targets were significantly associated with histological grade, tumor size, and stage, as well as proliferation score and MKI67 expression. Furthermore, a higher E2F targets score was significantly associated with higher intra-tumoral genomic heterogeneity and homologous recombination deficiency, suggesting greater tumor aggressiveness. In agreement, E2F targets score correlated with the pathological progression from normal liver, cirrhosis, dysplasia, to early and advanced HCC consistently in two cohorts (GSE6764 and GSE89377). Further, the abundance of hepatocytes, fibroblasts, adipocytes, and lymphatic endothelial cells were all significantly less in high E2F HCC, which reflects proliferative cancer. On the other hand, there was no relationship between E2F and mutation rates or neoantigens. High E2F was associated with high infiltration of anti-cancerous immune cells; CD8, CD4 memory, and Th1 cells, however, there was no difference in cytolytic activity, and it did not enrich any of immune response-related gene sets. High E2F HCC was associated with worse disease-free (DFS), disease-specific (DSS), and overall survival (OS), and this was the case in both early (I and II) and late (III and IV) stages. A multivariate analysis revealed that the E2F targets score was an independent prognostic factor for OS (HR=1.68, 95%CI= 1.15–2.46, p = 0.007) as well as DSS (HR=1.81, 95%CI=1.27–2.59, p = 0.001) in patients with HCC. Conclusions: We found that the E2F targets score not only indicates cell proliferation, but also is associated with cancer aggressiveness and worse survival. Our findings suggest a possible future use of the E2F targets score as a prognostic biomarker in patients with HCC.

Granular analysis of individual immune-related gene expression in a randomized phase I/II study of the oncolytic adenovirus, ONCOS-102, in combination with pemetrexed/cisplatin (P/C) in patients (pts) with unresectable malignant pleural mesothelioma (MPM).

e20536 Background: ONCOS-102 is an oncolytic adenovirus expressing granulocyte-macrophage colony stimulating factor (GM-CSF; Ad5/3-D24-GMCSF) that stimulates immune responses and modulates the tumor microenvironment, both as a monotherapy and in combination with programmed cell death protein 1 (PD-1) blockade. We present granular analyses of individual immune-related gene expression in response to ONCOS-102 in a randomized phase I/II study in MPM. Methods: Following a safety run-in (n = 6), 25 pts were randomized to receive ONCOS-102 (3 x 1011 virus particles in 2.5 mL) intratumorally under CT or US guidance on Days 1, 4, 8, 36, 78 and 120, plus six cycles of P/C starting on Day 22 (ONCOS+P/C), or six cycles of P/C only (control). Both treatment-naïve (1L) and previously treated patients were enrolled. Survival was followed up to 30 months. Tumor biopsies were collected at baseline and Day 36, and immunofluorescence for immune cell subsets, RNASeq and qPCR were performed. Results: In the pooled population (n = 31), median overall survival (OS) was 16.6 vs 18.3 months for ONCOS-102 + P/C vs control arms ( P&gt; 0.05). In 1L patients (n = 17) median OS was 20.3 vs 13.5 months ( P&gt; 0.05), and 30-month OS rate was 34.1% vs 0. A significant increase in tumour-infiltrating immune cells was observed (baseline to Day 36) with ONCOS-102 + P/C (n = 20) but not control (n = 11) using immunofluorescence and transcriptome analysis. CD8+, GranzymeB+ CD8+ cells, and CD8+:regulatory T-cell and M1:M2 macrophage ratios increased in ONCOS-102-treated pts (P≤0.016). A difference in tumor immune-infiltration was observed in ONCOS-102 + P/C pts stratified by survival at Month 18: while deregulation of complement pathways and inflammatory response was seen at baseline, at Day 36 adaptive immune response and T-cell activation were observed in pts who were alive but not in those deceased. Additionally, GM-CSF protein, likely derived from ONCOS-102, was transiently present in higher concentration at Days 4 and 8 in 18-month survivors vs deceased pts. Based on a previously described predictive inflammatory gene signature for treatment with checkpoint inhibitors in mesothelioma, a binomial model assessing baseline transcriptome levels demonstrated 89% accuracy in predicting 18-month survival. Conclusions: ONCOS-102 caused increased T-cell infiltration and upregulated immune response-related gene expression in tumor lesions. A trend for prolonged OS was observed in the 1L setting, only. Consistent with observations in PD-1 resistant melanoma, ONCOS-102-induced immune response in the tumor is hallmarked by a high prevalence of cytotoxic T-cells and depolarization of macrophages, which may support addition of checkpoint inhibitors to the combination of ONCOS-102 plus P/C in MPM. Clinical trial information: NCT02879669 .

Vorinostat-induced acetylation of RUNX3 reshapes transcriptional profile through long-range enhancer-promoter interactions in natural killer cells

Natural killer (NK) cells are an essential part of the innate immune system that helps control infections and tumors. Recent studies have shown that Vorinostat, a histone deacetylase (HDAC) inhibitor, can cause significant changes in gene expression and signaling pathways in NK cells. Since gene expression in eukaryotic cells is closely linked to the complex three-dimensional (3D) chromatin architecture, an integrative analysis of the transcriptome, histone profiling, chromatin accessibility, and 3D genome organization is needed to gain a more comprehensive understanding of how Vorinostat impacts transcription regulation of NK cells from a chromatin-based perspective. The results demonstrate that Vorinostat treatment reprograms the enhancer landscapes of the human NK-92 NK cell line while overall 3D genome organization remains largely stable. Moreover, we identified that the Vorinostat-induced RUNX3 acetylation is linked to the increased enhancer activity, leading to elevated expression of immune response-related genes via long-range enhancerpromoter chromatin interactions. In summary, these findings have important implications in the development of new therapies for cancer and immune-related diseases by shedding light on the mechanisms underlying Vorinostat's impact on transcriptional regulation in NK cells within the context of 3D enhancer network.