Tumor Necrosis Factor-related Apoptosis-inducing Ligand Research Articles

Microplastics have additive effects on cadmium accumulation and toxicity in Rice flower carp (Procypris merus)

Procypris merus, a local fish species found in Guangxi, China is often exposed to both microplastics (MPs) and Cd. However, it remains unclear how these two pollutants affect P. merus. Therefore, we investigated the effects of MPs on Cd accumulation in P. merus. To this end, P. merus was separately exposed to Cd and MPs (500 μg/L) or their combination for 14 days. We found that MPs enhanced Cd accumulation in liver and gills of P. merus. Further, both the single-contaminant (MP and Cd) and combined treatments resulted in lesions in these two tissues, with more severe damage associated with the combined treatment. Even though the effect of MP on the antioxidant defense system of P. merus was limited, the Cd-only and combined treatments considerably affected the antioxidant parameters of P. merus, with the combined treatment showing a stronger effect. GO and KEGG analyses revealed that the differentially expressed genes (DEGs; TNF-related apoptosis-inducing ligand receptor, trail-r) in the Cd-only treatment group were enriched for immune-related GO terms and cell growth and death related pathways, indicating that Cd toxicity affected immune defense in P. merus. The MP-only treatment downregulated DEGs (acyl-CoA synthetase long chain family member 1a, acsl1a) related to lipid metabolism, possibly leading to lipid accumulation in the liver. The combined treatment also upregulated DEGs (aspartate aminotransferase 1, ast 1) associated with immune-related GO terms and amino acid metabolism pathways, suggesting that it affected immune function in P. merus, thereby negatively impacting its health. Results indicated that MPs have additive effects on Cd accumulation and toxicity in rice flower carp. Consequently, MPs ingested by P. merus can promote Cd accumulation, more adverse effects on the health may occur after combined exposure, which can eventually reach humans through the food chain and pose potential risks to human health.

Targeted treatment of chondrosarcoma with a bacteriophage-based particle delivering a secreted tumor necrosis factor-related apoptosis-inducing ligand

Chondrosarcoma is a malignant cartilage-forming bone tumor which is inherently resistant to chemotherapy and radiotherapy leaving surgery as the only treatment option. We have designed a tumor-targeted bacteriophage (phage)-derived particle (PDP), for targeted systemic delivery of cytokine-encoding transgenes to solid tumors. Phage has no intrinsic tropism for mammalian cells; therefore, it was engineered to display a double cyclic RGD4C ligand on the capsid to target tumors. To induce cancer cell death, we constructed a transgene cassette expressing a secreted form of the cytokine tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). We detected high expression of αvβ3 and αvβ5 integrin receptors of the RGD4C ligand, and of the TRAIL receptor-2 in human chondrosarcoma cells (SW1353) but not in primary normal chondrocytes. The RGD4C.PDP-Luc particle carrying a luciferase reporter gene, Luc, effectively and selectively mediated gene delivery to SW1353 cells but not primary chondrocytes. Transduction of SW1353 cells with RGD4C.PDP-sTRAIL encoding a human sTRAIL, resulted in expression of TRAIL and subsequent cell dead without harming the normal chondrocytes. Intravenous administration of RGD4C.PDP-sTRAIL to mice with established human chondrosarcoma resulted in reduction of tumor size and tumor viability. Altogether, RGD4C.PDP-sTRAIL can be used to target systemic treatment of chondrosarcoma with the sTRAIL.

Spatial distribution of tumor-associated macrophages in an orthotopic prostate cancer mouse model.

Mounting evidence suggests that the immune landscape within prostate tumors influences progression, metastasis, treatment response, and patient outcomes. In this study, we investigated the spatial density of innate immune cell populations within NOD.SCID orthotopic prostate cancer xenografts following microinjection of human DU145 prostate cancer cells. Our laboratory has previously developed nanoscale liposomes that attach to leukocytes via conjugated E-selectin (ES) and kill cancer cells via TNF-related apoptosis inducing ligand (TRAIL). Immunohistochemistry (IHC) staining was performed on tumor samples to identify and quantify leukocyte infiltration for different periods of tumor growth and E-selectin/TRAIL (EST) liposome treatments. We examined the spatial-temporal dynamics of three different immune cell types infiltrating tumors using QuPath image analysis software. IHC staining revealed that F4/80+ tumor-associated macrophages (TAMs) were the most abundant immune cells in all groups, irrespective of time or treatment. The density of TAMs decreased over the course of tumor growth and decreased in response to EST liposome treatments. Intratumoral versus marginal analysis showed a greater presence of TAMs in the marginal regions at 3 weeks of tumor growth which became more evenly distributed over time and in tumors treated with EST liposomes. TUNEL staining indicated that EST liposomes significantly increased cell apoptosis in treated tumors. Additionally, confocal microscopy identified liposome-coated TAMs in both the core and periphery of tumors, highlighting the ability of liposomes to infiltrate tumors by "piggybacking" on macrophages. The results of this study indicate that TAMs represent the majority of innate immune cells within NOD.SCID orthotopic prostate tumors, and spatial density varies widely as a function of tumor size, duration of tumor growth, and treatment of EST liposomes.

Effect of coffee, tea and alcohol intake on circulating inflammatory cytokines: a two sample-Mendelian randomization study.

Despite the abundance of research examining the effects of coffee, tea, and alcohol on inflammatory diseases, there is a notable absence of conclusive evidence regarding their direct causal influence on circulating inflammatory cytokines. Previous studies have primarily concentrated on established cytokines, neglecting the potential impact of beverage consumption on lesser-studied but equally important cytokines. Information regarding the consumption of coffee, tea, and alcohol was collected from the UK Biobank, with sample sizes of 428,860, 447,485, and 462,346 individuals, respectively. Data on 41 inflammatory cytokines were obtained from summary statistics of 8293 healthy participants from Finnish cohorts. The consumption of coffee was found to be potentially associated with decreased levels of Macrophage colony-stimulating factor (β = -0.57, 95% CI -1.06 ~ -0.08; p = 0.022) and Stem cell growth factor beta (β = -0.64, 95% CI -1.16 ~ -0.12; p = 0.016), as well as an increase in TNF-related apoptosis-inducing ligand (β = 0.43, 95% CI 0.06 ~ 0.8; p = 0.023) levels. Conversely, tea intake was potentially correlated with a reduction in Interleukin-8 (β = -0.45, 95% CI -0.9 ~ 0; p = 0.045) levels. Moreover, our results indicated an association between alcohol consumption and decreased levels of Regulated on Activation, Normal T Cell Expressed and Secreted (β = -0.24, 95% CI -0.48 ~ 0; p = 0.047), as well as an increase in Stem cell factor (β = 0.17, 95% CI 0.02 ~ 0.31; p = 0.023) and Stromal cell-derived factor-1 alpha (β = 0.20, 95% CI 0.04 ~ 0.36; p = 0.013). Revealing the interactions between beverage consumption and various inflammatory cytokines may lead to the discovery of novel therapeutic targets, thereby facilitating dietary interventions to complement clinical disease treatments.

Mechanisms underlying reversed TRAIL sensitivity in acquired bortezomib-resistant non-small cell lung cancer cells.

Aim: The therapeutic targeting of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) death receptors in cancer, including non-small cell lung cancer (NSCLC), is a widely studied approach for tumor selective apoptotic cell death therapy. However, apoptosis resistance is often encountered. The main aim of this study was to investigate the apoptotic mechanism underlying TRAIL sensitivity in three bortezomib (BTZ)-resistant NSCLC variants, combining induction of both the intrinsic and extrinsic pathways. Methods: Sensitivity to TRAIL in BTZ-resistant variants was determined using a tetrazolium (MTT) and a clonogenic assay. A RT-qPCR profiling mRNA array was used to determine apoptosis pathway-specific gene expression. The expression of these proteins was determined through ELISA assays and western Blotting, while apoptosis (sub-G1) and cytokine expression were determined using flow cytometry. Apoptotic genes were silenced by specific siRNAs. Lipid rafts were isolated with fractional ultracentrifugation. Results: A549BTZR (BTZ-resistant) cells were sensitive to TRAIL in contrast to parental A549 cells, which are resistant to TRAIL. TRAIL-sensitive H460 cells remained equally sensitive for TRAIL as H460BTZR. In A549BTZR cells, we identified an increased mRNA expression of TNFRSF11B [osteoprotegerin (OPG)] and caspase-1, -4 and -5 mRNAs involved in cytokine activation and immunogenic cell death. Although the OPG, interleukin-6 (IL-6), and interleukin-8 (IL-8) protein levels were markedly enhanced (122-, 103-, and 11-fold, respectively) in the A549BTZR cells, this was not sufficient to trigger TRAIL-induced apoptosis in the parental A549 cells. Regarding the extrinsic apoptotic pathway, the A549BTZR cells showed TRAIL-R1-dependent TRAIL sensitivity. The shift of TRAIL-R1 from non-lipid into lipid rafts enhanced TRAIL-induced apoptosis. In the intrinsic apoptotic pathway, a strong increase in the mRNA and protein levels of the anti-apoptotic myeloid leukemia cell differentiation protein (Mcl-1) and B-cell leukemia/lymphoma 2 (Bcl-2) was found, whereas the B-cell lymphoma-extra large (Bcl-xL) expression was reduced. However, the stable overexpression of Bcl-xL in the A549BTZR cells did not reverse the TRAIL sensitivity in the A549BTZR cells, but silencing of the BH3 Interacting Domain Death Agonist (BID) protein demonstrated the importance of the intrinsic apoptotic pathway, regardless of Bcl-xL. Conclusion: In summary, increased sensitivity to TRAIL-R1 seems predominantly related to the relocalization into lipid rafts and increased extrinsic and intrinsic apoptotic pathways.

Polyphyllin I enhances tumor necrosis factor-related apoptosis-inducing ligand-induced inhibition of human osteosarcoma cell growth downregulating the Wnt/β-catenin pathway.

To investigate the synergistic effects of polyphyllin I (PPI) combined with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on the growth of osteosarcoma cells through downregulating the Wnt/β-catenin signaling pathway. Cell viability, apoptosis and cell cycle distribution were examined using cell counting kit-8 and flow cytometry assays. The morphology of cancer cells was observed with inverted phase contrast microscope. The migration and invasion abilities were examined by xCELLigence real time cell analysis DP system and transwell assays. The expressions of poly (adenosine diphosphate-ribose) polymerase, C-Myc, Cyclin B1, cyclin-dependent kinases 1, N-cadherin, Vimentin, Active-β-catenin, β-catenin, p-glycogen synthase kinase 3β (GSK-3β) and GSK-3β were determined by Western blotting assay. PPI sensitized TRAIL-induced decrease of viability, migration and invasion, as well as increase of apoptosis and cell cycle arrest of MG-63 and U-2 OS osteosarcoma cells. The synergistic effect of PPI with TRAIL in inhibiting the growth of osteosarcoma cells was at least partially realized through the inactivation of Wnt/β-catenin signaling pathway. The combination of PPI and TRAIL is potentially a novel treatment strategy of osteosarcoma.

Glutamine-mediated epigenetic regulation of cFLIP underlies resistance to TRAIL in pancreatic cancer

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent because it kills cancer cells while sparing normal cells. However, many cancers, including pancreatic ductal adenocarcinoma (PDAC), exhibit intrinsic or acquired resistance to TRAIL, and the molecular mechanisms underlying TRAIL resistance in cancers, particularly in PDAC, remain unclear. In this study, we demonstrated that glutamine (Gln) endows PDAC cells with resistance to TRAIL through KDM4C-mediated epigenetic regulation of cFLIP. Inhibition of glutaminolysis significantly reduced the cFLIP level, leading to TRAIL-mediated formation of death-inducing signaling complexes. Overexpression of cFLIP dramatically rescued PDAC cells from TRAIL/Gln deprivation-induced apoptosis. Alpha-Ketoglutarate (aKG) supplementation significantly reversed the decrease in the cFLIP level induced by glutaminolysis inhibition and rescued PDAC cells from TRAIL/Gln deprivation-induced apoptosis. Knockdown of glutamic-oxaloacetic transaminase 2, which facilitates the conversion of oxaloacetate and glutamate into aspartate and aKG, decreased aKG production and the cFLIP level and activated TRAIL-induced apoptosis. AKG-mediated epigenetic regulation was necessary for maintaining a high level of cFLIP. Glutaminolysis inhibition increased the abundance of H3K9me3 in the cFLIP promoter, indicating that Gln-derived aKG production is important for Jumonji-domain histone demethylase (JHDM)-mediated cFLIP regulation. The JHDM KDM4C regulated cFLIP expression by binding to its promoter, and KDM4C knockdown sensitized PDAC cells to TRAIL-induced apoptosis. The present findings suggest that Gln-derived aKG production is required for KDM4C-mediated epigenetic regulation of cFLIP, which leads to resistance to TRAIL.

In major dysmood disorder, physiosomatic, chronic fatigue and fibromyalgia symptoms are driven by immune activation and increased immune-associated neurotoxicity

Major depressive disorder (MDD) is accompanied by activated neuro-immune pathways, increased physiosomatic and chronic fatigue-fibromyalgia (FF) symptoms. The most severe MDD phenotype, namely major dysmood disorder (MDMD), is associated with adverse childhood experiences (ACEs) and negative life events (NLEs) which induce cytokines/chemokines/growth factors. To delineate the impact of ACE + NLEs on physiosomatic and FF symptoms in first episode (FE)-MDMD, and examine whether these effects are mediated by immune profiles. ACEs, NLEs, physiosomatic and FF symptoms, and 48 cytokines/chemokines/growth factors were measured in 64 FE-MDMD patients and 32 normal controls. Physiosomatic, FF and gastro-intestinal symptoms belong to the same factor as depression, anxiety, melancholia, and insomnia. The first factor extracted from these seven domains is labeled the physio-affective phenome of depression. A part (59.0%) of the variance in physiosomatic symptoms is explained by the independent effects of interleukin (IL)-16 and IL-8 (positively), CCL3 and IL-1 receptor antagonist (inversely correlated). A part (46.5%) of the variance in physiosomatic (59.0%) symptoms is explained by the independent effects of interleukin (IL)-16, TNF-related apoptosis-inducing ligand (TRAIL) (positively) and combined activities of negative immunoregulatory cytokines (inversely associated). Partial least squares analysis shows that ACE + NLEs exert a substantial influence on the physio-affective phenome which are partly mediated by an immune network composed of interleukin-16, CCL27, TRAIL, macrophage-colony stimulating factor, and stem cell growth factor. The physiosomatic and FF symptoms of FE-MDMD are partly caused by immune-associated neurotoxicity due to T helper (Th)-1 polarization and M1 macrophage activation and relative lowered compensatory immunoregulatory protection.

The Relationship Between Inflammatory Processes and Apoptosis in Lumbar Disc Degeneration

Degenerative Disc Disease (DDD) is a common health problem in the population. There are recent studies focusing on relationship between DDD and immunological factors. However, there is still a lack of data on the role of apoptosis in DDD pathophysiology. Therefore, we aimed to investigate the relationship between Modic-type changes and the apoptosis in DDD. Ninety adult male patients who presented with low back and/or radicular pain and were operated on due to lumbar disc herniation were included. Three groups were formed based on Modic type degeneration observed on magnetic resonance imaging. Specific parameters involved in the intrinsic and extrinsic pathways of apoptosis were assessed in excised disc materials using the enzyme-linked immunosorbent assay method. All three groups formed according to Modic degeneration types were homogenous in all variances. Cytochrome-C was significantly decreased only in the Modic type-3 group, whereas Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor-1, B-Cell Lymphoma-2 (Bcl-2) Homologous Antagonist Killer-1, Direct Inhibitor of Apoptosis-Binding Protein with Low Pi, and Bcl-2 Associated X Apoptosis Regulator levels were significantly different in both Modic type-2 and -3 groups. However, BH3 interacting domain death agonist and Bcl-2 levels were similar across all groups. In conclusion, this study suggests that Direct Inhibitor of Apoptosis-Binding Protein with Low Pi, cytochrome - c, Bcl-2 Associated X Apoptosis Regulator, Bcl-2 Homologous Antagonist Killer-1, and Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand Receptor-1proteins play important roles in the development and progression of DDD and are correlated with Modic types. Further studies are needed to explore the potential therapeutic role of inhibiting these apoptotic proteins in DDD.

Systemic Inflammatory Regulators Associated with Osteoporosis: A Bidirectional Mendelian Randomization Study.

To elucidate the precise upstream and downstream regulatory mechanisms of inflammatory factors in osteoporosis (OP) progression and to establish a causal relationship between inflammatory factors and OP. We conducted bidirectional Mendelian randomization (MR) analyses using data for 41 cytokines obtained from three independent cohorts comprising 8293 Finnish individuals. Estimated bone mineral density (eBMD) data were derived from 426,824 UK Biobank White British individuals (55% female) and fracture data from 416,795 UK Biobank participants of European ancestry. The inverse variance-weighted method was the primary MR analysis approach. We employed other methods as complementary approaches for mutual corroboration. To test for pleiotropy and heterogeneity, we used the MR-Egger regression, MR-pleiotropy residual sum and outlier global test, and the Cochrane Q test. Macrophage inflammatory protein (MIP)-1α and interleukin (IL)-12p70 expression associated negatively and causally with eBMD (β = -0.017 [MIP-1α], β = -0.011 [IL-12p70]). Conversely, tumor necrosis factor-related apoptosis-inducing ligand was associated with a decreased risk of fractures (Odds Ratio: 0.980). Additionally, OP influenced the expression of multiple inflammatory factors, including growth-regulated oncogene-α, interferon-gamma, IL-6, beta nerve growth factor, and IL-2. Finally, we discovered complex bidirectional causal relationships between IL-8, IL-10, and OP. Specific inflammatory factors may contribute to OP development or may be causally affected by OP. We identified a bidirectional causal relationship between certain inflammatory factors and OP. These findings provide new perspectives for early prediction and targeted treatment of OP. Larger cohort studies are necessary in the future to further validate these findings.

Abstract 347: Tumor necrosis factor related apoptosis inducing ligand (TRAIL)-induced cytokine production in TNBC promotes neutrophil chemotaxis and immune suppression

Abstract Background: TRAIL induces apoptosis in many preclinical cancer models including breast cancers and has been extensively studied as a potential cancer therapeutic. However, its efficacy in clinical trials is limited, suggesting an unknown modulatory mechanism responsible for lack of TRAIL activity in vivo. Here, we describe that TRAIL treatment elicits transcriptional changes in TNBC cells that alter the immune milieu. Method: We performed RNAseq of MDA-MB-231 cells treated with TRAIL for different time points, followed by validation with RT-PCR in various TNBC cells. RNAi, silencing key differentially regulated genes, along with RT-PCR, ELISA and CHIP assays, were used to validate RNAseq findings. Elucidation of the functional relevance of the outcome was supported both in vitro and in vivo by chemotaxis assay, cytotoxicity assay, RNAseq analysis of donor isolated neutrophils and intravital microscopy, CODEX analysis in TNBC xenografts from mice treated with the TRAIL respectively. Results: TRAIL treatment of the TNBC significantly induced expression of several cytokines, such as CXCLs 1, 2, 3, 8,11 and IL6, both in vitro and in vivo which are known to affect neutrophil function. Mechanistically, induction of these cytokines was predominantly mediated by death receptor 5 and caspase-8 protein, but not caspase-8 enzymatic activity. GSEA of the RNAseq data indicated that NFKB pathway was significantly enriched. Concordantly, we confirmed that both canonical NFKB1 and non-canonical NFKB2 pathways were activated by TRAIL in vitro and in vivo. However, the induction of the cytokine mRNAs was primarily dependent on the NFKB2 pathway. Neutrophils isolated from healthy human donors incubated with supernatants from TNBC cells in vitro indicated that TRAIL-induced CXCLs and IL6 significantly increased neutrophil chemotaxis. Additionally, CODEX analysis as well as intravital imaging confirmed that TRAIL treatment increases the number of neutrophils in the tumor. Preincubation of neutrophils with supernatants from TRAIL-treated TNBC significantly inhibited their cytotoxic effect against TNBCs. Further, transcriptome analysis of neutrophils incubated with either TRAIL or supernatant of TRAIL treated TNBC revealed significant enrichment of expression of inflammatory cytokine genes, immune modulating and immune checkpoint genes like PDL1. Functional studies with these neutrophils confirmed their suppressive effect on T cell function as well as the effect of TRAIL on decreased neutrophil apoptosis. Conclusion: Collectively, our study suggests the novel role of TRAIL-induced NFKB2-dependent cytokine production promoting neutrophil chemotaxis and immune suppression. This study implies that alterations in the innate immune system may modulate the effects of TRAIL on TNBC tumors. Citation Format: Manjari Kundu, Yoshimi Endo Greer, Lisa Ann Ridnour, David A Wink, Yeap S. Ng, Roberto Weigert, Stan Lipkowitz. Tumor necrosis factor related apoptosis inducing ligand (TRAIL)-induced cytokine production in TNBC promotes neutrophil chemotaxis and immune suppression [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 347.

Abstract 5173: TRAIL expression in tumor cells predicts and drives poor response to neoadjuvant immunochemotherapy in esophageal squamous cell carcinoma

Abstract Background: Neoadjuvant PD-1 blockade combined with chemotherapy has shown promising antitumor efficacy in the therapy of esophageal squamous cell carcinoma (ESCC). However, effective biomarkers and potential mechanisms of resistance to this treatment are still unclear. Methods: We prospectively collected pre- and post-neoadjuvant treatment ESCC tissues from 40 patients who received PD-1blockade (camrelizumab, 200mg) and chemotherapy (albumin-paclitaxel, 260mg/m2 and carboplatin, area under the curve = 5). Based on their pathological responses to neoadjuvant immunochemotherapy (NICT), patients were classified into “responder” and “non-responder” groups. All samples were subjected to bulk RNA-sequencing and whole-exome sequencing. Of note, samples from 14 patients in both groups were subjected to single cell RNA sequencing and TCR sequencing. We characterized and validated the changes in the immunological landscape of “responder” and “non-responder” groups using multi-omics sequencing data, multiplex immunofluorescence, in vitro and in vivo experiments. External validation cohorts of ESCC treated with NICT were analyzed to validate the predictive biomarkers for NICT. Results: We found that TNF-related apoptosis-inducing ligand (TRAIL) signaling was significantly higher in pre-treatment non-responders than in responders, and was inversely correlated with T cell proliferation and TCR expansion. TRAIL was mainly secreted by tumor cells and bound to its specific receptor DR5 in T cells, thereby inhibiting the activation of T cells. Functional co-culture experiments and T cell cytotoxicity assays revealed that TRAIL+ tumor cells mediated T cell suppression and caused CD8+ T cell dysfunction. Furthermore, TRAIL inhibition could improve the efficacy of anti-PD-1 in a xenografted mouse model. In bulk RNA-seq (n=40), TRAIL expression was associated with worse disease-free survival and overall survival, however, these results were not observed in the ESCC cohort treated with upfront surgery from the TCGA database, suggesting that the predictive value of TRAIL is specific to NICT. In an external validation cohort of ESCC patients treated with NICT (n=81), multivariable Cox regression analysis showed that high TRAIL expression was an independent risk factor for unfavorable outcomes. Importantly, TRAIL expression exhibited a higher accuracy in predicting NICT response compared with PD-L1 expression in ESCC (AUC of ROC curves: 0.886 vs 0.607, respectively P<0.001). Meanwhile, the serum concentrations of TRAIL (measured by ELISA) in pre-NICT ESCC patients were also significantly higher in non-responders (n=58). Conclusions: TRAIL expression in tumor cells is negatively correlated with the response of ESCC to NICT. TRAIL expression may serve as a promising biomarker to predict NICT response and guide NICT selection in ESCC patients. Citation Format: Weixiong Yang, Fang Wang, Jianping Guo, Junchao Cai, Zekang Wang, Yao Liu, Zengli Fang, Wenfang Chen, Lixia Xu, Shuishen Zhang, Bo Zeng, Zhenguo Liu, Sui Peng, Sai-Ching Jim Yeung, Chao Cheng. TRAIL expression in tumor cells predicts and drives poor response to neoadjuvant immunochemotherapy in esophageal squamous cell carcinoma [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 5173.

Abstract 4780: TRAIL-inducing imipridone ONC201/TIC10 demonstrates anti-neoplastic effects in colonic adenoma-derived organoids

Abstract Background: Colorectal cancer (CRC) is the second leading cause of cancer-related death inthe United States and screening is the most effective method to reduce mortality. Chemoprevention, which aims to prevent CRC through eradication or prevention of precursor colonic adenomas, represents an alternative strategy to address this problem. The imipridone dordaviprone (ONC201) has been shown to induce apoptosis in cancer cells through both upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and the integrated stress response. More recently, we have shown that ONC201 induces TRAIL and apoptosis in a dose-dependent manner and reduces adenoma formation in the Apcmin/+ CRC mouse model. We hypothesized that ONC201 would similarly enhance human colonic adenoma cell death through both TRAIL-dependent and TRAIL-independent mechanisms. We therefore evaluated the effects of ONC201 in two adenoma-derived organoid lines: one developed from an adenoma obtained from an individual with familial adenomatous polyposis (FAP) and the second from a sessile serrated adenoma (SSA). Methods: Sensitivity of the FAP and SSA organoid lines to ONC201 was assessed using CellTiter-Glo® luminescent cell viability assay. Organoids were plated in triplicates and treated with ONC201 (concentration ranging from 0 uM to 20 uM). Results were analyzed after 72 hours of incubation. Western blotting was performed on lysates generated from organoids treated with either 1.69 uM DMSO, 1.69 uM ONC201, or 3.38 uM ONC201 for FAP and 1.23 uM DMSO, 1.23 uM ONC201, or 2.46 uM ONC201 for SSA for 0, 24, or 48 hours. Co-culture experiments were performed with the adenoma-derived organoids (dyed using blue CMAC dye) and human NK-92MI cells (dyed with green CMFDA dye) treated with either 845 nM DMSO, 845 nM ONC201, or 1.69 uM ONC201 for FAP and 615 nM DMSO, 615 nM ONC201, or 1.23 uM ONC201 for SSA. Fluorescent images following administration of red live/dead dye were performed using DAPI, FITC, and cherry red channels on ImageXpress® at 0, 24, 48, and 72hours. Results: ONC201 demonstrated antineoplastic effects in adenoma-derived FAP and SSA organoids. Half maximal inhibitory concentrations (IC50) of ONC201 in FAP and SSA organoids were 1.69 uM and 1.23 uM, respectively. As was noted in rodent studies, the mechanism of action of ONC201 appears to be mediated through modulation of multiple pathways including the TRAIL pathway (TRAIL and DR5 were both upregulated) and the integrated stress response (ATF4 was upregulated). Co-culture with NK cells revealed an increase in NK-mediated organoid cell death. The ability of ONC201 to enhance NK-mediated killing is still being evaluated. Further analysis of the effects of ONC201 on stemness markers and on T-cell-mediated organoid cell killing are currently underway and may reveal other potential biomarkers and chemopreventive strategies for ONC201. Citation Format: Alexis J. Lannigan, Jasper Chan, Maximilian Schwermann, Lanlan Zhou, Varun V. Prabhu, Michael Dame, Dean Brenner, Wafik S. El-Deiry, Alexander G. Raufi. TRAIL-inducing imipridone ONC201/TIC10 demonstrates anti-neoplastic effects in colonic adenoma-derived organoids [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 4780.

Abstract 3036: Sensitivity of ONC201/TIC10 cancer therapeutic on neuroendocrine prostate cancer (NEPC) and neuroendocrine differentiation (NED)

Abstract Although androgen deprivation therapy and androgen receptor (AR) blockade are highly effective therapies for metastatic prostate cancer (PCa), most patients develop resistance, leading to castration-resistant prostate cancer (CRPC). A subset of CRPC shows neuroendocrine prostate cancer (NEPC) features, which are associated with poor prognosis and limited therapeutic strategies. NEPC is characterized by oncogenic driver activation and epigenetic changes, partly controlled by transcription factors like BRN2 and SOX2. AR has been shown to suppress BRN2 transcription, which is required for NEPC, and BRN2-dependent regulation of the NEPC marker SOX2 (Bishop et al., 2017). Dordaviprone (ONC201/TIC10) is a first-in-class small molecule that antagonizes DRD2, induces tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and activates the integrated stress response (ISR). Neuroendocrine tumors express significant DRD2 levels, suggesting ONC201 as a potential therapeutic strategy in NEPC or when drivers for disease progression are overexpressed (OE). PCa cell lines PC3, DU145, LNCaP, and 22RV1 are ONC201 sensitive, with IC50=2.87 μM, 3.18 μM, 1.31 μM, and 1.16 μM, respectively (Ding et al., 2023). To characterize the association of BRN2/SOX2 dysregulation with ONC201 sensitivity, we transiently OE BRN2/SOX2 in PCa. We hypothesized that markers of neuroendocrine differentiation (NED), activation of the ISR, TRAIL, and ClpP and dopamine receptor expression contribute to NEPC cell death and sensitivity to ONC201. We performed CellTiter-Glo experiments to assess viability after 96 hours ± BRN2 OE. DU145 cells with BRN2 OE increased their sensitivity to ONC201 (IC50=2.75 µM) compared to empty vector (IC50=3.03 µM). Colony formation assays were conducted in PCa and analyzed 7 days after ONC201 treatment to show ONC201 effect on PCa cell lines. DU145 was treated at 0.5, 1, and 2 µM of ONC201. There were 95 ± 3.22 colonies for the control group, 83 ± 5.22 colonies with 0.5 µM, 74 ± 3.64 colonies with 1 µM, and 8 ± 2.03 colonies with 2 µM ONC201. DU145 showed a significant reduction in colony-forming ability when treated with 1 and 2 uM ONC201 compared to the control, with P values of <0.05 and <0.0001, respectively. We OE BRN2 and observed changes in NED markers and ISR pathway. In PC3, FoxO1 and ATF4 levels increased. In DU145, FoxO1, ENO2, PGP9.5, CgA, and ATF4 levels decreased. We reveal that DU145 OE at 48 hours does not result in greater expression of NEPC markers. BRN2 OE significantly increased PCa cell sensitivity to ONC201 and was evident without an increase in common phenotypical markers used for NEPC assessment (FoxO1, ENO2, PGP9.5, CgA). Expression change was not shown when PCa had an integral AR pathway. Ongoing efforts are observing ONC201 treatment effects on NED markers and the TRAIL pathway due to BRN2 OE and showing increased sensitivity of PCa to ONC201 with BRN2 OE. Citation Format: Elizabeth C. Ding, Maximilian Pinho-Schwermann, Shengliang Zhang, Connor Purcell, Wafik S. El-Deiry. Sensitivity of ONC201/TIC10 cancer therapeutic on neuroendocrine prostate cancer (NEPC) and neuroendocrine differentiation (NED) [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 3036.

Abstract 5675: An intronic SINE-VNTR-Alu element insertion in CASP8 alters gene expression and confers resistance to induction of cell death in gastrointestinal cancer

Abstract Transposable elements are a major source of variation in the human genome. SINE-VNTR-Alus (SVAs), the hominid-specific and youngest active retrotransposon family, are highly polymorphic within the human germline and can contribute to disease risk and progression. In fact, polymorphic SVAs have previously been linked to several human diseases, including rare cases of hereditary cancer syndromes such as neurofibromatosis type I and Lynch syndrome. These examples illustrate that the mobility of SVAs in the genome can significantly impact gene function. Here, we sought to evaluate the potential of SVAs inserting into known tumor suppressors and oncogenes. To this end, we surveyed genomic data from Pan-Cancer Analysis of Whole Genomes (PCAWG) and identified 1956 unique germline SVA insertions in the introns of 1933 genes. Among these, we discovered a highly prevalent polymorphic SVA_E (subfamily) in an intronic region of CASP8. Given caspase-8’s canonical role in apoptosis, we hypothesized that this SVA may impact both caspase-8 expression as well as cell death in cancer. We first created a panel of colon and gastric cancer cell lines genotyped by PCR for the presence or absence of the CASP8-SVA (SVA+/+: RKO, NCI-N87, HT29, NCI-H747, OCUM-1; SVA-/-: HCT116, LoVo, SNU-407, 23132/87, KM12). RT-qPCR analysis showed significantly higher CASP8 expression in SVA+/+ cell lines, which was concordant with caspase-8 protein expression by immunoblot. Treatment of these cell lines with cytotoxic chemotherapy (FOLFOXIRI) in vitro demonstrated increased resistance to cell death in SVA+/+ cell lines. Direct induction of caspase-8-mediated apoptosis via TRAIL (TNF-related apoptosis-inducing ligand) revealed decreased TRAIL-mediated cell death in SVA+/+ cell lines; interestingly, caspase-8 enzymatic activity was also decreased in SVA+/+ cell lines upon TRAIL stimulation despite increased pro-caspase-8 expression. To further isolate the effects of the CASP8-SVA, we generated an isogenic cell line (RKO CASP8-SVA knockout) model via CRISPR-Cas9. Deletion of the CASP8-SVA redemonstrated decreased CASP8 expression at the mRNA and protein levels. Treatment with FOLFOXIRI in the CASP8-SVA KO cell line exhibited increased sensitivity compared to empty vector control. In summary, our findings indicate that an intronic SVA insertion in CASP8 increases gene and protein expression but decreases canonical caspase-8 activity, which was associated with increased resistance to FOLFIRINOX or TRAIL induced cell death. Additional interrogation of SVA-induced effects on transcriptional regulation (e.g., epigenetic effects) as well as non-canonical functions of caspase-8 (e.g., activation of NF-κB) is ongoing. To our knowledge this is the first report describing the mechanistic effects of a germline SVA insertion in the context of cancer and therapeutic resistance. Citation Format: Eric W. Lin, Joshua R. Kocher, Chong Chu, Peter J. Park, David T. Ting. An intronic SINE-VNTR-Alu element insertion in CASP8 alters gene expression and confers resistance to induction of cell death in gastrointestinal cancer [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 5675.

Abstract 5180: Pharmacodynamic biomarker analysis of gastrointestinal cancer patients treated with metronomic chemotherapy in the Italian COMET Trial

Abstract Metronomic, or continuous low-dose, chemotherapy produces antitumor effects through multiple mechanisms. These include inhibition of angiogenesis and activation of the immune system. At AACR 2017 we presented a small, proof-of-concept, multiplex analysis of 14 cytokines specifically chosen by their association with activation of the immune system. The 14 cytokines were evaluated in 31 out of 38 patients with advanced refractory gastrointestinal tumors, enrolled in a phase II clinical trial of metronomic UFT (a 5-fluorouracil prodrug; 100 mg/twice per day p.o.) and cyclophosphamide (500 mg/mq2 i.v. bolus on day 1 and then 50 mg/day p.o.) plus celecoxib (200 mg/twice a day p.o.); the Italian COMET trial (Clinical Trial ID NCT02926911; Allegrini et al. Angiogenesis 2012: 15(2):275-86). We evaluated baseline (day 0) plasma levels with levels at days 28 and 56, and tested for correlations with observed Progression Free Survival, which we then reported (Valenzuela et al. Clin Exp Med. 2021: 21(1):149-159). We now present a comprehensive analysis of 75 cytokines in 34 of the 38 patients. Furthermore we extend the analysis to include baseline (day 0), days 28 and 56, and days 84 and 112 of the COMET trial, and we also correlated specific cytokines levels with Overall Survival. After an estimation of optimal cut-off points of plasma levels of the cytokines, survival analysis was performed, and log-rank tests and cox regression analyses were applied. We show that pre-treatment plasma level <435.46pg/ml of macrophage-derived chemokine (MDC), was a predictive marker for those patients with better progression-free survival at 3 and 6 months (log-rank test p=0.0046; Cox regression analysis p=0.016). Moreover, baseline plasma levels of three cytokines, including monocyte chemoattractant protein 2 (MCP-2), monocyte chemoattractant protein 4 (MCP-4) and TNF-related apoptosis-inducing ligand (TRAIL), were associated with an improved overall survival. MCP-2 values >26.21pg/ml (log-rank test p=0.0012; Cox regression analysis p=0.007), MCP-4 >72.97pg/ml (log-rank test p=0.012; Cox regression analysis p=0.019) and TRAIL >60.73pg/ml (log-rank test p=0.0059; Cox regression analysis p=0.011) were predictive markers for subjects with a better overall survival at 6 and 9 months. These results indicate that a methodology based on the detection of optimal cut-off points and survival analysis allowed the selection of potential biomarkers, such as MDC or MCPs. We propose that the systematic use of these statistical strategies, aimed at detecting potential biomarkers, could help to identify those patients that are likely to benefit from metronomic chemotherapy schedules. This would allow the stratification of such patients for future metronomic chemotherapy trials, and the potential identification of patients that should instead be offered alternative therapeutic strategies. Citation Format: Maria L. Manca, Diana Prospero, Saeedeh Darvishi, Arianna Bandini, Marta Banchi, Paola Orlandi, Hector Padilla, Alejandro Sanchez, Claire E. Perucho, Nydia de Avila, Hazel Padilla, Brianna E. Ortega, Ryley D. Stewart, Magdiel Santillan, Valeria Lopez, Robert Kirken, Giacomo Allegrini, Guido Bocci, Giulio Francia. Pharmacodynamic biomarker analysis of gastrointestinal cancer patients treated with metronomic chemotherapy in the Italian COMET Trial [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 5180.

Abstract 4604: Imipridones ONC201 and ONC212 exhibit anti-tumor activity in biliary tract cancers and synergy when combined with trametinib and olaparib in vitro

Abstract Background: Biliary tract cancers (BTCs) are an aggressive group of malignancies affecting 220,000 new individuals globally each year. Despite advances in immunotherapy and targeted therapies, 5-year survival remains at 2% for those with metastatic disease. Thus, new therapies are desperately needed. The novel imipridone class of antineoplastic agents show promising activity in preclinical pancreatic ductal adenocarcinoma models; however, have not been investigated in BTC. Dordaviprone (ONC201) is a dopamine receptor D2 and mitochondrial protease ClpP modulator that induces apoptosis in cancer cells through both upregulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and the integrated stress response. ONC212 is a second-generation imipridone that targets orphan GPCR 132 and ClpP, leading to reduced oxidative phosphorylation from ClpX suppression. We hypothesized that the ONC201 and ONC212 would demonstrate antitumor effects in BTCs, both alone, and in combination with MEK and PARP inhibition. Methods: Two established BTC cell lines, RBE and HuCCT1, were used for these experiments. Sensitivity of RBE and HuCCT1 cells to ONC201, ONC212, MEK inhibition (trametinib), and PARP inhibition (olaparib) was assessed using CellTiter-Glo® luminescent cell viability assay. Each cell line was plated on 96-well plates and treated with ONC201 concentrations ranging from 0uM to 40uM for RBE and 0uM to 32uM for HuCCT1; ONC212 ranging from 0nM to 752nM for RBE and 0nM to 640nM for HuCCT1; trametinib ranging from 0nM to 24nM for RBE and 0nM to 16nM for HuCCT1; and olaparib ranging from 0uM to 256uM for RBE and 0uM to 124uM for HuCCT1. Results were analyzed after 72 hours of incubation. Synergy studies were conducted in both cell lines, combining each imipridone with either trametinib or olaparib, and SynergyFinder® was used to evaluate synergy. Western blotting was performed on lysates generated from treated cell lines. Results: Both ONC201 and ONC212 demonstrated antineoplastic effects in two BTC cell lines. Half maximal inhibitory concentrations (IC50) of ONC201 in RBE and HuCCT1 cell lines were 2.5uM and 2.0uM, respectively. The IC50 of ONC212 in RBE and HuCCT1 was 47nM and 39nM, respectively, which was similar to IC50s demonstrated with cytotoxic chemotherapy (gemcitabine IC50 = 7.57nM (RBE) and 9.84nM (HuCCT1)). Both lines also demonstrated sensitivity to trametinib (IC50 = 5.9nM (RBE) and 3.7nM (HCCT1)), and to a lesser extent olaparib (IC50 = 64uM (RBE) and 31uM (HuCCT1)). Although synergy was noted with several combinations, this was most striking with the combination of ONC212 and either trametinib or olaparib. Further analysis of key signal transduction pathways, including MAPK and PI3K, as well as analysis of mechanisms of cell-death through apoptosis, autophagy, ferroptosis, and necroptosis is underway Citation Format: Grace Sun, Alexis J. Lannigan, Jasper Chan, Maximilian Schwermann, Varun V. Prabhu, Lanlan Zhou, Wafik S. El-Deiry, Alexander Grenander Raufi. Imipridones ONC201 and ONC212 exhibit anti-tumor activity in biliary tract cancers and synergy when combined with trametinib and olaparib in vitro [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 4604.

Circulating cytokines and vascular dementia: A bi-directional Mendelian randomization study

Inflammatory responses are associated with the development of vascular dementia (VaD). Circulating cytokines modulate the inflammatory response and are important for the immune system. To further elucidate the role of the immune system in VaD, we used Mendelian randomization (MR) to comprehensively and bi-directionally assess the role of circulating cytokines in VaD.Using state-of-the-art genome-wide association studies, we primarily assessed whether different genetic levels of 41 circulating cytokines affect the risk of developing VaD and, in turn, whether the genetic risk of VaD affects these circulating cytokines. We used inverse variance weighting (IVW) and several other MR methods to assess the bidirectional causality between circulating cytokines and VaD, and performed sensitivity analyses.Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) was inversely associated with VaD risk [odds ratio (OR): 0.74, 95 % confidence interval (CI): 0.60–0.92, P = 0.007, 0.007]. VaD was associated with seven circulating cytokines: macrophage inflammatory protein 1b (MIP-1 beta) [OR: 1.05, 95 % CI: 1.01–1.08, P = 0.009], Interleukin-12p70 (IL-12) [OR: 1.04, 95 % CI: 1.00–1.08, P = 0.047], Interleukin-17 (IL-17) [OR: 1.04, 95 % CI: 1.00–1.07, P = 0.038], Interleukin-7 (IL-7) [OR: 1.07, 95 % CI: 1.02–1.12, P = 0.009], Interferon gamma (IFN-γ) [OR: 1.03, 95 % CI: 1.00–1.07, P = 0.046], Granulocyte-colony stimulating factor (GCSF) [OR: 1.06, 95 % CI: 1.02–1.09, P = 0.001], Fibroblast growth factor (FGF) [P = 0.001], and Fibroblast growth factor (FGF) [P = 0.001]. Fibroblast growth factor basic (FGF-Basic) [OR: 1.04, 95 % CI: 1.01–1.08, P = 0.02] were positively correlated.Circulating cytokines are associated with VaD, and further studies are needed to determine whether they are effective targets for intervention to prevent or treat VaD.

Abstract 7226: Synergy between ONC201 and temozolomide on ATF4 integrated stress response in glioblastoma

Abstract Glioblastoma multiforme (GBM) is the most common primary brain tumor in adults, with a median survival of 15 months. The standard of care involves surgical resection followed by chemoradiotherapy. Even with the full course of treatment, GBM is uncurable, and patients eventually relapse. Although temozolomide (TMZ) is the first-choice chemotherapeutic for GBM, tumor drug resistance limits the effects of the treatment. There is a growing need for new therapies with enhanced efficacy on GBM tumors. Dordaviprone (ONC201) is a TNF-related apoptosis inducing ligand (TRAIL)-inducing compound that is increasingly being studied for GBM treatment. This imipridone small molecule induces apoptosis by activating the integrated stress response, which involves transcription factors ATF4 and CHOP. Subsequently, these transcription factors upregulate proapoptotic protein TRAIL and its receptor, Death Receptor 5 (DR5). This study aims to investigate the combinatory effects of chemotherapeutics TMZ and ONC201 on GBM. Preliminary studies examined the cell viability of GBM cell line U251MG after dual treatment of 0 to 10uM of ONC201 and 0 to 200uM of TMZ using the CellTiter-Glo assay. Synergy was observed between the two drugs at higher concentrations of TMZ. This synergistic interaction was further characterized using western blots. When 0, 2, and 4uM of ONC201 were administered alone or in combination with 0, 60, and 120uM of TMZ, greater ATF4 expression was observed with the dual treatments than with the monotherapies. Subsequent western blots were conducted using three GBM cell lines (U251MG, T98G, and SNB19-GFP) at three time points (8, 24, and 48 hours). All cell lines were treated with 0 and 5uM of ONC201 alone or in combination with 0 and 200uM of TMZ. For all three cell lines, ATF4 expression increased in the combined presence of TMZ and ONC201. However, the effects of TMZ on ONC201-induced ATF4 expression were most pronounced in U251MG and least conclusive in T98G. Additionally, the time points in which this increase in ATF4 expression occurred varied among cell lines. CHOP expression was also probed and was seen to increase with the combination treatment in U251MG. These observations suggest a potential synergistic relationship between chemotherapeutics ONC201 and TMZ in the ATF4 integrated stress response pathway in GBM. By exploring the effects of this dual treatment on upstream and downstream factors in the pathway, we can identify, characterize, and potentially target the root cause of this synergy. Citation Format: Josephine Chen, Tyler J. Roady, Lanlan Zhou, Wafik S. El-Deiry. Synergy between ONC201 and temozolomide on ATF4 integrated stress response in glioblastoma [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 7226.

Abstract 3602: TRAIL-NK-92 MI as a novel adoptive therapy for castration-resistant prostate cancer (CRPC): Preliminary in vitro results

Abstract Introduction: Natural killer (NK) cell tumor infiltration and higher expression of activating receptors NKp30 and NKp46 are associated with improved clinical outcomes in prostate cancer (PC). Activation of NK cells by androgen receptor inhibitors enhanced the killing of PC cell lines (Schwermann 2023). These findings support the therapeutic potential of strategies inducing NK cell activation to treat CRPC. NK cells induce apoptosis of cancer cells via TNF-related apoptosis-inducing ligand) (TRAIL) binding of death receptor 5 (DR5), which is overexpressed in PC cells. This is the first report of adoptive therapy utilizing high expression of TRAIL as a novel therapeutic strategy. Methods: We transduced NK-92-MI and CD4+ (Jurkat) cell lines with human TRAIL (pLVX-EF1alpha-TRAIL-IRES-eGFP).Transduced cells were pre-treated with simvastatin or atorvastatin (4uM) for 36h, followed by stimulation (IL-2 100 IU/ml and IL-12 100 ng/ml) for 2h. The eGFP-expressing cells were then sorted by FACS. PC (PC3, DU145, LNCaP, and 22Rv1), fibroblast (IMR-90), and brain endothelial (hCMEC/D3) cell lines were used in co-culture experiments with non-transduced and transduced NK cells and CD4+ cells. These experiments were quantified using the ImageXpress Confocal HT instrument. Viability assays were performed using Annexin-PI and flow cytometry staining for caspases 3/7. Results: Transduced NK-92 MI cells with TRAIL displayed increased surface expression of TRAIL (control[C]: 4.38%±1; TRAIL-overexpression[NK-TRAIL]: 78.54%±3.76; p<0.001). Co-culture of PC cell lines with TRAIL-NK-92 MI cells resulted in significant tumor-induced apoptosis compared to co-culture with non-transduced NK cells after 48 hours (LNCaP + NK [C]: 16.52%±5, LNCaP + NK-TRAIL: 73.19%±6; PC [C]: 14.4%±3, PC3 + NK-TRAIL: 79%±8; 22Rv1 [C]: 16.78%±1.7, 22Rv1 + NK-TRAIL: 76%±8; DU145 [C]: 13%±4, DU145 + NK-TRAIL: 59.1%±10; p<0.001). This enhanced cytotoxic function was also observed in CD4+ cells expressing high levels of TRAIL. Annexin-PI staining as well as Caspase 3 and 7 showed increased apoptosis in PC co-cultured with NK-TRAIL. Co-culture of NK-TRAIL cells did not affect the viability of the non-malignant cells (IMR-90 and hCMEC/D3) over 48h (IMR-90 + NK: 4.6%±1, IMR-90 + NK-TRAIL: 4.47±2, p=0.9; hCMEC/D3 + NK: 2.7%±0.8, hCMEC/D3 + NK-TRAIL: 2.2±0.5, p>0.99). These results align with the lower expression of DR5 on non-malignant cells compared to PC cell lines (IMR-90: 1.46%±0.5, hCMEC/D3: 1.9%±0.8, LNCaP: 92.3%±4.5). Conclusion: Transduced NK-92 MI cells with human TRAIL-induced higher levels of apoptosis of PC cell lines compared with non-transduced NK cells in co-culture experiments. The results highlight the potential of TRAIL-expressing adoptive cell therapy for CRPC. Engaging adoptive therapies with specific targets (anti-PSMA CAR) might increase the therapeutic potential of this strategy. Citation Format: Maximilian Schwermann, Praveen Srinivasan, William MacDonald, Vida Tajiknia, Andrea Schmidt, Shengliang Zhang, Lindsey Carlsen, Andrew George, Connor Purcell, Shaolei Lu, Matthew Hadfield, Anthony Mega, Benedito Carneiro, Wafik El-Deiry. TRAIL-NK-92 MI as a novel adoptive therapy for castration-resistant prostate cancer (CRPC): Preliminary in vitro results [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 3602.