Tumor Necrosis Factor-related Apoptosis Research Articles

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.

Inhibition of Chk1 stimulates cytotoxic action of platinum-based drugs and TRAIL combination in human prostate cancer cells.

Checkpoint kinase 1 (Chk1) plays an important role in regulation of the cell cycle, DNA damage response and cell death, and represents an attractive target in anticancer therapy. Small-molecule inhibitors of Chk1 have been intensively investigated either as single agents or in combination with various chemotherapeutic drugs and they can enhance the chemosensitivity of numerous tumor types. Here we newly demonstrate that pharmacological inhibition of Chk1 using potent and selective inhibitor SCH900776, currently profiled in phase II clinical trials, significantly enhances cytotoxic effects of the combination of platinum-based drugs (cisplatin or LA-12) and TRAIL (tumor necrosis factor-related apoptosis inducing ligand) in human prostate cancer cells. The specific role of Chk1 in the drug combination-induced cytotoxicity was confirmed by siRNA-mediated silencing of this kinase. Using RNAi-based methods we also showed the importance of Bak-dependent mitochondrial apoptotic pathway in the combined anticancer action of SCH900776, cisplatin and TRAIL. The triple drug combination-induced cytotoxicity was partially enhanced by siRNA-mediated Mcl-1 silencing. Our findings suggest that targeting Chk1 may be used as an efficient strategy for sensitization of prostate cancer cells to killing action of platinum-based chemotherapeutic drugs and TRAIL.

Circulating cytokines and alcoholic liver disease: a two-sample bidirectional Mendelian randomization study

Background Increased inflammation in the liver during ethanol exposure is a major feature of alcoholic liver disease (ALD). An important contributing component to the development of ALD is the inflammatory response brought on by immunological response, however the connection between individual circulating cytokines and ALD is still unclear. To ascertain the causation, we conducted a two-sample bidirectional Mendelian randomization research. Methods We extracted 41 cytokines and growth factors of 8293 Europeans and ALD cases of the same ethnicity (1416 cases and 217,376 controls) from the Genome-Wide Association Studies (GWAS) database for two-sample bidirectional MR analysis. Results Our analyses suggest that higher interleukin-7 (IL-7) levels are associated with an increased risk of ALD (p = 0.028, OR = 1.191,95% CI = 1.019–1.392), while tumor necrosis factor related apoptosis inducing ligand (TRAIL) is a protective factor for ALD (p = 0.032, OR = 0.863, 95% CI = 0.754–0.988) which can reduce the risk of disease occurrence. In addition, genetically predicted ALD does not affect the expression of circulating cytokines regulators. Conclusions Our study supports that cytokines play a pivotal role in the pathogenesis of ALD. To determine the mechanisms and pathways of action of these biomarkers, further basic research is required to ensure their clinical suitability for preventing and treating ALD.

Expression of Tumor Necrosis Factor-Related Apoptosis- Inducing Ligand Conjugated to the scFV Region of Anti-CD20 Antibody Using a Small Ubiquitin-related Modifier

Expression of Tumor Necrosis Factor-Related Apoptosis- Inducing Ligand Conjugated to the scFV Region of Anti-CD20 Antibody Using a Small Ubiquitin-related Modifier

Tumor necrosis factor-related apoptosis- inducing ligand polymorphism.

Tumor necrosis factor-related apoptosis- inducing ligand polymorphism.

Hyperthermia maintains death receptor expression and promotes TRAIL-induced apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand activates apoptotic pathways and could potentially be used in anticancer treatments. However, oral squamous cell carcinoma cells are known to be resistant to tumor necrosis factor-related apoptosis-inducing ligand-induced cell death. It has been previously reported that hyperthermia upregulates tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in other cancers. As such, we evaluated whether hyperthermia upregulates tumor necrosis factor-related apoptosis-inducing ligand-mediated apoptosis in a tumor necrosis factor-related apoptosis-inducing ligand-resistant oral squamous cell carcinoma cell line. The oral squamous cell carcinoma cell line HSC3 was cultured and divided into hyperthermia and control groups. We investigated the antitumor effects of recombinant human tumor necrosis factor-related apoptosis-inducing ligand using cell proliferation and apoptosis assays. Additionally, we measured death receptor 4 and 5 levels, and determined death receptor ubiquitination status, as well as E3 ubiquitin ligase targeting of death receptor in both hyperthermia and control groups before recombinant human tumor necrosis factor-related apoptosis-inducing ligand administration. Treatment with recombinant human tumor necrosis factor-related apoptosis-inducing ligand produced greater inhibitory effects in the hyperthermia group than in the control group. Moreover, death receptor protein expression in the hyperthermia group was upregulated on the cell surface (and overall), although death receptor mRNA was downregulated. The half-life of death receptor was several hours longer in the hyperthermia group; concomitantly, E3 ubiquitin ligase expression and death receptor ubiquitination were downregulated in this group. Our findings suggested that hyperthermia enhances apoptotic signaling by tumor necrosis factor-related apoptosis-inducing ligand via the suppression of death receptor ubiquitination, which upregulates death receptor expression. These data suggest that the combination of hyperthermia and tumor necrosis factor-related apoptosis-inducing ligand has implications in developing a novel treatment strategy for oral squamous cell carcinoma.

#3665 DCR2 PROMOTES TUBULAR MALADAPTIVE REPAIR BY INHIBITING KETONE BODY–INDUCED FOXO3 SIGNALING AFTER ACUTE KIDNEY INJURY

Abstract Background and Aims The proximal tubule has a capacity for repair after acute kidney injury (AKI), but this capacity is limited, especially in severe AKI. Tubular maladaptive repair after AKI leads to chronic kidney disease (CKD), even to end-stage renal disease (ESRD). However, the mechanism of maladaptive repair is not fully clarified, and there is a lack of effective treatment for AKI in clinic. Our previous study found that decoy receptor 2 (DcR2), a trans-membrane receptor of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), was specifically expressed renal tubules and did not have the ability of proliferation in AKI, suggesting DcR2 may be associated with maladaptive repair of tubular cells. This study aims to investigate the role and mechanism of DcR2-positive tubular cells in the repair of AKI. Method The DcR2-GFP lineage trace mice,KSP-creDcR2f/f mice (Distal tubular DcR2 Conditional Knockout CKO) mice and GGT1-creDcR2f/f (proximal tubular DcR2 Conditional Knockout CKO) mice were constructed, and three AKI mouse models (moderate and severe ischemia-reperfusion injury and cisplatin-induced AKI) were conducted. Light microscopic examination of paraffin-embedded sections stained with haematoxylin and eosin, periodic acid–Schiff stain and Masson stain. Confocal analysis the co-expression of DcR2-GFP and proximal tubular markers(AQP1, Villin), distal markers (AQP2), failed repair markers (Vcam1, Dcdc2), proliferative markers (Ki-67, Edu, PCNA), Kim1, differentiated markers (pax2, sox9, six2), senescent markers (P16, P21, SA-β-gal), senescent phenotype (IL-6, TGF-β1) and fibrotic markers (a-SMA, collagen I, Fibronectin). And wild type (WT) mice and DcR2 CKO mice were used to compare the degree of kidney injury, renal function and tubular repair after AKI. Furthermore, quantitative proteomics and bioinformatics analyzed the downstream molecules of DcR2 in renal tissues from WT-AKI and CKO-AKI, and validated studied were done. Results In this study, we found decoy receptor DcR2 was highly expressed in renal tubules and associated with kidney damage in AKI patients and mouse models. DcR2-GFP transgenic mice verified that DcR2 was specifically expressed in proximal tubular epithelial cells (PTECs) after AKI. The levels of Scr, BUN and urinary DcR2 and renal acute and chronic injury scores were significantly lower inGGT1-creDcR2f/f -AKI than that of WT-AKI. Meanwhile, the proliferative markers, senescent markers and area of renal fibrosis and fibrotic markers expression was decreased inGGT1-creDcR2f/f mice compared with WT. However, the above effects were not obviously improved in KSP-creDcR2f/f mice after AKI. These results suggested that proximal tubular DcR2 knockout alleviated kidney injury and promote tubular repair after AKI. Consistent with findings in vivo, DcR2 promoted cell senescence and inhibited cell proliferation in hypoxia-reoxygenation and cisplatin-treated primary PTECs. Further quantitative proteomics and validated studies showed Hmgcs2, a key enzyme for ketone synthesis, was increased in GGT1-creDcR2f/f mice compared with WT. And the levels of urinary and renal β-hydroxybutyrate (β-OHB) were higher inGGT1-creDcR2f/f mice, suggesting DcR2 affects the synthesis of β-hydroxybutyrate through regulating the expression of Hmgcs2. Inhibition or deletion of Hmgcs2 aggravated kidney damage and repressed renal repair, but administration with β-OHB rescued these phenomena. Subsequently, PTEC-specific DcR2/Hmgcs2 double deletion decreased β-OHB levels, which inhibited FOXO3 signaling by regulating histone acetylation, thereby boosting tubular maladaptive repair after AKI. Conclusion DcR2 promotes maladaptive repair of tubular cells by regulating Hmgcs2-induced β-OHB production, and β-OHB affects FOXO3 signaling by regulating histone acetylation. The findings suggests that DcR2/Hmgcs2/ketogenesis/FOXO3 signaling mediates tubular maladaptive repair, and DcR2 could serve as a promising target for improving renal repair and AKI prognosis.

Abstract 2539: Tumor necrosis factor related apoptosis inducing ligand (TRAIL) induces cytokine release via the alternative NFKB2 pathway in triple negative breast cancer cells (TNBC) and modulates neutrophil chemotaxis

Abstract 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 that there are unknown modulatory mechanisms responsible for its lack of TRAIL activity in vivo. We hypothesized that TRAIL treatment elicits transcriptional changes in TNBC cells that alter the immune milieu, modulating the therapeutic efficacy. To investigate the hypothesis, we performed RNAseq analysis of MDA-MB231 cells treated with TRAIL for different time points, followed by validation with RT-PCR in various TNBC cells. TRAIL treatment of the TNBC significantly induced expression of a number of cytokines, such as CXCL1, CXCL2, CXCL3, CXCL8, CXCL11, IL6, 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. Gene Set Enrichment Analysis of the RNAseq indicated that NFKB pathway-mediated TNF-alpha signaling was significantly enriched. Concordantly, we confirmed that both canonical NFKB1 and non-canonical NFKB2 pathways were activated by TRAIL. However, siRNA knockdown experiments indicated that 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’ (1, 2, 3, 8) and IL6 significantly increased neutrophil chemotaxis in a NFKB2-dependent manner. Moreover, neutrophils pre-incubated with supernatants from TRAIL-treated TNBC significantly inhibited its cytotoxic effect in TNBCs in a NFKB2-dependent pathway. Further RNAseq and RT-PCR of neutrophils incubated with by either TRAIL or supernatant of MDA-MB231 cells treated with TRAIL revealed significant enrichment of inflammatory pathway-related genes as well as increased expression of immune modulating cytokines. These results suggested that TRAIL exerts a pro-inflammatory role towards immune cells in tumor microenvironment. CODEX (CO-Detection by indEXing) analysis of in vivo TNBC xenografts from mice treated with the TRAIL agonist MEDI3039 confirmed that TRAIL treatment increases the number of neutrophils in the tumor. Using TNBC organoids and humanized mice models, the changes in tumor immune environment caused by TRAIL are currently under investigation. Collectively, our study suggests the novel role of TRAIL-induced NFKB2-dependent cytokine production that affects neutrophil functions, leading to modulation of the immune response in TNBC. Citation Format: Manjari Kundu, Yoshimi Endo Greer, Lisa A. Ridnour, David A. Wink, Stan Lipkowitz. Tumor necrosis factor related apoptosis inducing ligand (TRAIL) induces cytokine release via the alternative NFKB2 pathway in triple negative breast cancer cells (TNBC) and modulates neutrophil chemotaxis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2539.

Anti-tumor Effects of IL-1β Induced TRAIL-Expressing hUCMSCs on Embelin Treated Breast Cancer Cell Lines.

Human umbilical cord mesenchymal stem cells (hUCMSCs) have high therapeutic value in cancer treatment. We have found that pre-activating hUCMSCs with IL-1β promotes tumor necrosis factor-related apoptosis inducing ligand (TRAIL) expression and facilitates anti-tumor effect. Furthermore, embelin has been found to induce apoptosis of different cancer cell lines by upregulating the expression of TRAIL receptor 1 (DR4) and TRAIL receptor 2 (DR5). This study investigated whether IL-1β induced TRAIL-expressing hUCMSCs, in combination with low-dose embelin, could further induce apoptosis in breast cancer cell lines. MTT assay was used to examine the cytotoxicity of embelin in MDA-MB-231 and MCF-7. To detect the interested protein expression in cells, Western blot and cell immunofluorescence were used to double-confirm the observed results. Annexin V/PI apoptosis assay was detected by flow cytometry to analyze the apoptosis rate of embelin treated breast cancer cell lines and the effect of co-culturing with breast cancer cells and hUCMSCs. Using Western blot and immunofluorescence, we found that breast cancer cell lines treated with low-dose embelin (2.5-5 μM) increased the expression of apoptosis-related receptor DR4, DR5 and the cleaved caspase 8, 9 and 3. Moreover, TRAIL expression was enhanced in IL-1β induced hUCMSCs. Combining these observations, we expected that coculturing IL-1β induced hUCMSCs with low dose embelin treated MDA-MB-231 and MCF-7 cells might enhance the apoptosis of breast cancer cells. We confirmed via flow cytometry that coculture of IL-1β induced TRAIL-expressing hUCMSCs and embelin treated MDA-MB-231 and MCF-7 cells enhances the apoptosis rate of these breast cancer cells. We found that embelin upregulated the expression of DR4 and DR5 to increase the TRAIL-mediated apoptosis in breast cancer cell lines. Low dose embelin treated breast cancer cell lines in combination with IL-1β induced TRAIL-expressing hUCMSCs may become a potential anti-tumor therapy.

Nanoscale Organization of TRAIL Trimers using DNA Origami to Promote Clustering of Death Receptor and Cancer Cell Apoptosis.

Through inducing death receptor (DR) clustering to activate downstream signaling, tumor necrosis factor related apoptosis inducing ligand (TRAIL) trimers trigger apoptosis of tumor cells. However, the poor agonistic activity of current TRAIL-based therapeutics limits their antitumor efficiency. The nanoscale spatial organization of TRAIL trimers at different interligand distances is still challenging, which is essential for the understanding of interaction pattern between TRAIL and DR. In this study, a flat rectangular DNA origami is employed as display scaffold, and an "engraving-printing" strategy is developed to rapidly decorate three TRAIL monomers onto its surface to form DNA-TRAIL3 trimer (DNA origami with surface decoration of three TRAIL monomers). With the spatial addressability of DNA origami, the interligand distances are precisely controlled from 15 to 60 nm. Through comparing the receptor affinity, agonistic activity and cytotoxicity of these DNA-TRAIL3 trimers, it is found that ≈40 nm is the critical interligand distance of DNA-TRAIL3 trimers to induce death receptor clustering and the resulting apoptosis.Finally, a hypothetical "active unit" model is proposed for the DR5 clustering induced by DNA-TRAIL3 trimers.

Docetaxel Enhances Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Mediated Apoptosis in Prostate Cancer Cells via Epigenetic Gene Regulation by Enhancer of Zeste Homolog 2.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent because of its tumor selectivity and its ability to induce apoptosis in cancer cells while sparing most normal cells. We evaluated whether docetaxel enhances TRAIL-mediated apoptosis in prostate cancer (PCa) cells and its mechanism. LNCap-LN3, PC3, and DU 145 PCa cell lines were used to investigate the effects of TRAIL with docetaxel treatment (dosages, 1, 3, 5, and 10 nmol). To evaluate the mechanism, death receptor 4 (DR4), DR5, enhancer of zeste homolog 2 (EZH2) and E2F1 levels were assessed in PCa cells. Hormone-sensitive LNCap-LN3 showed apoptosis in proportion to the concentration of docetaxel. Castration-resistant PC3 and DU 145 showed no change irrespective of the docetaxel concentration. However, combinations of docetaxel (2 nM) and TRAIL (100 ng/mL) had a significant effect on apoptosis of DU 145 cells. In DU 145 cells, docetaxel reduced EZH2 and elevated expression of DR4. The decrease of EZH2 by docetaxel was correlated with the E2F1 level, which was considered as the promoter of EZH2. DZNep reduced EZH2 and elevated DR4 in all PCa cells. Additionally, DZNep-enhanced TRAIL mediated reduction of PCa cell viability. Docetaxel and the EZH2 inhibitor reduced EZH2 and elevated expression of DR4 in all PCa cell lines. Docetaxel-enhanced TRAIL mediated apoptosis in PCa via elevation of DR4 through epigenetic regulation by EZH2. To improve the efficacy of TRAIL for PCa treatment, adding docetaxel or EZH2 inhibitors to TRAIL may be promising.

Mechanisms underlying synergism between circularized tumor necrosis factor-related apoptosis inducing ligand and bortezomib in bortezomib-sensitive or -resistant myeloma cells.

Mechanisms underlying interactions between a novel, clinically relevant circularized tumor necrosis factor-related apoptosis inducing ligand (TRAIL) agonist, circularly permuted TRAIL (CPT) have been examined in multiple myeloma (MM) cells sensitive or resistant to bortezomib (BTZ). Various MM cell lines for example, U266, including those resistant to bortezomib-resistant U266 cells were exposed to low nanomolar concentrations of bortezomib±CPT and apoptosis monitored. Circularly permuted TRAIL and bortezomib synergistically induced apoptosis in both BTZ-naïve and -resistant cells. The regimen up-regulated DR4 receptor internalization in MM cells, known to modulate both NF-κB and extrinsic apoptotic pathways. CPT/BTZ disrupted the non-canonical NF-κB pathway, reflected by tumor necrosis factor (TNF) receptor associated factors 3 (TRAF3) up-regulation, NF-κB inducing kinase down-regulation, diminished p52 and p50 processing, and B-cell lymphoma-extra large (BCL-XL) down-regulation, but failed to inactivate the canonical NF-κB pathway, reflected by unchanged or increased expression of phospho-p65. The regimen also sharply increased extrinsic apoptotic pathway activation. Cells exhibiting TRAF3 knock-down, dominant-negative Fas-associated protein with death domain, knock-down of caspase-8, BCL-2/BCL-XL, or exposure to a caspase-9 inhibitor displayed markedly reduced CPT/BTZ sensitivity. Concordant results were observed in bortezomib-resistant cells. The regimen was also active in the presence of stromal cells and was relatively sparing toward normal CD34+ hematopoietic cells. Finally, ex vivo results revealed synergism in primary MM primary cells, including those BTZ, and the CPT/BTZ regimen significantly decreased tumor growth in a patient-derived MM xenograft model. These results indicate that the CPT/BTZ regimen acts via the non-canonical NF-κB as well as intrinsic/extrinsic apoptotic pathways to induce cell death in MM cells, and may represent an effective strategy in the setting of bortezomib resistance.

Abstract 1315: Tumor necrosis factor related apoptosis inducing ligand fosters myeloid derived suppressor cell mediated tumor immune evasion in cholangiocarcinoma

Abstract Background and Aims: Tumor necrosis factor related apoptosis inducing ligand (TRAIL) is predominantly expressed on immune cells. Although TRAIL biology has garnered considerable attention as a potential anti-cancer strategy, TRAIL agonists have had very limited anti-cancer activity in humans. TRAIL signaling can attenuate the T lymphocyte response. However, this potential immunosuppressive function of TRAIL has not been examined in cancer biology. Cholangiocarcinoma (CCA), a highly lethal biliary tract cancer, has a dense immunosuppressive microenvironment. Accordingly, CCA provides a model to examine the potential immune regulatory function of TRAIL in cancer biology. Methods: Using a syngeneic, orthotopic murine model of CCA (PMID: 29464042), murine CCA cells (SB cells) that express both Trail and Trail receptor (Tr) were implanted into livers of WT and Tr-/- mice. Hence, in this model the host immune cells express Trail but not the receptor; therefore, they would be capable of inducing TRAIL-mediated apoptosis in CCA cells but would be resistant to TRAIL-mediated immunosuppression. After 4 weeks of tumor growth, mice were sacrificed, and tumors were characterized using flow cytometry. Results: We observed that Tr-/- mice had a significant reduction in tumor burden compared to WT mice. Moreover, tumor bearing Tr-/- mice had a significant increase in cytotoxic T lymphocytes (CTLs) and enhanced CTL effector function. Myeloid-derived suppressor cells (MDSCs) were significantly decreased in Tr-/- tumors compared to WT tumors. Furthermore, implantation of SB cells devoid of Trail (SB-Trail-/-) into WT mice resulted in a significant reduction in tumor burden and MDSC infiltration. In vitro functional studies employing SB cells deficient in Tr (SB-Tr-/-) and MDSCs were carried out. There was a significant reduction in proliferation and immunosuppressive function of MDSCs when cocultured with SB-Tr-/- compared to SB cells. These results indicate that TRAIL-TR fosters MDSC growth and immunosuppressive function. In conclusion, we have demonstrated that Tr-/- mice have a significant reduction in CCA tumor burden and MDSC infiltration. Consequently, Tr-/- tumors have enhanced CTL infiltration and function. These data suggest that the TRAIL-TR system mediates tumors immune evasion via MDSCs, and direct targeting of TRAIL on CCA cells is a potential anti-tumor strategy. Citation Format: Emilien Loeuillard, Jingchun Yang, Haidong Dong, Gregory J. Gores, Sumera I. Ilyas. Tumor necrosis factor related apoptosis inducing ligand fosters myeloid derived suppressor cell mediated tumor immune evasion in cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1315.

Antiproliferative activity of moringa oleifera's leaves ethanolic extract and docetaxel on lncap cell line and on the level of tumor necrosis factor-related apoptosis-inducing ligand

Background: Cancer is abnormal cells growth that is responsible for the majority of global deaths and cancer is expected to rank as leading cause of death and the single most important barrier to increasing life expectancy in every country of the world in the 21st century. According to the estimates from the world health organization in 2015, cancer is the first or second leading cause of death before age 70 years in 91 of 172 countries, and it ranks third or fourth in an additional 22 countries. Many anti-cancer drugs in current use such as paclitaxel, vincristine, and vinblastine are naturally derived agents. The aim of this study is to determine the effects of ethanolic leaves extract of Moringa oleifera and docetaxel each separately and in combinations on the proliferation of LNCaP cell line, and on the level of tumor necrosis factor related apoptosis induced ligand (TRAIL). Methods: This work was performed in the laboratory of cancer research at college of medicine\ university of Babylon during the period from January 2021 to October 2021. The study was approved by the ethics committee in the college of medicine at university of Babylon.

Fusion of an EGFR-antagonistic affibody enhances the anti-tumor effect of TRAIL to EGFR positive tumors

Tumor necrosis factor-related apoptosis ligand (TRAIL) is a promising antitumor agent candidate for its selective proapoptotic activity to various tumor cells. However, TRAIL showed limited efficacy in clinical trials despite of good tolerability. One important reason might be attributed to the poor tumor-homing ability of TRAIL. Herein, we designed an EGFR-targeting TRAIL (Z-TRAIL) by genetically fusing an EGFR-antagonistic affibody (ZEGFR:1907) to the N-terminus of TRAIL. Z-TRAIL was produced as a soluble protein with high yield in E. coli and it maintained the trimeric state of active TRAIL. Under the EGFR-binding mediated by ZEGFR:1907, Z-TRAIL showed a ∼5 to 20-fold enhancement of cytotoxicity compared to TRAIL on tumor cells in vitro. Furthermore, fusion to ZEGFR:1907 endowed TRAIL with a ∼1.8-fold increase of tumor uptake and a dramatical stronger apoptosis-inducing ability in the mice bearing EGFR-overexpressing A431 tumor xenografts. More importantly, Z-TRAIL exhibited significantly enhanced antitumor efficacy against whether EGFR high-expressing or low-expressing tumors than TRAIL in vivo. In addition, repeated injection of high-dose Z-TRAIL did not show obvious acute toxicity in mice. These results demonstrated that the newly engineered Z-TRAIL might be a promising agent for targeted therapy of EGFR-expressing tumors.

TRAIL-sensitizing Cytochalasins from the Endophytic Fungus Phoma multirostrata.

Seven undescribed cytochalasins, multirostratins K - Q (2: -8: ), together with one known analogue, cytochalasin Z3 (1: ), were isolated from the culture of Phoma multirostrata XJ-2-1, an endophytic fungus obtained from the root of Parasenecio albus. Their structures with absolute configurations were determined by 1D and 2D NMR, high-resolution electrospray ionization mass spectrometry (HRESIMS), electronic circular dichroism (ECD), single-crystal X-ray crystallography, and chemical methods. The structure of ascochalasin was revised from Δ 13 to Δ 21 by detailed analysis of the NMR data and by comparison with the data for 7: . In a TRAIL (tumor necrosis factor related apoptosis inducing ligand)-resistance-overcoming experiment, co-treatment of 2: or 6: with TRAIL reduced the cell viability of A549 cells by 30.3% and 27.5% at 10 µM, respectively.

POS-064 DcR2 mediates the maladaptive repair of acute kidney injury by regulating the synthesis the β-hydroxybutyrate

Acute kidney injury (AKI) is a common clinical emergency and critical illness, and has a high mortality rate among hospitalized patients. The repair and regeneration of renal tubular cells determine the prognosis of AKI. Our previous study found that decoy receptor 2 (DcR2), a trans-membrane receptor of tumor necrosis factor-related apoptosis inducing ligand (TRAIL), was specifically expressed renal tubules and did not have the ability of proliferation in AKI, suggesting DcR2 may be associated with regeneration and repair of tubular cells. This study aims to investigate the role and mechanism of DcR2-positive tubular cells in the repair of AKI. The DcR2-GFP lineage trace mice,KSP-creDcR2f/fmice (Distal tubular DcR2 Conditional Knockout CKO) mice and GGT1-creDcR2f/f(proximal tubular DcR2 Conditional Knockout CKO) mice and Ischemia-Reperfusion (I/R) Injury models were constructed. The models were divided into moderated (ischemia 25min) and severe (ischemia 35min) injury. Light microscopic examination of paraffin-embedded sections stained with haematoxylin and eosin, periodic acid–Schiff stain and Masson stain. Confocal analysis the co-expression of DcR-GFP and proximal tubular markers(AQP1, Villin), distal markers (AQP2), failed repair markers (Vcam1, Dcdc2), proliferative markers (Ki-67, Edu, PCNA), Kim1, differentiated markers (pax2, sox9, six2), senescent markers (P16, P21, SA-β-gal), senescent phenotype (IL-6, TGF-β1) and fibrotic markers (a-SMA, collagen I, Fibronectin). And wild type (WT) mice and DcR2 CKO mice were used to compare the degree of kidney injury, renal function and tubular repair after I/R injury. Furthermore, quantitative proteomics and bioinformatics analyzed the downstream molecules of DcR2 in renal tissues from WT-AKI and CKO-AKI, and validated studied were done. The DcR2-GFP were mainly expressed proximal tubular cells in AKI. DcR2-GFP positive cells were co-expressed failed repair markers, senescent markers and co-localization with fibrotic markers. And DcR2-GFP positive cells were not expressed Kim1, proliferative markers and differentiated markers, suggesting that DcR2-positve tubules were maladaptive repair cells. The levels of Scr, BUN and urinary DcR2 and renal acute and chronic injury scores were significantly lower inGGT1-creDcR2f/f-AKI than that of WT-AKI. Meanwhile, the area of renal fibrosis and fibrotic markers expression was decreased inGGT1-creDcR2f/fmice compared with WT. However, the above phenomenon of KSP-creDcR2f/f–AKI were not obviously improved. These results suggested that proximal tubular DcR2 gene knockout alleviated kidney injury and promote tubular repair. Additionally, quantitative proteomics and validated studies showed HMGCS2, a key enzyme for ketone synthesis, was increased in GGT1-creDcR2f/fmice compared with WT. And the levels of urinary and serum β-hydroxybutyrate were higher inGGT1-creDcR2f/fmice, suggesting DcR2 affects the synthesis of β-hydroxybutyrate through regulating the expression of HMGCS2. DcR2-positive cells were failed-repair tubules in AKI. And DcR2 promotes maladaptive repair of tubular cells through regulating the expression of HMGCS2 and affects the synthesis of β-hydroxybutyrate, suggesting that DcR2 may be a potential intervention target during the progression of AKI, and β-hydroxybutyrate play a critical role in the repair of AKI.

Enhanced extrinsic apoptosis of therapy-induced senescent cancer cells using a death receptor 5 (DR5) selective agonist.

Genotoxic agents are widely used anti-cancer therapies because of their ability to interfere with highly proliferative cells. An important outcome of these interventions is the induction of a state of permanent arrest also known as cellular senescence. However, senescent cancer cells are characterized by genomic instability and are at risk of escaping the growth arrest to eventually facilitate cancer relapse. The tumor necrosis factor related apoptosis inducing ligand (TRAIL) signals extrinsic apoptosis via Death Receptors (DR) 4 and 5, while Decoy Receptors (DcR) 1 and 2, and Osteoprotegerin (OPG) are homologous to death receptors but incapable of transducing an apoptotic signal. The use of recombinant TRAIL as an anti-cancer strategy in combination with chemotherapy is currently in development, and a major question remains whether senescent cancer cells respond to TRAIL. Here, we show variable sensitivity of cancer cells to TRAIL after senescence induction, and upregulation of both pro-apoptotic and anti-apoptotic receptors in therapy-induced senescent cancer cells. A DR5-selective TRAIL variant (DHER), unable to bind to DcR1 or OPG, was more effective in inducing apoptosis of senescent cancer cells compared to wild-type TRAIL. Importantly, no apoptosis induction was observed in non-cancerous cells, even at the highest concentrations tested. Our results suggest that targeting DR5 can serve as a novel therapeutic strategy for the elimination of therapy-induced senescent cancer cells.

32. Host Immune-Protein Signature Combining TRAIL, IP-10 and CRP for Early and Accurate Prediction of Severe COVID-19 Outcome

BackgroundAccurately identifying COVID-19 patients at-risk to deteriorate remains challenging. Dysregulated immune responses impact disease progression and development of life-threatening complications. Tools integrating host immune-protein expression have proven useful in determining infection etiology and hold potential for prognosticating disease severity.MethodsAdults with COVID-19 were enrolled at medical centers in Israel, Germany, and the United States (Figure 1). Severe outcome was defined as intensive care unit admission, non-invasive or invasive ventilation, or death. Tumor necrosis factor related apoptosis inducing ligand (TRAIL), interferon gamma inducible protein-10 (IP-10) and C-reactive protein (CRP) were measured using an analyzer providing values within 15 minutes (MeMed Key®). A signature indicating the likelihood of severe outcome was derived generating a score (0-100).Description of derivation cohort RT-PCR, reverse transcription polymerase chain reaction.ResultsBetween March and November 2020, 518 COVID-19 patients were enrolled, of whom 394 were eligible, 29% meeting a severe outcome. Age ranged between 19-98 (median 61.5), with 59.1% male. Patients meeting severe outcomes exhibited higher levels of CRP and IP-10 and lower levels of TRAIL (Figure 2; p < 0.001). Likelihood of severe outcome increased significantly (p < 0.001) with higher scores. The signature’s area under the receiver operating characteristic curve (AUC) was 0.86 (95% confidence interval: 0.81-0.91). Performance was not confounded by age, sex, or comorbidities and was superior to IL-6 (AUC 0.77; p = 0.033) and CRP (AUC 0.78; p < 0.001). Clinical deterioration proximal to blood draw was associated with higher signature score. Scores of patients meeting a first outcome over 3 days after blood draw were significantly (p < 0.001) higher than scores of non-severe patients (Figure 3). Moreover, the signature differentiated patients who further deteriorated after meeting a severe outcome from those who improved (p = 0.004) and projected 14-day survival probabilities (p < 0.001; Figure 4).TRAIL, IP-10, CRP and the severity signature score are differentially expressed in severe and non-severe COVID-19 infection Dots represent patients and boxes denote median and interquartile range (IQR)The signature score of patients meeting a severe outcome on or after the day of blood draw is significantly (p < 0.001) higher than the signature score of non-severe patients. Dots represents patients and boxes denote median and IQRKaplan-Meier survival estimates for signature score bins ConclusionThe derived signature combined with a rapid measurement platform has potential to serve as an accurate predictive tool for early detection of COVID-19 patients at risk for severe outcome, facilitating timely care escalation and de-escalation and appropriate resource allocation. Disclosures Alon Angel, n/a, MeMed (Employee, Shareholder) Niv Samuel Mastboim, BSc, MeMed (Employee, Shareholder) Oded Shaham, PhD, MeMed (Employee, Shareholder) Tahel Ilan Ber, MD, MeMed (Employee, Shareholder) Roy Navon, MSc, MeMed (Employee, Shareholder) Einav Simon, PhD, MeMed (Employee, Shareholder) Michal Rosenberg, PhD, MeMed (Employee) Yael Israeli, PhD, MeMed (Employee) Mary Hainrichson, PhD, MeMed (Employee, Shareholder) Noa Avni, PhD, MeMed (Employee) Eran Reiner, MD, MeMed (Employee) Kfir Oved, MD, PhD, MeMed (Board Member, Employee, Shareholder) Ilya Kagan, MD, MeMed (Scientific Research Study Investigator) Shaul Lev, M.D, MeMed (Scientific Research Study Investigator) Dror Diker, MD, MeMed (Scientific Research Study Investigator) Amir Jarjou’i, MD, MeMed (Scientific Research Study Investigator) Ramzi Kurd, MD, MeMed (Scientific Research Study Investigator) Guy Danziger, MD, MeMed (Scientific Research Study Investigator) Cihan Papan, MD, MeMed (Scientific Research Study Investigator) Sergey Motov, MD, MeMed (Scientific Research Study Investigator) Maanit Shapira, Ph.D, MeMed (Scientific Research Study Investigator) Tanya Gottlieb, PhD, MeMed (Employee, Shareholder) Eran Eden, PhD, MeMed (Board Member, Employee, Shareholder)

Rationale behind using valproic acid for Non-Hodgkin lymphoma: a biomolecular perspective.

Non-Hodgkin lymphoma (NHL) is a hematological malignancy with a high rate of relapse and refractory cases. It is believed to be caused by resistance to standard treatment modalities. Valproic acid (VPA), previously used as a broad-spectrum anticonvulsant drug, has been proposed for NHL owing to its action of epigenetic modification by inhibiting histone deacetylase. However, VPA studies on NHL are limited. This review describes the rationale behind the use of VPA for NHL treatment, particularly focusing on its molecular mechanism of action. This is a narrative review. The literature search strategy for indexed Scopus articles was performed randomly using PubMed and MEDLINE as the primary sources. No specific term was used. Several mechanisms are responsible for NHL development. VPA can modulate these mechanisms via epigenetic and nonepigenetic modifications. It may also have an impact on the proteins responsible for treatment resistance. The mechanisms of action of VPA in NHL are as follows: the induction of cell cycle arrest via the upregulation of cyclin-dependent protein kinase inhibitors; induction of Apo2 ligand or tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis; inactivation of B-cell lymphoma 6; inhibition of Janus kinase/signal transducer and activator of transcription, phosphoinositide 3-kinase/Akt, and nuclear factor kappa B signaling pathways; upregulation of tumor antigen as the primary target of immunotherapy; and strengthening of tumor immunosurveillance. Based on its biomolecular mechanism of action, VPA appears to be a promising initial treatment before initiating the standard treatment in patients with NHL to overcome resistance.