cIAP2 Levels Research Articles

CIAP-2 protein is upregulated by human papillomavirus in oropharyngeal cancers: role in radioresistance in vitro

BackgroundHigh-risk human papillomaviruses are the causal agents of a subset of head and neck cancers. A previous transcriptomic analysis showed that cIAP2 protein, involved in cell survival and apoptosis, is upregulated in OKF6 oral cells that express HPV16 E6/E7. In addition, cIAP2 promotes radioresistance, a very important concern in HNC treatment. However, cIAP2 increase has not yet been evaluated in oropharyngeal carcinomas (OPCs), nor has been the role of cIAP2 in HNC radioresistance.MethodsWe carried out a descriptive-analytical retrospective study in 49 OPCs from Chilean patients. We determined the expression of cIAP2 at transcript and proteins levels using reverse-transcriptase -polymerase chain reaction and immunohistochemistry, respectively. HPV and p16 expression were previously analyzed in these specimens. In addition, SCC-143 HNC cells ectopically expressing HPV16 E6/E7 were analyzed for cIAP2 expression and after transfection with a siRNA for HPV16 E6/E7 knocking down.ResultsWe found a statistically significant association between HPV presence and cIAP2 expression (p = 0.0032 and p = 0.0061, respectively). An association between p16 and cIAP2 levels was also found (p = 0.038). When SCC-143 cells were transfected with a construct expressing HPV16 E6/E7, the levels of cIAP2 were significantly increased (p = 0.0383 and p = 0.0115, respectively). Conversely, HPV16 E6 and E7 knocking down resulted in a decrease of cIAP2 levels (p = 0.0161 and p = 0.006, respectively). Finally, cIAP2 knocking down in HPV16 E6/E7 cells resulted in increased apoptosis after exposure to radiation at 4 and 8 Gy (p = 0.0187 and p = 0.0061, respectively).ConclusionThis study demonstrated for the first time a positive relationship between HPV presence and cIAP2 levels in OPCs. Additionally, cIAP2 knocking down sensitizes HNC cells to apoptosis promoted by radiation. Therefore, cIAP2 is a potential therapeutic target for radiation in HPV-driven HNC.

Chimeric antigen receptor dendritic cells targeted delivery of a single tumoricidal factor for cancer immunotherapy

BackgroundChimeric antigen receptor (CAR)-T cells have been used to treat blood cancers by producing a wide variety of cytokines. However, they are not effective in treating solid cancers and can cause severe side-effects, including cytokine release syndrome. TNFα is a tumoricidal cytokine, but it markedly increases the protein levels of cIAP1 and cIAP2, the members of inhibitor of apoptosis protein (IAP) family of E3 ubiquitin ligase that limits caspase-induced apoptosis. Degradation of IAP proteins by an IAP antagonist does not effectively kill cancer cells but enables TNFα to strongly induce cancer cell apoptosis. It would be a promising approach to treat cancers by targeted delivery of TNFα through an inactive adoptive cell in combination with an IAP antagonist.MethodsHuman dendritic cells (DCs) were engineered to express a single tumoricidal factor, TNFα, and a membrane-anchored Mucin1 antibody scFv, named Mucin 1 directed DCs expressing TNFα (M-DCsTNF). The efficacy of M-DCsTNF in recognizing and treating breast cancer was tested in vitro and in vivo.ResultsMucin1 was highly expressed on the surface of a wide range of human breast cancer cell lines. M-DCsTNF directly associated with MDA-MB-231 cells in the bone of NSG mice. M-DCsTNF plus an IAP antagonist, SM-164, but neither alone, markedly induce MDA-MB-231 breast cancer cell apoptosis, which was blocked by TNF antibody. Importantly, M-DCsTNF combined with SM-164, but not SM-164 alone, inhibited the growth of patient-derived breast cancer in NSG mice.ConclusionAn adoptive cell targeting delivery of TNFα combined with an IAP antagonist is a novel effective approach to treat breast cancer and could be expanded to treat other solid cancers. Unlike CAR-T cell, this novel adoptive cell is not activated to produce a wide variety of cytokines, except for additional overexpressed TNF, and thus could avoid the severe side effects such as cytokine release syndrome.

Phase I study of TQB3728 tablets, an oral inhibitor of apoptosis proteins in patients with advanced solid tumor.

e13104 Background: Apoptosis proteins are frequently upregulated in human cancers and are associated with resistance to therapeutic agents and poor outcomes. TQB3728 antagonizes the function of inhibitor of apoptosis proteins (IAPs), thereby inhibiting NF-кB signaling pathways and promoting cancer cell death. This first-in-human study aims to evaluate the maximum-tolerated dose (MTD), safety, pharmacokinetics, pharmacodynamics, and antitumor activity of TQB3728 in patients with advanced solid tumors after the failure of standard therapy. Methods: This study combined an accelerated dose titration design with a 3+3 dose escalation design. Three dosage schedules were explored. First of all, TQB3728 was given orally once a week, on a 28-day cycle (DS1). The starting dose of 25mg was escalated by 100% in a single patient until the third dose, then diverted to a 3+3 dose escalation design. The other two dosage schedules were TQB3728 given once daily on days 1-14 every 3 weeks (DS2) and daily every 4 weeks (DS3). The primary endpoint was safety, maximum tolerated or maximum administered dose (MTD), and the secondary endpoints were pharmacokinetics, pharmacodynamics, and tumor response per RECIST v1.1. Results: As of Dec 15, 2023, 23 patients (pts) received at least one dose of TQB3728 (dose range, 25 to 600mg). Based on the pharmacokinetics and safety of DS1, then DS2 (dose range, 100 to 200mg) and DS3 (dose range, 100 to 200mg) were investigated. The median follow-up period was 8.5m (range, 0.5 to 25m). No dose-limited toxicity and cytokine release syndrome were observed. The main treatment-related adverse events (TRAEs) were nausea (56.5%), lymphopenia (56.5%), dizziness (39.1%), hypersomnia (34.8%), increased aspartate aminotransferase (30.4%), increased alanine aminotransferase (26.1%), vomiting (26.1%), anemia (21.7%), decreased appetite (21.7%), and grade≥3 TRAE were lymphopenia (8.7%), anemia (4.4%). As for DS1, the average Tmax and t1/2 were 2 h and 9.6 h, respectively. The exposure did not increase with the dose above 150mg. Moreover, the average Tmax was about 2.3 h and 0.8 h, and t1/2 was 6.6 h and 7 h in DS2 and DS3, respectively. There was no significant accumulation with continuous medication. Additionally, Plasma MCP-1 increased at 3-6h postdose at high dose; cIAP-1 levels in PBMCs were undetected. Two pts (8.7%) had stable disease >6 weeks as the best treatment response. Conclusions: TQB3728 was well tolerated at doses up to 600mg but had limited anti-tumor activity in advanced solid tumor. According to the preliminary results, TQB3728 administered orally in combination with conventional chemo-radiotherapy is under exploration. Clinical trial information: NCT04523285 .

Abstract CT140: A phase 1 study of the IAP inhibitor xevinapant (Debio 1143) to evaluate food effect and drug-drug interactions with a proton pump inhibitor in healthy volunteers

Abstract INTRODUCTION: Xevinapant is an oral IAP (inhibitor of apoptosis protein) inhibitor designed to restore cancer cell sensitivity to apoptosis, thereby enhancing the efficacy of chemoradiotherapy (CRT) and radiotherapy (RT). Xevinapant 200 mg/d (d1-14, Q3W) + CRT showed clinical benefit vs placebo + CRT in a phase 2 study of patients with locally advanced squamous cell carcinoma of the head and neck (LA SCCHN; NCT02022098). Two phase 3 studies are currently ongoing evaluating xevinapant in combination with CRT (TrilynX; NCT04459715) or RT (XRay Vision; NCT05386550) in patients with LA SCCHN, with dosing under fasting conditions or without regard to food, respectively. METHODS: A 2-stage study (Debio 1143-107) was conducted to assess the effect of food on 2 different formulations of xevinapant and the effect of co-administration with the proton pump inhibitor pantoprazole to evaluate the effect of high gastric pH on bioavailability of xevinapant. Stage 1 was an open-label, randomized, 4-arm, crossover study with 26 healthy volunteers (HV) treated with a single oral dose of xevinapant 200 mg in fasted or fed (high-fat diet) conditions with 2 different formulations. Stage 2 was a randomized, 2-way crossover study with 12 HV treated with a single oral dose of xevinapant 200 mg with or without pantoprazole. Pharmacokinetics (PK) and pharmacodynamics (PD) samples (Ciap1 levels in peripheral blood mononuclear cells [PBMCs]) were collected over 72 h post dose. The utilization of HV to address clinical pharmacology aspects of xevinapant was justified by holistic assessment of the toxicology, safety, and pharmacology profiles. RESULTS: Following a single dose of xevinapant 200 mg, exposures in HV under fasted conditions were consistent with those observed previously in cancer patients. Xevinapant formulations were bioequivalent and were considered safe and well tolerated in both fasted and fed conditions. Food delayed Tmax and decreased xevinapant Cmax by 39% but did not meaningfully alter the extent of absorption of xevinapant, as evidenced by no change in AUC, which is the most relevant exposure metric for efficacy based on xevinapant’s mechanism of action. Stage 2 data indicated that the PK of xevinapant was not affected by treatment with pantoprazole. Ciap1 levels in PBMCs decreased at least 4-fold compared to the baseline for all treatments and were reduced up to 72 h post single dose. No relevant differences in PD were observed across treatments. PK simulations indicated no relevant change in AUC and no impact on target engagement at steady state with food. CONCLUSION: These data indicate that xevinapant can be administered without regard to food or gastric acid reducing agents, which would be advantageous for patient convenience and compliance. Citation Format: Valérie Nicolas-Métral, Elisabeth Rouits, Camille Riff, Dany Spaggiari, Norbert Wiedemann, Pierre Colin, Eleanor Harrison-Moench, Rafael Crabbé, Heidi Nauwelaerts, Andreas Schroeder, Carolina Haefliger, Yulia Vugmeyster. A phase 1 study of the IAP inhibitor xevinapant (Debio 1143) to evaluate food effect and drug-drug interactions with a proton pump inhibitor in healthy volunteers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT140.

Abstract 5088: Evaluating TNF-receptor associated factor 2 (TRAF2) as a targetable driver of immune resistance in LKB1 deficient non-small cell lung cancer

Abstract STK11 is a serine-threonine kinase and tumor suppressor lost in about 25% of NSCLC. It potently regulates a network of downstream effector kinases in the AMPK family, thereby exerting major effects on cell signaling, metabolic activity, and epigenetic patterning. Clinically loss of STK11 in NSCLC is associated with poor response to immune checkpoint blockade. Several mechanisms of immune resistance have been identified involving both tumor intrinsic pathways as well as changes in the immune microenvironment. Dysregulation of cellular inhibitor of apoptosis proteins, specifically cIAP1 has been identified as a specific resistance mechanism, which was shown to also influence the STING pathway in the context of STK11 loss. However, there is a gap in understanding how loss of STK11 influences cIAP1 signaling.We evaluated cIAP1, its TRAF family member binding partners, and downstream NF-kB and immune signaling using a multiomic approach. Using CPTAC multiomic data of 110 lung adenocarcinomas, we observe discordance between protein vs mRNA expression for cIAP1, which shows no significant association with STK11 loss in mRNA, but is significantly over-expressed at the protein level (P<1e-4). Conversely, we identify TRAF2 as the only TRAF member whose expression is increased in STK11-deficient NSCLC, with concordant increase in both mRNA (P<1e-8) and protein (P<1e-10). Further, on the protein level TRAF2 and cIAP1 levels were highly correlated (P<1e-12), strongly suggesting that TRAF2 is the proximal factor driving dysregulation of cIAP1 signaling in these tumors. We also observe a correlated pattern of phosphorylation sites within cIAP1, TRAF2, and other NF-kB signaling members that we putatively link to STK11 loss. We confirm that TRAF2 mRNA is consistently over-expressed in STK11 deficient NSCLC across multiple additional transcriptomic datasets together comprising >2000 tumors. Across this large dataset we observe that among STK11-deficient tumors, TRAF2 is anti-correlated with NF-kB signaling and immune infiltration signatures, a relationship that is strikingly absent among the STK11-WT subset. Using GEMM cell lines (KRAS, TP53, STK11 GEMM; Winslow lab, Stanford), we derived STK11 mutant vs WT isogenic pairs and performed T-cell co-culture experiments using ovalbumin antigen and OT-I CD8 T-cells. This showed that inhibition of cIAP1 using birinapant was synergistic to CD8 cytotoxicity, as previously known, but the synergy was much more pronounced in STK11 mutant vs STK11-WT context (Bliss index 38, P<1e-10). Together, these data demonstrate that the link between STK11-loss and cIAP1 dysregulation is likely through over-expression of TRAF2, and support the strategy of targeting TRAF2/cIAP1 to overcome immune resistance STK11-deficient NSCLC. Citation Format: Bahareh Nourmohammadi, Joseph M. Amann, Rahul Shivahare, Qin Ma, Zihai Li, David P. Carbone, Jacob Kaufman. Evaluating TNF-receptor associated factor 2 (TRAF2) as a targetable driver of immune resistance in LKB1 deficient non-small cell lung 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 5088.

Expression and clinical significance of Capase3 and C-IAP1/2 in ectopic endometrium of patients with endometriosis.

To analyze the expression of capase3 and C-IAP1/2 in endometriosis and their relationship with clinical practice. Ectopic and eutopic endometrial tissues were collected from women with endometriosis, and the control tissues were obtained from regular patients without endometriosis. The expression level of capase3 and C-IAP1/2 was detected by immunohistochemistry; in addition, the relationship between C-IAP1/2 expression and the clinical stage of endometriosis was analyzed. The expression of Caspase-3 in ectopic and eutopic endometrium was significantly lower than that in the control group (P < 0.005). The expression of C-IAP1/2 in ectopic and situ endometrium was significantly higher than in the control group (P < 0.005). The expression of C-IAP1/2 in stage III-IV was higher than in stage I-II endometriosis. The positive expression of c-IAP1 protein in ectopic endometrial tissue was negatively correlated with the positive expression of Caspase-3 protein in ectopic endometrial tissue (P<0.001). The low expression of Caspase-3 and the high expression of C-IAP1/2 in the ectopic endometrium and eutopic endometrial tissues compared with the controls. The results indicated that the apoptotic ability of eutopic and ectopic endometrium is weakened, which is involved in the pathogenesis of endometriosis. It is expected to be a target for the diagnosis and therapy of this disease. At the same time, the expression level of C-IAP1/2 is related to the stage of endometriosis, which can guide clinical treatment.

The Effect of Xevinapant Combined with Ionizing Radiation on HNSCC and Normal Tissue Cells and the Impact of Xevinapant on Its Targeted Proteins cIAP1 and XIAP

The poor prognosis of HNSCC is partly due to treatment resistance. The SMAC mimetic Xevinapant is a promising new approach to targeted cancer therapy. Xevinapant inhibits cIAP1/2 and XIAP, leading to apoptosis, necroptosis and inhibition of prosurvival signaling. Combining Xevinapant with IR could improve therapeutic potential. The effect of Xevinapant in combination with IR on HNSCC and healthy tissue cells was investigated. Cell growth, cell death, clonogenic survival and DNA double-strand breaks (DSBs) were studied, and intracellular cIAP1 and XIAP levels were evaluated. Xevinapant had cytostatic and cytotoxic, as well as radiosensitizing, effects on the malignant cells, while healthy tissue cells were less affected. Apoptotic and necrotic cell death was particularly affected, but the increase in residual DSBs and the reduced survival implied an additional effect of Xevinapant on DNA damage repair and other cell inactivation mechanisms. cIAP1 and XIAP levels varied for each cell line and were affected by Xevinapant and IR treatment. There was an association between higher IAP levels and increased cell death. Xevinapant appears to be a potent new drug for HNSCC therapy, especially in combination with IR. IAP levels could be an indicator for impaired DNA damage repair and increased susceptibility to cellular stress.

TRIM56 promotes malignant progression of glioblastoma by stabilizing cIAP1 protein

BackgroundThe tripartite motif (TRIM) family of proteins plays a key role in the developmental growth and therapeutic resistance of many tumors. However, the regulatory mechanisms and biological functions of TRIM proteins in human glioblastoma (GBM) are not yet fully understood. In this study, we focused on TRIM56, which emerged as the most differentially expressed TRIM family member with increased expression in GBM.MethodsWestern blot, real-time quantitative PCR (qRT-PCR), immunofluorescence (IF) and immunohistochemistry (IHC) were used to study the expression levels of TRIM56 and cIAP1 in GBM cell lines. Co-immunoprecipitation (co-IP) was used to explore the specific binding between target proteins and TRIM56. A xenograft animal model was used to verify the tumor promoting effect of TRIM56 on glioma in vivo.ResultsWe observed elevated expression of TRIM56 in malignant gliomas and revealed that TRIM56 promoted glioma progression in vitro and in a GBM xenograft model in nude mice. Analysis of the Human Ubiquitin Array and co-IPs showed that cIAP1 is a protein downstream of TRIM56. TRIM56 deubiquitinated cIAP1, mainly through the zinc finger domain (amino acids 21–205) of TRIM56, thereby reducing the degradation of cIAP1 and thus increasing its expression. TRIM56 also showed prognostic significance in overall survival of glioma patients.ConclusionsTRIM56-regulated post-translational modifications may contribute to glioma development through stabilization of cIAP1. Furthermore, TRIM56 may serve as a novel prognostic indicator and therapeutic molecular target for GBM.

Identification of cerebrospinal fluid biomarker candidates for anti-N-methyl-D-aspartate receptor encephalitis: High-throughput proteomic investigation.

Although the diagnosis is mainly dependent on the detection of anti-N-methyl-D-aspartate receptor (NMDAR) antibodies in cerebrospinal fluid (CSF) and/or serum, there was no direct correlations between anti-NMDAR antibody titers in CSF and disease severity and prognosis in anti-NMDAR encephalitis patients. Here, we aimed to extensively identify CSF biomarkers related to the occurrence, development, and prognosis of anti-NMDAR encephalitis using a high-throughput proteomic approach. A CSF cytokine antibody array containing 80 cytokines and inflammatory mediators related to immune and inflammatory responses was applied to identify biomarker candidates in individual CSF samples from a well-characterized cohort comprising patients with anti-NMDAR encephalitis (n = 6) and controls (n = 6). Validation and specific detection were performed in an extended cohort consisting of anti-NMDAR encephalitis patients (n = 13), controls (n = 13), and viral encephalitis (n = 13) by enzyme-linked immunosorbent assay (ELISA). Additionally, the levels of some inflammatory proteins in three groups in cohort 2 reported in previous literatures that may be involved in the development of anti-NMDAR encephalitis were also tested by ELISA. Correlations between candidate biomarkers and clinical characteristics of anti-NMDAR encephalitis patients were analyzed. Three differentially expressed cytokines and inflammatory mediators were screened from the 80-cytokine array in cohort 1. Functional enrichment analysis results suggested that these differentially expressed proteins were related to autophagy, immune/inflammatory responses, cell death, and other processes. In cohort 2, the elevations of cellular inhibitor of apoptosis protein-1 (cIAP-1), macrophage colony-stimulating factor (MCSF), CXC chemokine ligand 13 (CXCL13), and nucleotide binding oligomerization domain-like receptor protein 3 (NLRP3) in anti-NMDAR encephalitis were validated by ELISA. Linear regression revealed that the levels of CSF CXCL13 and cIAP-1 were positively correlated with the highest modified Rankin scale (mRS) score in the acute phase (p < 0.05). The level of cIAP-1 was positively correlated with the anti-NMDAR Encephalitis One-Year Functional Status (NEOS) score (p < 0.05). These biomarkers show promising functions to evaluate severity or prognosis of anti-NMDAR encephalitis. The biological processes of immune/inflammatory responses, altered levels of autophagy, and the tumornecrosis factor (TNF) signal pathway may be involved in the pathophysiology of anti-NMDAR encephalitis to some extent.

BV6 enhances apoptosis in Lung cancer cells by ameliorating caspase expressions through attenuation of XIAP, cIAP-1, and cIAP-2 proteins

The present study aimed to investigate the inhibitory role of second mitochondria determined activator of caspases mimetic on inhibitor of apoptosis proteins (IAPs) and regulation of caspases in nonsmall cell lung cancer cell line. Dimethyl sulfoxide and 3-(4, 5-dimethyl thizol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay was done to determine the IC50 of BV6 using NCI-H23 cell line. The levels of mRNA of X-linked IAP (XIAP), cellular IAP (cIAP-1), cIAP-2, caspase-6, and caspase-7 in H23 cell line were evaluated by a quantitative real-time polymerase chain reaction, while their protein expressions were tested using western blotting. Two doses of BV6 dependently downregulated the expression of mRNA of XIAP (P = 0.002, P= 0.0003 vs. untreated), cIAP-1 (P = 0.05, P = 0.005 vs. untreated), and cIAP-2 (P = 0.001, P = 0.0002 vs. untreated), respectively, while the compound upregulated the mRNA expression of caspase-6 (P = 0.001, P < 0.0001 vs. untreated) and caspase-7 (P = 0.001, P = 0.0004 vs. untreated), respectively. Dose dependent of BV6 treatment significantly decreased the protein level of XIAP (P = 0.003, P = 0.007 vs. untreated), cIAP-1 (P = 0.02, P = 0.01 vs. untreated), and cIAP-2 (P = 0.008,P = 0.008 vs. untreated), respectively. However, the compound increased the protein level of caspase-6 and caspase-7 when compared to untreated control (P = 0.006,P = 0.001) and (P = 0.01, P = 0.001), respectively. The result showed that BV6 treatment reduced the level of mRNA of XIAP, cIAP-1, and cIAP-2 and increased the gene expression of caspase-6 and caspase-7 in NCI-H23 cell line. Therefore, the study revealed that BV6 could be used in future as additional therapeutics in lung cancer.

Endogenous TRAIL-R4 critically impacts apoptotic and non-apoptotic TRAIL-induced signaling in cancer cells.

Binding of TRAIL to its death domain-containing receptors TRAIL-R1 and TRAIL-R2 can induce cell death and/or pro-inflammatory signaling. The importance of TRAIL and TRAIL-R1/R2 in tumor immune surveillance and cancer biology has meanwhile been well documented. In addition, TRAIL has been shown to preferentially kill tumor cells, raising hope for the development of targeted anti-cancer therapies. Apart from death-inducing receptors, TRAIL also binds to TRAIL-R3 and TRAIL-R4. Whereas TRAIL-R3 is lacking an intracellular domain entirely, TRAIL-R4 contains a truncated death domain but still a signaling-competent intracellular part. It is assumed that these receptors have anti-apoptotic, yet still not well understood regulatory functions.To analyze the significance of the endogenous levels of TRAIL-R4 for TRAIL-induced signaling in cancer cells, we stably knocked down this receptor in Colo357 and MDA-MB-231 cells and analyzed the activation of apoptotic and non-apoptotic pathways in response to treatment with TRAIL.We found that TRAIL-R4 affects a plethora of signaling pathways, partly in an opposite way. While knockdown of TRAIL-R4 in Colo357 strongly increased apoptosis and reduced clonogenic survival, it inhibited cell death and improved clonogenic survival of MDA-MB-231 cells after TRAIL treatment. Furthermore, TRAIL-R4 turned out to be an important regulator of the expression of a variety of anti-apoptotic proteins in MDA-MB-231 cells since TRAIL-R4-KD reduced the cellular levels of FLIPs, XIAP and cIAP2 but upregulated the levels of Bcl-xL. By inhibiting Bcl-xL with Navitoclax, we could finally show that this protein mainly accounts for the acquired resistance of MDA-MB-231 TRAIL-R4-KD cells to TRAIL-induced apoptosis. Analyses of non-apoptotic signaling pathways revealed that in both cell lines TRAIL-R4-KD resulted in a constitutively increased activity of AKT and ERK, while it reduced AKT activity after TRAIL treatment. Furthermore, TRAIL-R4-KD potentiated TRAIL-induced activation of ERK and p38 in Colo357, and NF-κB in MDA-MB-231 cells. Importantly, in both cell lines the activity of AKT, ERK, p38 and NF-κB after TRAIL treatment was higher in TRAIL-R4-KD cells than in respective control cells.Thus, our data provide evidence for the important regulatory functions of endogenous TRAIL-R4 in cancer cells and improve our understanding of the very complex human TRAIL/TRAIL-R system.

P1278: ACTIVATION OF THE NECROPTOSIS PATHWAY PLAYS A ROLE IN SYNERGISM WITH COMBINATION OF THE IAP ANTAGONIST, TOLINAPANT AND HYPOMETHYLATING AGENTS (HMA) IN IN VITRO MODELS OF T-CELL LYMPHOMA (TCL).

Background: Peripheral T-cell lymphoma (PTCL) are a highly aggressive and heterogeneous group of non-Hodgkin lymphomas that often carry a poor prognosis with standard chemotherapy. Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In an ongoing Phase 2 trial (NCT02503423), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma. HMAs have also shown clinical responses in some subsets of PTCL. Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in pre-clinical studies. We have also shown that decitabine and tolinapant as single agent induced necroptosis in mouse TCL cell lines with the activation of the RIPK1/RIPK3/MLKL necroptosis pathway. In addition, re-expression of RIPK3 in CT26 cells by CRISPR increased lytic cell death on treatment with tolinapant indicating its essential role in the necroptosis pathway. Aims: We explored the sensitivity of a range of TCL lines to tolinapant, established the synergy coefficient and interrogated the role of necroptosis in the mechanism of synergy between tolinapant and drugs active against PTCL, the HMAs; azacytidine and decitabine Methods: A panel of 10 human TCL lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10 ng/ml of TNFa. For combination studies, each drug was tested at the IC10, IC20 and IC40 concentrations, in the presence or absence of TNFa. Synergy coefficient was calculated using the Excess over Bliss model with SynergyFinder. Additionally, the on-target effects of the drugs were measured by analyzing levels of the IAPs, DNMTs and key apoptosis and necroptosis markers, such as PARP and RIPK3 by Western blotting. Results: TCL Lines demonstrate varying sensitivities to tolinapant with the most sensitive cell line, ALK+ ALCL SUP-M2, having an IC50 as low as 20 nM ± 1 nM while a resistant CTCL cell line HH had an IC50 of over 20 µM. In combination experiments using both cell lines, tolinapant plus azacytidine or decitabine displayed a very high degree of synergism; Average synergy score of 50.9 and 49.8 with azacytidine and 22.6 and 31.3 with decitabine for HH and SUP-M2, respectively. Of note, a high degree of synergism was also seen with concentrations 10 fold lower of azacytidine or 100 fold lower of decitabine, added daily (Figure 1). The combination of tolinapant and the HMAs led to a decrease in the levels of cIAP1 and DNMT3a in TCL lines, demonstrating on-target activity of tolinapant and the HMAs, respectively. In addition, both azacytidine and decitabine increased the levels of RIPK3 up to 3-4 fold and 6-8 fold respectively, in a concentration dependent manner at 72Hrs by western blot analysis, indicating activation of the necroptosis pathway. Image:Figure 1: 3D synergy plots showing average synergy scores for decitabine in combination with tolinapant in the CTCL line HH.Summary/Conclusion: Tolinapant has demonstrated varying cytotoxic effects against a range of TCL lines as a single agent. Tolinapant, in combination with the HMAs, azacytidine and decitabine, displays a very high degree of synergism, in vitro, even at very low concentrations of both HMA drugs. The HMAs were able to activate the necroptosis pathway, as shown by the increase in RIPK3. Thus, activation of the necroptosis pathway, is a possible mechanism for the high degree of synergism displayed when HMAs are used in combination with tolinapant in the TCL lines in vitro. These data provide the rationale to initiate clinical trials with the combination of tolinapant and decitabine.

P1293: COMBINING THE IAP ANTAGONIST, TOLINAPANT, WITH A DNA HYPOMETHYLATING AGENT ENHANCES ANTI-TUMOUR MECHANISMS IN PRECLINICAL MODELS OF T-CELL LYMPHOMA.

Background: Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In a Phase 2 trial (NCT02503423), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma (Samaniego et al., Hematological Oncology, 2019). Hypomethylating agents (HMAs) have also shown clinical responses in some subsets of PTCL (Lemonnier et al., Blood, 2019). Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in preclinical studies. Aims: Here we have undertaken a biomarker-driven approach to understand the potential for induction of immunogenic forms of cell death, such as necroptosis, by rational combination of our clinical compounds in preclinical models of TCL. Methods: On-target effects of decitabine and tolinapant were measured by analysing levels of DNMT1 and cIAP1, respectively, by Western blotting in mouse and human cell lines. Levels of key necroptosis biomarkers (RIPK3, MLKL) were also monitored by Western blotting to provide evidence of lytic cell death contributing to a potential immune response. Karpas-299 cells genetically-manipulated to express RIPK3 were used to demonstrate involvement of necroptosis in drug-induced cell death (Cytotox NIR) in vitro. Cell death was monitored by viability (CellTiterGlo) or real-time microscopy (IncuCyte) assays. Levels of key immunomodulatory mediators or DAMPS were measured in tissue culture supernatants and mouse plasma. Levels of methylation in RIPK3 promoter regions were measured by pyrosequencing after bisulfite conversion. Comparative changes in gene expression were measured by RT-qPCR. Results: TCL cell lines treated with tolinapant, decitabine or both led to depletion of cIAP1 and DNMT1 in TCL cell lines, demonstrating target engagement of both agents. The combination of tolinapant and decitabine synergistically reduced viability of some human T-cell lymphoma cell lines. Some cell lines, including Karpas-299, were resistant to tolinapant treatment and showed low expression of RIPK3, which was found to be due to promoter methylation. Increased expression of RIPK3 in Karpas-299 by genetic manipulation or by decitabine treatment resulted in enhanced lytic cell death upon tolinapant treatment. Decitabine and tolinapant treatments resulted in expression of cytokines, chemokines and DAMPs, suggesting potential for immune activation and the effects were enhanced when combined. Furthermore, normally silenced cancer/testis antigen expression was increased by decitabine, potentially increasing the immunogenicity of the cells. Evaluation of the combination of agents in mouse models suggested that increased necroptosis signal and immune-potentiating biomarker modulation can be achieved in vivo. Summary/Conclusion: These data demonstrate that hypomethylating agents enhance immunogenic cell death induced by tolinapant through the re-expression of genes in the necroptotic pathway. In addition, modulation of cytokine response and cancer/testis antigen expression could enhance anti-tumour response. These findings provide a strong rationale to explore this combination clinically.

Abstract 5337: Tolinapant (ASTX660) enhances the anti-leukemic activity of Venetoclax and Dexamethasone in T cell acute lymphoblastic leukemia (T-ALL)

Abstract Background: Inhibitors of apoptosis proteins (IAPs) are overexpressed in ALL leading to resistance to apoptosis and chemo-resistance. Tolinapant, a non-peptidomimetic antagonist of the cellular and X-linked inhibitors of apoptosis (cIAP1/2 and XIAP) being evaluated in Phase 1/2 clinical trial [NCT02503423] showed single-agent activity in T-cell lymphoma. Objectives: Herein, we analyzed the preclinical activity of tolinapant in combination with ABT199 and Dexamethasone (DEX) in T-ALL (in vitro and ex-vivo patient derived xenografts (PDX). Results: Using a panel of 8 T-ALL cell lines we analyzed the single agent activity of tolinapant. Loucy and SUP-T11 were most sensitive (IC50 = 190-309 nM). CCRF-CEM and ALL-SIL showed moderate sensitivity (IC50 = 10-18 µM) while Jurkat, MOLT16, MOLT4 and PF382 were resistant. Western blotting (WB) showed decreased levels of cIAP1and cIAP2 with no change in XIAP in response to tolinapant as a single agent. We next investigated the efficacy of tolinapant with Bcl2 inhibitor ABT199. The combination of ABT199 and ASTX660 was synergistic in Loucy cell line with combination index (CI) of 0.14. Cell death was increased to 64±3% in combination v/s 27±0.9% in ABT199. WB showed decreased cIAP2 and increased levels of cleaved caspase 7 and cleaved caspase 9 in combination compared to single agents, suggesting increased apoptosis. Treatment of PDX-derived cells with tolinapant and ABT199 was more effective than monotherapy in inducing apoptosis in CD45+ bulk (46±0.7% to 63±6%, p&amp;lt;0.0001) and leukemia initiating cells (LICs, CD45+ CD7+ CD19- CD34+) (39±3% to 54±8%, p=0.003). Next, we checked the effect of combination of DEX with tolinapant in T-ALL cell lines. CCRF-CEM cell line is from a relapsed patient and is resistant to DEX. The combination was synergistic with CI of 0.26 and cell death of 50±4% compared to 20±3% by DEX. A triple combination of DEX, ABT199 and tolinapant increased apoptotic response to 82.9±1%, compared to DEX+ABT199 dual combination where apoptosis was induced to 52.4±2%. Strong synergy in terms of both cytoreduction as well as apoptosis induction was observed in SUP-T11 cells. Tolinapant sensitized SUP-T11 cells to DEX with ED50 value of 542 nM in combination compared to ~2 µM with DEX treatment. Simultaneous analysis of cell proliferation, stress response and DNA damage using single-cell proteomics analysis showed downregulation of proliferation (Ki-67), stress response (ATF4, LC3B) and increased levels of cleaved PARP, cleaved caspase 3 suggesting increased apoptosis in combination of tolinapant with DEX. Treatment of PDX-derived cells with tolinapant enhanced the cytotoxic effect of DEX in CD45+ bulk (58±2% to 71±0.1%, p&amp;lt;0.0001) and LICs (62±2% to 78±0.9%, p&amp;lt;0.0001). Conclusion: Tolinapant synergizes with the anti-leukemic activity of ABT199 and DEX, establishing a therapeutic rationale for IAP antagonist in T-ALL. Citation Format: Priyanka Sharma, Sujan Piya, Huaxian Ma, Natalia Baran, Muharrem Muftuoglu, Mahesh Basyal, Vivian Ruvolo, George Ward, Tomoko Smyth, Martin J. Sims, Michael Andreeff, Gautam Borthakur. Tolinapant (ASTX660) enhances the anti-leukemic activity of Venetoclax and Dexamethasone in T cell acute lymphoblastic leukemia (T-ALL) [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 5337.

The combination of the IAP antagonist, tolinapant and hypomethylating agents (HMA) is highly synergistic in in vitro models of T-cell lymphoma (TCL).

e15088 Background: Peripheral T-cell lymphoma (PTCL) are a highly aggressive and heterogeneous group of non-Hodgkin lymphomas that often carry a poor prognosis with standard chemotherapy. Tolinapant is a potent antagonist of inhibitors of apoptosis proteins (cIAP1/2 and XIAP). In a phase 2 trial (NCT02503423), tolinapant showed activity against heavily pre-treated patients with TCL. We explored the sensitivity of a range of TCL lines to tolinapant, established the synergy coefficient and interrogated the mechanism of synergy between tolinapant and drugs active against PTCL including romidepsin, pralatrexate, and the HMAs; azacytidine and decitabine. Methods: A panel of 10 human TCL lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10 ng/ml of TNFα. For combination studies, each drug was tested, in the presence or absence of TNFα. Synergy coefficient was calculated using Excess over Bliss. Additionally, the on-target effects of the drugs were measured by analyzing levels of the IAPs, acetylated histones, DNMTs and key apoptosis and necroptosis markers by Western blotting. Results: TCL Lines demonstrated varying sensitivities to tolinapant with the most sensitive cell line, ALK+ ALCL SUP-M2, having an IC50 as low as 20 nM ± 1 nM while a resistant CTCL cell line HH had an IC50 of over 20 µM. In combination experiments using both cell lines, tolinapant plus azacytidine or decitabine displayed the highest degree of synergism, compared to romidepsin or pralatrexate. Of note, a high degree of synergism was also seen with concentrations 10 folds lower of azacytidine or 100 folds lower of decitabine, added daily (Table). The combination of tolinapant and the HMAs led to a decrease in the levels of cIAP1 and DNMT3a in TCL lines, demonstrating on-target activity of tolinapant and the HMAs, respectively. In addition, both azacytidine and decitabine increased the levels of RIPK3 and MLKL by western blot analysis, indicating activation of the necroptosis pathway. Conclusions: Tolinapant has demonstrated varying cytotoxic effects against a range of TCL lines as a monotherapy and displays a very high degree of synergism in combination with the HMAs, azacytidine and decitabine, also at low concentration, in vitro. The HMAs were also able to activate the necroptosis pathway, providing a possible mechanism for the high degree of synergism displayed when combined with tolinapant in the TCL lines in vitro. These data provide the rationale to initiate phase I trials with the combination of tolinapant and HMAs. Average synergy scores at 48 hrs for drugs used in combination with tolinapant in a CTCL line HH and ALK+ ALCL line SUP-M2. Above 10 indicates synergy.[Table: see text]

Expression levels of cellular inhibitor of apoptosis proteins and colitogenic cytokines are inversely correlated with the activation of interferon regulatory factor 4.

Cellular inhibitors of apoptosis proteins 1 (cIAP1) and 2 (cIAP2) are involved in signaling pathways mediated by Toll-like receptors (TLRs) and tumor necrosis factor (TNF)-α. Excessive activation of TLRs and TNF-α underlies the immunopathogenesis of Crohn's disease (CD) and ulcerative colitis (UC). However, the roles played by cIAP1 and cIAP2 in the development of CD and UC remain poorly understood. In this study, we attempted to clarify the molecular link between cIAP1/cIAP2 and colonic inflammation. Human monocyte-derived dendritic cells (DCs) treated with siRNAs specific for cIAP1 or cIAP2 exhibited reduced pro-inflammatory cytokine responses upon stimulation with TLR ligands. Expression of cIAP1 and cIAP2 in human DCs was suppressed in the presence of interferon regulatory factor 4 (IRF4). This effect was associated with inhibition of cIAP1 and cIAP2 polyubiquitination. To verify these in vitro findings, we created mice overexpressing IRF4 in DCs and showed that these mice were resistant to trinitrobenzene sulfonic acid-induced colitis as compared with wild-type mice; these effects were accompanied by reduced expression levels of cIAP1 and cIAP2. Pro-inflammatory cytokine production by mesenteric lymph node cells upon stimulation with TLR ligands was reduced in mice with DC-specific IRF4 overexpression as compared with that in wild-type mice. Finally, in clinical samples of the colonic mucosa from patients with CD, there was a negative relationship between the percentage of IRF4+ DCs and percentages of cIAP1+ or cIAP2+ lamina propria mononuclear cells. These data suggest that the colitogenic roles of cIAP1 and cIAP2 are negatively regulated by IRF4.

Exploratory window-of-opportunity trial to investigate the tumor pharmacokinetics/pharmacodynamics of the IAP antagonist Debio 1143 in patients with head and neck cancer.

Inhibitor of apoptosis proteins (IAPs) regulate apoptosis and modulate NF‐κB signaling thereby driving expression of genes involved in immune/inflammatory responses. The orally available IAP antagonist Debio 1143 has potential to enhance tumor response to chemoradiotherapy and/or immunotherapy. Patients with pre‐operative squamous cell carcinomas of the head and neck (SCCHN) received: Debio 1143 monotherapy (200 mg/day [D]1–15 +/‐ 2); Debio 1143 (200 mg/day D1–15 +/‐ 2) plus cisplatin (40 mg/m2 D 1 and 8); cisplatin alone (40 mg/m2 D 1 and 8; EudraCT: 2014‐004655‐31). Pharmacokinetic/pharmacodynamic effects were assessed in plasma and resected tumors. Primary end point; effect of Debio 1143 on cellular IAP‐1 (cIAP‐1). Levels of cIAP‐1/‐2, X‐linked inhibitor of apoptosis protein (XIAP), tumor infiltrating lymphocytes (TILs), including CD8+ T cells, programmed cell death protein 1 (PD‐1), PD‐ligand 1 (PD‐L1), and gene expression were also analyzed. Twenty‐three of 26 patients completed treatment. In the Debio 1143 monotherapy cohort (n = 13), mean tumor concentrations of Debio 1143 were 18‐fold (maximum 55.2‐fold) greater than in plasma, exceeding the half‐maximal inhibitory concentration for cIAPs and XIAP by 100 to 1000‐fold, with significant engagement/degradation of cIAP‐1 (p < 0.05). Overall, levels of CD8+ TILs, PD‐1, and PD‐L1 positive immune cells increased significantly (p < 0.05) following Debio 1143 treatment. Changes were observed in the expression of genes related to NF‐κB signaling. Treatments were well‐tolerated. Debio 1143 penetrated SCCHN tumors, engaged cIAP‐1, and induced immune inflammatory changes in the tumor microenvironment. Based on the mode of action demonstrated here and in previous studies, these data support future combinations of Debio 1143 with immune‐checkpoint agents.

Tolinapant (ASTX660), a Non-Peptidomimetic Antagonist of cIAP1/2 and XIAP, and the HDAC Inhibitor Romidepsin Are Synergistic in in Vitro Models of T Cell Lymphoma

Background: The PTCL are a heterogeneous group of non-Hodgkin lymphomas originating from mature T-lymphocytes. They are aggressive diseases, often resistant to conventional chemotherapy. Despite the fact that a number of new agents have been approved, treatment paradigms tailored to the biology of the disease have yet to emerge. Tolinapant (ASTX660) is a potent antagonist of both cellular and X-linked inhibitors of apoptosis proteins (cIAP1/2 and XIAP), and is presently in phase I/II trials in patients with advanced solid tumors and lymphomas (NCT02503423). IAP antagonists enhance tumor necrosis factor (TNF) receptor superfamily mediated apoptosis (Ward GA, et al. Mol Cancer Ther. 2018), are potent anti-tumor immune enhancers and induce markers of immunogenic cell death such as damage associated molecular patterns (DAMPs; Ye W, et al, Oncoimmunology, 2020). Objectives: We explored the sensitivity of a range of T-cell lymphoma (TCL) cell lines to tolinapant. We establish the synergy coefficient between tolinapant and the HDAC inhibitor, romidepsin, and interrogated the molecular basis of their synergistic interaction. Methods: A panel of human T-cell lymphoma cell lines were tested in proliferation assays (CellTiterGlo) for sensitivity to tolinapant in the presence or absence of 10ng/ml of TNF alpha. For combination studies, with tolinapant and romidepsin, each drug was tested at the IC10 and IC40 concentrations in the presence or absence of TNF alpha. Synergy scores using the Excess over Bliss (EOB) model were calculated using SynergyFinder (Aleksandr Ianevski et al; Nucleic Acids Research, 2020). Additionally, the effects of tolinapant and romidepsin on the IAPs and caspases were analyzed by western blots. TNFR1 receptor expression and induction of DAMPs were also analyzed by flow cytometry. Results: TCL Lines demonstrated varying sensitivities to tolinapant in the presence or absence of TNF alpha. The most sensitive cell lines, ALK+ ALCL and SUP-M2, had IC50 concentrations ranging from 200nM ± 100nM to 20nM ± 1nM in the absence or presence of TNF alpha, respectively, at 24, 48 and 72hrs, while a resistant CTCL cell line HH had an IC50 concentration of over 20mM, even in the presence of TNF alpha. Interestingly, using western blot analysis, we found that the presence of TNF alpha increased the levels of cIAP1 in the tolinapant sensitive SUP-M2 cell line, but not in the resistant HH cell line. However, there was a concentration dependent decrease in cIAP1 but not in XIAP in both cell lines treated with tolinapant. Flow cytometry analysis demonstrated that tolinapant increases the expression of TNFR1 and DAMPs in a dose dependent manner on the sensitive SUP-M2, but not in the resistant HH cells. In combination experiments, using the EOB model, tolinapant plus romidepsin was found to be synergistic in the absence of TNF alpha, at 36hrs, in both the sensitive cell line SUP-M2 and the resistant cell line HH. In the presence of TNF alpha, synergism was seen only in the sensitive cell line SUP-M2 and antagonistic in the HH cell line (Fig. 3). In the tolinapant plus romidepsin treated samples, cIAP1 levels decreased in the SUP-M2 cell line, in the absence of TNF alpha, however, addition of TNF alpha did not alter the levels of cIAP1 in the SUP-M2 cells. The cIAP1 levels decreased in the HH cells treated with the combination, in both the presence or absence of TNF alpha (Figure). Our findings indicate that the synergy of the tolinapant plus romidepsin is not dependent on the presence of TNF alpha. Conclusion: Tolinapant has demonstrated potent cytotoxic effects against a broad range of TCL lines both as a monotherapy and in combination with the HDAC Inhibitor, romidepsin. In in vitro studies, T cell lymphoma cell lines demonstrated varying sensitivity to tolinapant with certain cell lines being more resistant, even in the presence of TNF alpha. Interestingly, the addition of romidepsin appeared to overcome the intrinsic resistance to tolinapant in the absence of TNF alpha. These data provide the rationale to continue to explore the combination of tolinapant and romidepsin in vivo and to investigate additional combinations with T-cell specific agents (e.g. pralatrexate, belinostat, azacitidine and decitabine). [Display omitted] DisclosuresSmyth: Astex Pharmaceuticals: Current Employment. Sims: Astex Pharmaceuticals: Current Employment. Loughran: Kymera Therapeutics: Membership on an entity's Board of Directors or advisory committees; Bioniz Therapeutics: Membership on an entity's Board of Directors or advisory committees; Keystone Nano: Membership on an entity's Board of Directors or advisory committees; Dren Bio: Membership on an entity's Board of Directors or advisory committees. Marchi: Kyowa Kirin: Honoraria; Myeloid Therapeutics: Honoraria; Astex: Research Funding; BMS: Research Funding; Merck: Research Funding; Kymera Therapeutics: Other: Scientific Advisor.

Combining the IAP Antagonist Tolinapant with a DNA Hypomethylating Agent Enhances Immunogenic Cell Death in Preclinical Models of T-Cell Lymphoma

Introduction: Tolinapant is a potent, non-peptidomimetic antagonist of cIAP1, cIAP2 and XIAP. In ongoing Phase 2 trial (NCT02503423), tolinapant has shown activity against highly pre-treated peripheral and cutaneous T-cell lymphoma (Samaniego et al., Hematological Oncology, 2019). Hypomethylating agents (HMAs) have also shown clinical responses in some subsets of PTCL (Lemonnier et al., Blood, 2019). Both HMAs and IAP antagonists show immunomodulatory anti-cancer potential in pre-clinical studies. A Phase 1 clinical study investigating the combination of tolinapant and ASTX727 (oral decitabine) in AML is currently in progress (NCT04155580). Here we have undertaken a biomarker-driven approach to understand the potential for induction of immunogenic forms of cell death (ICD), such as necroptosis, by rational combination of our clinical compounds in pre-clinical models of T-cell lymphoma (TCL).Methods: On-target effects of decitabine and tolinapant were measured by analysing levels of DNMT1 and cIAP1, respectively, by Western blotting in mouse and human cell lines. Levels of key apoptosis, necroptosis or pyroptosis biomarkers were also monitored by Western blotting to provide evidence of lytic cell death contributing to a potential immune response. RIPK3- or MLKL-knockout cell lines were generated by CRISPR to demonstrate involvement of necroptosis in drug-induced cell death in a T-cell lymphoma cell line (BW5147.G.1.4) in vitro. Cell death was monitored by viability (CellTiterGlo) or real-time microscopy (IncuCyte) assays. Levels of key inflammatory mediators or DAMPS were measured in tissue culture supernatants and mouse plasma by Luminex assay (Ampersand).Results: Combined treatment of tolinapant and decitabine led to depletion of cIAP1 and DNMT1 in TCL cell lines, demonstrating on-target activity of tolinapant and decitabine, respectively. The combination of tolinapant and decitabine acted synergistically in mouse and human T-cell lymphoma cell lines to reduce viability in proliferation assays.Necroptosis was induced by decitabine or tolinapant alone in mouse TCL cell lines with robust activation of the RIPK1/RIPK3/MLKL necroptosis pathway when caspase activity was inhibited, and the combination of both agents enhanced loss of viability.Furthermore, we demonstrated decitabine treatment led to re-expression of both RIPK3 and MLKL in mouse cell lines, supporting published evidence that methylation can silence these key biomarkers (Koo et al., Cell Research, 2015; Koch et al., Neoplasia, 2021). Enhanced release of chemokine, cytokine and DAMPs was demonstrated with the combination of agents in vitro and in vivo.By removal of key necroptosis pathway components using CRISPR, we confirmed the importance of this lytic cell death pathway by demonstrating that RIPK3 -/- and MLKL -/- T-cell lymphoma (BW5147.G.1.4) cell lines had reduced necroptosis potential after treatment with tolinapant or decitabine alone or in combination; and demonstrate reduced release of inflammatory mediators in vitro.Finally, our in vivo evaluation of the combination of agents in mouse syngeneic models suggested that increased anti-tumour activity and immune-potentiating systemic biomarker modulation can be achieved with a tolerated dosing regimen of both compounds.Conclusion: These data demonstrate that decitabine enhances immunogenic cell death induced by tolinapant through the re-expression of genes in the necroptotic pathway. This finding provides strong rationale to explore this combination clinically. DisclosuresSims: Astex Pharmaceuticals: Current Employment. Davis: Astex Pharmacueticals: Current Employment. Smyth: Astex Pharmaceuticals: Current Employment.

The SMAC mimetic AT-101 exhibits anti-tumor and anti-metastasis activity in lung adenocarcinoma cells by the IAPs/ caspase-dependent apoptosis and p65-NFƙB cross-talk.

Objective(s):The Inhibitors of Apoptosis (IAPs) regulate initiator and effector phases of caspase mediated apoptosis. This study evaluates the effects of SMAC mimetic AT-101 in regulation of IAPs/caspases/NFƙB-p65 in an adenocarcinoma cell line.Materials and Methods:MTT assay was performed in the NCI-H522 cell line. Flow cytometry was used for detecting cell cycle, apoptosis, and NFƙB-p65 regulation. Effects of AT-101 on IAPs and caspases were determined by quantitative real time-PCR and western blotting. AutoDock-VINA was used for computational analysis. Results:AT-101 reduced the cell proliferation of NCI-H522 with a GI50 value of 7 μM. The compound arrested adenocarcinoma cells in the G1 phase of the cell cycle and increased early and late phase apoptosis while decreasing tumor-cell trans-migration. AT-101 treatment to NCI H522 at a concentration of 0.35 μM decreased XIAP, cIAP-1, and cIAP-2 mRNA levels to 4.39±0.66, 1.93±0.26, and 2.20±0.24 folds, respectively. Increased dose of AT-101 at 0.7 μM concentration further decreased XIAP, cIAP-1, and cIAP-2 mRNA levels to 2.44±0.67, 1.46±0.93, and 0.97±0.10 folds, respectively. Similar effects of a dose-dependent decrease in the protein expressions of XIAP, cIAP-1, and cIAP-2 were observed with AT-101 treatments, while a dose-responsive increase in the mRNA and protein expression levels of caspase 6 and caspase 7 was observed in the NCI-H522 cell line. The compound exhibited binding affinity (-6.1 kcal/mol) and inhibited NFƙB-p65 in these cells. Conclusion:AT-101 had anti-tumor efficacy against lung adenocarcinoma cells which could be mediated through IAPs/caspase-dependent apoptosis and NFƙB-p65 cross talk. Results from this study suggests a signal cross talk between IAPs and NFkB and open new channels for further investigations in therapeutic intervention against lung cancer management.