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Heart failure is a major cause of mortality following myocardial infarction. Neutrophils are among the first immune cells to accumulate in the infarcted region. Although beneficial functions of neutrophils in heart injury are now appreciated, neutrophils are also well known for their ability to exacerbate inflammation and promote tissue damage. Myocardial infarction induces hypoxia, where hypoxia-inducible factors (HIFs) are activated and play critical roles in cellular functions. In this context, the role of Hif2α in neutrophils during myocardial infarction is unknown. Here, we demonstrate that neutrophil Hif2α deletion markedly attenuates myocardial infarct size, improves cardiac function, reduces neutrophil survival and tissue accumulation, and correlates with increased macrophage engulfment rates. Mechanistic studies revealed that Hif2α promotes neutrophil survival through binding to hypoxia response element (HRE) in the promoter region of Birc2 to regulate expression of the prosurvival factor, cellular inhibitor of apoptosis protein-1 (cIAP1). Inhibition of cIAP1 in neutrophils using the pharmacological agent, Birinapant resulted in increased cell death, establishing a critical role of cIAP1 downstream of Hif2α in neutrophil survival. Taken together, our data demonstrate a protective effect of Hif2α deletion in neutrophils on cardiac injury outcomes through modulation of neutrophil cell survival. NEW & NOTEWORTHY Hif2α in neutrophils increases infarct size, cardiac dysfunction, and ventricular scar after myocardial infarction. Hif2α in neutrophils supports neutrophil survival via cIAP-1 signaling and delays macrophage engulfment.

Caspase 9 (CASP9) is a well-known initiator caspase of intrinsic apoptosis. In humans, cIAP1 binds and induces degradation of the activated form of CASP9, but not pro-CASP9. In fish, the activity and regulation of CASP9 remain unknown. In this work, using flounder Paralichthys olivaceus as a representative species, we examined the regulatory mechanism of CASP9 in teleost. P. olivaceus CASP9 (PoCASP9) induced robust apoptosis, which was inhibited by P. olivaceus cIAP1 (PocIAP1). Unlike human cIAP1, PocIAP1 bound both pro- and active PoCASP9 and induced their degradation via the RING domain-involved proteasome pathway. In humans, the adaptor molecule CRADD cannot interact with CASP9. In contrast, P. olivaceus CRADD (PoCRADD) bound both pro- and active PoCASP9 via CARD-CARD interaction and enhanced apoptosis by promoting the cellular levels of pro- and active PoCASP9. Furthermore, PoCRADD abrogated the inhibition of PoCASP9 by PocIAP1 by preventing PocIAP1-PoCASP9 interaction. Together these results reveal a CASP9 regulation mechanism in teleost that differs from that in humans and demonstrate that teleost CASP9 is tightly and directly controlled by both negative and positive regulators that exert a regulation effect both before and after CASP9 activation. These findings advance our understanding of the regulation of CASP9-mediated apoptosis in vertebrates., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Introduction: The purpose of this research was to determine how the P53/microRNA-34a (miR-34a)/survivin pathway contributes to oxaliplatin-induced (L-OHP) cell inhibition in gastric cancer., Methods: The BGC-823 gastric cancer cells were selected, and we examined their viability following treatment with L-OHP at different concentrations and time periods. The expression levels of miR-34a, P53, and survivin in the cells were determined., Results: In the 12- and 24-h groups, drug concentration of 15 μg/cm² (p < .005 in both) significantly lowered cell viability. In comparison to the control group, miR-34a mRNA expression, P53 mRNA expression, and protein expression were all significantly greater in the 24-h group (p = .0324, p = .0069, p = .0260, respectively), but survivin mRNA and protein expressions were significantly lower than those in the control group (p = .0338, p = .0032, respectively). There was a significant decrease in gastric cancer cells in the miR-34a overexpression group (p = .0020), a significant increase in P53 mRNA and protein expression compared to the control group (p = .0080, p = .0121, respectively), and a significant decrease in survivin mRNA and protein expression compared to the control group. (p = .0213, p = .0069, respectively)., Conclusion: Oxaliplatin inhibits tumor growth, invasion, and metastasis by upregulating miR-34a, activating the expression of the upstream P53 gene, and driving the downregulation of survivin (P53/miR-34a/survivin axis) in BGC-823 gastric cancer cells., (© 2024 The Author(s). Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Background/aim: Glioblastoma is the most aggressive form of brain tumor and has a dismal prognosis; therefore, novel therapeutic approaches based on the mechanisms underlying its aggressive nature are urgently required. A growing body of evidence suggests that neurotransmitters play a key role in modulating the biology of glioblastoma; however, the role of melanocortins remains unclear., Materials and Methods: The effects of bremelanotide, a melanocortin receptor agonist, alone or in combination with chemotherapeutic agents, on survivin expression and cell viability were investigated in human glioblastoma cell lines., Results: Bremelanotide reduced survivin expression and induced cell death in glioblastoma cells at concentrations that were not toxic to normal human cells, and both of these effects were canceled in the presence of an antagonist of melanocortin receptors 3 and 4. Bremelanotide-induced cell death was prevented by the forced over-expression of survivin in glioblastoma cells, suggesting that bremelanotide induces glioblastoma cell death by inhibiting the expression of survivin. Bremelanotide also promoted cell death induced by chemotherapeutic agents, such as temozolomide and osimertinib., Conclusion: The present results identified melanocortin receptors 3 and 4 as novel and viable therapeutic targets for glioblastoma. Activation of these receptors by bremelanotide may inhibit the expression of survivin, thereby sensitizing glioblastoma cells to cell death., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

SIRT6, an evolutionarily conserved histone deacetylase, has been identified as a novel direct downstream target of Akt/FoxO3a and a tumor suppressor in colon cancer in our previous research. Nevertheless, the precise mechanisms through which SIRT6 hinders tumor development remain unclear. To ascertain whether SIRT6 directly impacts Survivin transcription, a ChIP assay was conducted using an anti-SIRT6 antibody to isolate DNA. YM155 was synthesized to explore Survivin's role in mitochondrial apoptosis, autophagy and tumor progression. Our investigation into the regulation of Survivin involved real-time fluorescence imaging in living cells, real-time PCR, immunohistochemistry, flow cytometry, and xenograft mouse assays. In this current study, we delved into the role of SIRT6 in colon cancer and established that activated SIRT6 triggers mitochondrial apoptosis by reducing Survivin expression. Subsequent examinations revealed that SIRT6 directly binds to the Survivin promoter, impeding its transcription. Notably, direct inhibition of Survivin significantly impeded colon cancer proliferation by inducing mitochondrial apoptosis and autophagy both in vitro and in vivo. More interestingly, Survivin inhibition reactivated the Akt/FoxO3a pathway and elevated SIRT6 levels, establishing a positive feedback loop. Our results identify Survivin as a novel downstream transcriptional target of SIRT6 that fosters tumor growth and holds promise as a prospective target for colon cancer therapy., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Bisphenol A (BPA) has been a substantial additive in plastics until the reports on its adverse effects have led to its restrictions and replacement. Monitoring studies document the increasing occurrence of bisphenol analogs, however, data on their effects and risks is still insufficient. Based on the indications that BPA might contribute to ovarian cancer pathogenesis, we examined effects of the analogs AF (BPAF), S (BPS) and F (BPF) (10 -9 -10 -4  M) on the Caov-3 epithelial cancer cells, including the impact on cell viability, proliferation, oxidative stress, and production and expression of several factors and genes related to ovarian cancer. At environmentally relevant doses, bisphenols did not exert significant effects. At the highest concentration, BPAF caused varied alterations, including decreased cell viability and proliferation, caspase activation, down-regulation of PCNA and BIRC5, elevation of IL8, VEGFA, MYC, PTGS2 and ABCB1 expressions. Only BPA (10 -4  M) increased IL-6, IL-8 and VEGFA output by the Caov-3 cells. Each bisphenol induced generation of reactive oxygen species and decreased superoxide dismutase activity at the highest concentration. Although the effects were observed only in the supraphysiological doses, the results indicate that certain bisphenol analogs might affect several ovarian cancer cell characteristics and merit further investigation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

AT-IAP (1-{6-[(4-fluorophenyl)methyl]-3,3-dimethyl-1H,2H,3H-pyrrolo[3,2-b]pyridin-1-yl}-2-[(2R,5R)-5-methyl-2-{[(3R)-3-methylmorpholin-4-yl]methyl}piperazin-1-yl]ethan-1-one) was identified as a novel potent non-alanine small molecule dual inhibitor of cIAP1/XIAP protein. AT-IAP was assessed in preclinical species, demonstrating favorable bioavailability in rodent species and oral efficacy at 30 mg/kg in MDA-MB-231 mouse xenograft models. The major metabolic route of AT-IAP was identified to be CYP3A driven, resulting in low oral exposure in non-human primate (NHP) studies, given the comparatively high expression of equivalent CYP3A. AT-IAP metabolite identification studies determined ring opening of the morpholino or piperazine moiety. An extensive campaign of optimisation resulted in increased polarity by the addition of a hydroxymethyl, which led to the identification of tolinapant (1-(6-[(4-Fluorophenyl)methyl]-5-(hydroxymethyl)-3,3-dimethyl-1 H,2 H,3 H-pyrrolo[3,2- b]pyridin-1-yl)-2-[(2 R,5 R)-5-methyl-2-([(3R)-3-methylmorpholin-4-yl]methyl)piperazin-1-yl]ethan-1-one) with reduced CYP3A metabolism. Tolinapant showed oral bioavailability in rodents and NHP in the range 12-34% at 5 mg/kg. Non-linear pharmacokinetics in NHP were observed at oral doses in the range 5-75 mg/kg. Pharmacodynamic modulation and efficacy were demonstrated in A375 mouse xenograft models at dose ranges between 5 and 20 mg/kg. On-target engagement, as measured by reduction of cIAP 1 protein levels, was confirmed in NHP surrogate tissues and applied to target activity assessments in tolinapant phase1/2 clinical trials., (© 2024 ASTEX PHARMACEUTICALS. Pharmacology Research & Perspectives published by British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics and John Wiley & Sons Ltd.)

Survivin holds significant importance as a member of the inhibitor of apoptosis protein (IAP) family due to its predominant expression in tumours rather than normal terminally differentiated adult tissues. The high expression level of survivin in tumours is closely linked to chemotherapy resistance, heightened tumour recurrence, and increased tumour aggressiveness and serves as a negative prognostic factor for cancer patients. Consequently, survivin has emerged as a promising therapeutic target for cancer treatment. In this review, we delve into the various biological characteristics of survivin in cancers and its pivotal role in maintaining immune system homeostasis. Additionally, we explore different therapeutic strategies aimed at targeting survivin.

Purpose: The abnormal growth factors-induced epithelial-mesenchymal transition (EMT) in retinal pigment epithelial (RPE) cells was known as a vital pathogenesis of proliferative vitreoretinopathy (PVR). This study aims to explore how survivin inhibition affects EMT induced by epidermal growth factor (EGF) in RPE cells., Methods: Human primary RPE cells were identified in vitro. EMT in RPE cells was induced by EGF. Inhibition of survivin in RPE cells was accomplished through the use of a survivin inhibitor (YM155) and survivin siRNA. The viability, proliferation and migration of RPE cells was detected by methylthiazol tetrazolium assay, bromodeoxyuridine labeling assay, and wound healing assay, respectively. The EGF receptor /mitogen-activated protein kinase (EGFR/MAPK) proteins and EMT-related proteins were measured by western blot and immunofluorescence assay., Results: EGF induced significant EMT in RPE cells, activated the phosphorylation of EGFR/MAPK signaling proteins, and caused changes to EMT-related proteins. YM155 suppressed RPE cells' viability, proliferation, and migration; induced the phosphorylation of EGFR, JNK, and P38MAPK; and down regulated EGFR and phosphorylated ERK. YM155 also increased expression of E-cadherin and ZO-1 proteins and reduced expression of N-cadherin, Vimentin, and α-SMA proteins. The EGF-induced increase of RPE cell proliferation and migration was constrained by survivin inhibition. Moreover, survivin inhibition in RPE cells suppressed the EGF-caused phosphorylation of EGFR/MAPK proteins and attenuated the EGF-induced reduction of E-cadherin and ZO-1 proteins and increase of N-cadherin, Vimentin, and α-SMA proteins., Conclusions: Survivin inhibition attenuates EGF-induced EMT of RPE cells by affecting the EGFR/MAPK signaling pathway. Survivin might be a promising target for preventing PVR., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Zhu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Chimeric 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., Methods: Human 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-DCs TNF ). The efficacy of M-DCs TNF in recognizing and treating breast cancer was tested in vitro and in vivo., Results: Mucin1 was highly expressed on the surface of a wide range of human breast cancer cell lines. M-DCs TNF directly associated with MDA-MB-231 cells in the bone of NSG mice. M-DCs TNF 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-DCs TNF combined with SM-164, but not SM-164 alone, inhibited the growth of patient-derived breast cancer in NSG mice., Conclusion: An 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., (© 2024. The Author(s).)

Background: APG-1387 is a novel second mitochondrial-derived activator of caspases mimetic, small-molecule inhibitor targeting inhibitor of apoptosis proteins. We report results from two phase I trials evaluating the tolerability, safety, and antitumor activity of APG-1387 monotherapy and APG-1387 plus toripalimab [a programmed cell death 1 (PD-1) inhibitor] for advanced solid tumors., Patients and Methods: Participants aged ≥18 years who had histologically confirmed advanced solid tumors with no appropriate standard of care (or refractory to standard care) were eligible. Patients received escalating intravenous doses of APG-1387 alone or combined with fixed-dose toripalimab (240 mg every 3 weeks) in a '3 + 3' design. Primary endpoints were dose-limiting toxicities (DLTs) and maximum tolerated dose (MTD) in the monotherapy trial, and recommended phase II dose (RP2D) in the combination therapy trial. Secondary endpoints included the pharmacokinetic and pharmacodynamic profiles and preliminary efficacy in both trials., Results: In the monotherapy trial, 28 subjects were enrolled and received ≥1 treatment cycle. No DLT was reported among the 28 subjects, and the MTD was not reached. One participant (3.6%) had a grade ≥3 treatment-related adverse event (TRAE) of alanine aminotransferase elevation. In efficacy analysis of 23 participants, none achieved an objective response, and the disease control rate was 21.7%. In the combination trial, 22 subjects were enrolled and included in all analyses. There was one DLT of grade 3 lipase elevation. The MTD was not reached. Four grade ≥3 TRAEs occurred in three participants (13.6%), with the most common being lipase elevation (n = 2). The RP2D was 45 mg weekly. The objective response rate was 13.6%, with complete response achieved in one subject, and the disease control rate was 54.5%., Conclusions: APG-1387 45 mg weekly plus toripalimab was well tolerated and is recommended for further study, with preliminary clinical activity observed in study participants with advanced solid tumors., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Acute respiratory distress syndrome (ARDS)/acute lung injury (ALI) is a severe complication of sepsis characterized by acute respiratory distress, hypoxemia, and diffuse bilateral pulmonary infiltrates. The regulation of RIPK1 is an important part of the inflammatory response, and cIAP1/2 serves as the E3 ubiquitin ligase for RIPK1. In this study, we investigated the effect and mechanism of cIAP1/2 inhibition on sepsis-induced lung injury. Our results showed that cIAP1/2 inhibition can alleviate sepsis-induced lung injury and reduce the inflammatory response, which is accompanied by downregulation of RIPK1 phosphorylation and ubiquitination. Additionally, cIAP1/2 inhibition led to the up-regulation of programmed cell death, including apoptosis, necroptosis, and pyroptosis, and inhibiting these three cell death pathways can further reduce the inflammatory response, which is similar to the recently discovered programmed cell death pathway PANoptosis. Our findings suggest that cIAP1/2 and PANoptosis inhibition may be a new strategy for treating sepsis-induced lung injury and provide important references for further exploring the mechanism of sepsis-induced lung injury and identifying new therapeutic targets., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Inhibitors of Apoptosis proteins (IAPs) were discovered through experiments aimed at rescuing apoptosis in insects. Classically associated with the inhibition of apoptosis, the IAP member Survivin also regulates cell cycle progression and is an essential component of the Chromosomal Passenger Complex (CPC), responsible for chromosomal segregation. Although undetectable in most adult tissues, Survivin is expressed in Adult Stem Cells (ASCs) and plays a crucial role in their maintenance. Survivin is overexpressed in most cancers, contributing to their clonal expansion. As a result, it has been proposed as a possible anticancer target for nearly two decades. In this discussion, we will explore the rationale behind Survivin as a therapeutic target, focusing on common cancer types such as carcinomas, sarcomas, and leukemias. We will delve into the modulation of Survivin by cancer pro-survival cell signaling, the association between SNPs and tumorigenesis, and its regulation by miRNAs. Finally, we will compare cell growth, clonogenic capacity, and apoptosis, along with different strategies for Survivin inhibition, including gene expression and protein activity modulation., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare. All co-authors have seen and agree with the contents of the manuscript and there is no financial interest to report., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Metabolic reprogramming and abnormal glucose metabolism are hallmarks of head and neck squamous cell carcinoma (HNSCC). Certain oncogenes can promote cancer-related metabolic changes, but understanding their crosstalk in HNSCC biology and treatment is essential for identifying predictive biomarkers and developing target therapies., Methods: We assessed the value of survivin/BIRC5 as a radioresistance factor potentially modulated by glucose for predicting therapeutic sensitivity and prognosis of HNSCC in a cohort of 32 patients. Additionally, we conducted in vitro experiments to explore the role of survivin/BIRC5 in glucose metabolism concerning radiation response., Results: Tumoral BIRC5 expression is associated with serum glucose and predicts locoregional disease-free survival and lower BIRC5 mRNA levels are associated with better outcomes. Upregulation of BIRC5 by radiation depends on glucose levels and provokes a pro-tumoral and radioresistant phenotype in surviving cells., Conclusions: Survivin/BIRC5 might be independently associated with the risk of recurrence in patients with HNSCC., (© 2024 The Authors. Head & Neck published by Wiley Periodicals LLC.)

Chemotherapeutic agents for treating colorectal cancer (CRC) primarily induce apoptosis in tumor cells. The ubiquitin-proteasome system is critical for apoptosis regulation. Deubiquitinating enzymes (DUBs) remove ubiquitin from substrates to reverse ubiquitination. Although over 100 DUB members have been discovered, the biological functions of only a small proportion of DUBs have been characterized. Here, we aimed to systematically identify the DUBs that contribute to the development of CRC. Among the DUBs, ubiquitin-specific protease 36 (USP36) is upregulated in CRC. We showed that the knockdown of USP36 induces intrinsic and extrinsic apoptosis. Through gene silencing and coimmunoprecipitation techniques, we identified survivin and cIAP1 as USP36 targets. Mechanistically, USP36 binds and removes lysine-11-linked ubiquitin chains from cIAP1 and lysine-48-linked ubiquitin chains from survivin to abolish protein degradation. Overexpression of USP36 disrupts the formation of the XIAP-second mitochondria-derived activator of caspase complex and promotes receptor-interacting protein kinase 1 ubiquitination, validating USP36 as an inhibitor to intrinsic and extrinsic apoptosis through deubiquitinating survivin and cIAP1. Therefore, our results suggest that USP36 is involved in CRC progression and is a potential therapeutic target., Competing Interests: Conflict of interests The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Second mitochondrial-derived activator of caspase (SMAC), also known as direct inhibitor of apoptosis-binding proteins with low pI (Diablo), is known as a pro-apoptotic mitochondrial protein released into the cytosol in response to apoptotic signals. We recently reported SMAC overexpression in cancers as essential for cell proliferation and tumor growth due to non-apoptotic functions, including phospholipid synthesis regulation. These functions may be associated with its interactions with partner proteins. Using a peptide array with 768 peptides derived from 11 selected SMAC-interacting proteins, we identified SMAC-interacting sequences. These SMAC-binding sequences were produced as cell-penetrating peptides targeted to the cytosol, mitochondria, or nucleus, inhibiting cell proliferation and inducing apoptosis in several cell lines. For in vivo study, a survivin/baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5)-derived peptide was selected, due to its overexpression in many cancers and its involvement in mitosis, apoptosis, autophagy, cell proliferation, inflammation, and immune responses, as a target for cancer therapy. Specifically, a SMAC-targeting survivin/BIRC5-derived peptide, given intratumorally or intravenously, strongly inhibited lung tumor growth, cell proliferation, angiogenesis, and inflammation, induced apoptosis, and remodeled the tumor microenvironment. The peptide promoted tumor infiltration of CD-8 + cells and increased cell-intrinsic programmed cell death protein 1 (PD-1) and programmed cell death ligand 1 (PD-L1) expression, resulting in cancer cell self-destruction and increased tumor cell death, preserving immune cells. Thus, targeting the interaction between the multifunctional proteins SMAC and survivin represents an innovative therapeutic cancer paradigm., Competing Interests: Declaration of interests The authors declare that there are no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

We aimed to explore the role of regulating Smac expression levels in the occurrence and development of colon cancer through in vitro and in vivo experiments. Colon cancer cells HT-29 were cultured and transfected into different groups. qRT-PCR was used to detect the expression level of Smac in cells; Flow cytometry was used to detect the apoptotic ability of each group of cells; Western blot was used to detect the protein expression of Smac and apoptosis-related factors Survivin and Caspase-3; The nude mouse tumorigenesis experiment was conducted to detect the regulatory effect of regulating Smac expression levels on the growth of colon cancer transplanted tumors in vivo. In comparison to the FHC group, the HT-29 group exhibited a decrease in Smac expression. The si-Smac group, when compared with the si-NC group, showed significant reductions in Smac mRNA and protein levels, weaker cell apoptosis, increased Survivin, and decreased Caspase-3 expression. Contrarily, the oe-Smac group, against the oe-NC group, displayed increased Smac mRNA and protein levels, enhanced apoptosis, reduced Survivin, and elevated Caspase-3 expression. In nude mice tumor transplantation experiments, the LV-sh-Smac group, as opposed to the LV-sh-NC group, had tumors with greater volume and weight, reduced Smac and Caspase-3, and increased Survivin expression. In contrast, the LV-oe-Smac group, compared with the LV-oe-NC group, showed tumors with decreased volume and mass, increased expressions of Smac and Caspase-3, and decreased Survivin. Smac is lowly expressed in colon cancer. Upregulation of Smac expression can inhibit the occurrence and development of colon cancer, possibly by inhibiting Survivin expression and promoting Caspase-3 expression, thereby enhancing the pro-apoptotic function.

Lung adenocarcinoma (LUAD) is a subtype of lung cancer with high incidence and mortality globally. Emerging evidence suggests that circular RNAs (circRNAs) exert critical functions in human cancers, including LUAD. CircRNA&#95;100549 (circ&#95;100549) has been reported to be significantly upregulated in non-small cell lung cancer (NSCLC) samples, while its role in modulating LUAD progression remains to be explored. The current study aims at investigating the functional roles of circ&#95;100549 in LUAD and its downstream molecular mechanism. First, we found that the expression of circ&#95;100549 was higher in LUAD cell lines. Loss-of-function assays verified that depletion of circ&#95;100549 repressed LUAD cell proliferation but accelerated cell apoptosis. Furthermore, in vivo experiments demonstrated that silencing of circ&#95;100549 suppressed tumor growth. Subsequently, based on database analysis, we carried out a series of experiments to explore the mechanisms and effects of circ&#95;100549 underlying LUAD progression, including RNA-binding protein immunoprecipitation (RIP), RNA/DNA pull-down, luciferase reporter, and chromatin immunoprecipitation (ChIP) assays. The results indicated that circ&#95;100549 serves as a ceRNA by sponging miR-95-5p to upregulate BPTF expression, thus upregulating BIRC6 expression at a transcriptional level in LUAD. In summary, our study demonstrated that circ&#95;100549 facilitates LUAD progression by upregulating BIRC6 expression., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Upregulation of the multidrug efflux pump ABCB1/MDR1 (P-gp) and the anti-apoptotic protein BIRC5/Survivin promotes multidrug resistance in various human cancers. GDC-0152 is a DIABLO/SMAC mimetic currently being tested in patients with solid tumors. However, it is still unclear whether GDC-0152 is therapeutically applicable for patients with ABCB1-overexpressing multidrug-resistant tumors, and the molecular mechanism of action of GDC-0152 in cancer cells is still incompletely understood. In this study, we found that the potency of GDC-0152 is unaffected by the expression of ABCB1 in cancer cells. Interestingly, through in silico and in vitro analysis, we discovered that GDC-0152 directly modulates the ABCB1-ATPase activity and inhibits ABCB1 multidrug efflux activity at sub-cytotoxic concentrations (i.e., 0.25×IC 50 or less). Further investigation revealed that GDC-0152 also decreases BIRC5 expression, induces mitophagy, and lowers intracellular ATP levels in cancer cells at low cytotoxic concentrations (i.e., 0.5×IC 50 ). Co-treatment with GDC-0152 restored the sensitivity to the known ABCB1 substrates, including paclitaxel, vincristine, and YM155 in ABCB1-expressing multidrug-resistant cancer cells, and it also restored the sensitivity to tamoxifen in BIRC5-overexpressing tamoxifen-resistant breast cancer cells in vitro. Moreover, co-treatment with GDC-0152 restored and potentiated the anticancer effects of paclitaxel in ABCB1 and BIRC5 co-expressing xenograft tumors in vivo. In conclusion, GDC-0152 has the potential for use in the management of cancer patients with ABCB1 and BIRC5-related drug resistance. The findings of our study provide essential information to physicians for designing a more patient-specific GDC-0152 clinical trial program in the future., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Purpose: Oral Squamous Cell Carcinoma (OSCC) is the 6th most common cancer worldwide. It is generally aggressive and closely associated with chemoresistance and poor survival. There is accumulating evidence for the involvement of inhibitors of apoptosis proteins (IAPs), including IAP1 and XIAP, in mediating chemotherapy resistance in OSCC. Various strategies for targeting IAPs have been designed and tested in recent years and several small molecule IAP inhibitors are in clinical trials as monotherapies as well as in combination with radiotherapy and chemotherapy. The purpose of this study was to evaluate and compare the efficacy and biological activity of three IAP inhibitors both as stand-alone and sensitising agents to cisplatin in a preclinical model of squamous cell carcinoma of the tongue., Methods: Cisplatin-sensitive SCC4 and -resistant SCC4cisR cells were utilised in this study. Apoptosis was evaluated by flow cytometric analysis of Annexin V/Propidium Iodide-stained cells. Expression of IAP proteins was determined by western blotting and knockdown of cIAP1, livin and XIAP was conducted by transfection of cells with siRNA., Results: We establish for the first time the therapeutic efficacy of the Smac mimetic, BV6 and the XIAP inhibitor Embelin, for OSCC. Both of these IAP targeting agents synergistically enhanced cisplatin-mediated apoptotic cell death in resistant cells which was mediated in part by depletion of XIAP. In addition, knockdown of XIAP using siRNA enhanced cisplatin-mediated cell death, demonstrating the importance of targeting XIAP in this sensitisation., Conclusion: These findings provide pre-clinical evidence that IAP inhibition may be a valuable therapeutic option in OSCC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The hypomethylation agent decitabine (DAC), in combination with other apoptosis inducers, is considered a potential modality for cancer treatment. We investigated the mechanism underlying the combined cytotoxicity of DAC and YM155 in acute myeloid leukemia (AML) cells because of increasing evidence that YM155 induces apoptosis in cancer cells. Co-administration of DAC and YM155 resulted in synergistic cytotoxicity in AML U937 cells, which was characterized by the induction of apoptosis, NOXA-dependent degradation of MCL1 and survivin, and depolarization of mitochondria. Restoration of MCL1 or survivin expression attenuated DAC/YM155-induced U937 cell death. DAC initiated AKT and p38 MAPK phosphorylation in a Ca 2+ /ROS-dependent manner, thereby promoting autophagy-mediated degradation of β-TrCP mRNA, leading to increased Sp1 expression. DAC-induced Sp1 expression associated with Ten-eleven-translocation (TET) dioxygenases and p300 was used to upregulate the expression of SLC35F2. Simultaneously, the activation of p38 MAPK induced by DAC, promoted CREB-mediated NOXA expression, resulting in survivin and MCL1 degradation. The synergistic cytotoxicity of DAC and YM155 in U937 cells was dependent on elevated SLC35F2 expression. Additionally, YM155 facilitated DAC-induced degradation of MCL1 and survivin. A similar mechanism explained DAC/YM155-mediated cytotoxicity in AML HL-60 cells. Our data demonstrated that the synergistic cytotoxicity of DAC and YM155 in AML cell lines U937 and HL-60 is dependent on AKT- and p38 MAPK-mediated upregulation of SLC35F2 and p38 MAPK-mediated degradation of survivin and MCL1. This indicates that a treatment regimen that amalgamates YM155 and DAC may be beneficial for AML., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Bivalent Smac mimetics have been shown to possess binding affinity and pro-apoptotic activity similar to or more potent than that of native Smac, a protein dimer able to neutralize the anti-apoptotic activity of an inhibitor of caspase enzymes, XIAP, which endows cancer cells with resistance to anticancer drugs. We design five new bivalent Smac mimetics, which are formed by various linkers tethering two diazabicyclic cores being the IAP binding motifs. We built in silico models of the five mimetics by the TwistDock workflow and evaluated their conformational tendency, which suggests that compound 3, whose linker is n-hexylene, possess the highest binding potency among the five. After synthesis of these compounds, their ability in tumour cell growth inhibition and apoptosis induction displayed in experiments with SK-OV-3 and MDA-MB-231 cancer cell lines confirms our prediction. Among the five mimetics, compound 3 displays promising pro-apoptotic activity and deserves further optimization., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Cancer selective apoptosis remains a therapeutic challenge and off-target toxicity has limited enthusiasm for this target clinically. Sigma-2 ligands (S2) have been shown to enhance the cancer selectivity of small molecule drug candidates by improving internalization. Here, we report the synthesis of a novel drug conjugate, which was created by linking a clinically underperforming SMAC mimetic (second mitochondria-derived activator of caspases; LCL161), an inhibitor (antagonist) of inhibitor of apoptosis proteins (IAPinh) with the sigma-2 ligand SW43, resulting in the new chemical entity S2/IAPinh. Drug potency was assessed via cell viability assays across several pancreatic and ovarian cancer cell lines in comparison with the individual components (S2 and IAPinh) as well as their equimolar mixtures (S2 + IAPinh) both in vitro and in preclinical models of pancreatic and ovarian cancer. Mechanistic studies of S2/IAPinh-mediated cell death were investigated in vitro and in vivo using syngeneic and xenograft mouse models of murine pancreatic and human ovarian cancer, respectively. S2/IAPinh demonstrated markedly improved pharmacological activity in cancer cell lines and primary organoid cultures when compared to the controls. In vivo testing demonstrated a marked reduction in tumor growth rates and increased survival rates when compared to the respective control groups. The predicted mechanism of action of S2/IAPinh was confirmed through assessment of apoptosis pathways and demonstrated strong target degradation (cellular inhibitor of apoptosis proteins-1 [cIAP-1]) and activation of caspases 3 and 8. Taken together, S2/IAPinh demonstrated efficacy in models of pancreatic and ovarian cancer, two challenging malignancies in need of novel treatment concepts. Our data support an in-depth investigation into utilizing S2/IAPinh for the treatment of cancer., (© 2024. The Author(s).)

Cell death and proliferation are at a glance dichotomic events, but occasionally coupled. Caspases, traditionally known to execute apoptosis, play non-apoptotic roles, but their exact mechanism remains elusive. Here, using Drosophila intestinal stem cells (ISCs), we discovered that activation of caspases induces massive cell proliferation rather than cell death. We elucidate that a positive feedback circuit exists between caspases and JNK, which can simultaneously drive cell proliferation and cell death. In ISCs, signalling from JNK to caspases is defective, which skews the balance towards proliferation. Mechanistically, two-tiered regulation of the DIAP1 inhibitor rpr , through its transcription and its protein localization, exists. This work provides a conceptual framework that explains how caspases perform apoptotic and non-apoptotic functions in vivo and how ISCs accomplish their resistance to cell death., (© 2024 Sulekh et al.)

Procaspase 9 is the initiator caspase for apoptosis, but how its levels and activities are maintained remains unclear. The gigantic Inhibitor-of-Apoptosis Protein BIRC6/BRUCE/Apollon inhibits both apoptosis and autophagy by promoting ubiquitylation of proapoptotic factors and the key autophagic protein LC3, respectively. Here we show that BIRC6 forms an anti-parallel U-shaped dimer with multiple previously unannotated domains, including a ubiquitin-like domain, and the proapoptotic factor Smac/DIABLO binds BIRC6 in the central cavity. Notably, Smac outcompetes the effector caspase 3 and the pro-apoptotic protease HtrA2, but not procaspase 9, for binding BIRC6 in cells. BIRC6 also binds LC3 through its LC3-interacting region, probably following dimer disruption of this BIRC6 region. Mutation at LC3 ubiquitylation site promotes autophagy and autophagic degradation of BIRC6. Moreover, induction of autophagy promotes autophagic degradation of BIRC6 and caspase 9, but not of other effector caspases. These results are important to understand how the balance between apoptosis and autophagy is regulated under pathophysiological conditions., (© 2024. The Author(s).)

Overexpression of inhibitor of apoptosis (IAP) proteins is associated with poor prognosis. In multiple myeloma (MM), the IAP inhibitors (IAPi), LCL161, have been evaluated in preclinical and clinical settings but are not fully effective. Among IAPs, XIAP has the strongest anti-apoptotic function with direct binding activity to caspases and cIAP1 and cIAP2 are positive regulator of NF-κB signaling. Prior IAPi such as LCL161 has high affinity to cIAP1 and cIAP2 resulting in inferior inhibiting activity against XIAP. A novel dimeric IAPi, AZD5582 (C 58 H 78 N 8 O 8 ), have high binding potency to XIAP with EC50 dose of 15 nM, enabling to simultaneous inhibit XIAP and cIAP1/2. AZD5582 monotherapy showed cell growth inhibition for all MM cell lines, MM1S, RPMI8226, U266 and KMS-5 and induced apoptosis. AZD5582 further showed anti-proliferation effect under the IL-6 additional condition and inhibited JAK-STAT signaling triggered by IL-6. AZD5582 combined with carfilzomib therapy showed a synergistic effect. Enhanced apoptosis was also observed in combination therapy. Synergistic effect was further observed with other conventional therapeutics. Simultaneous XIAP and cIAP1/2 inhibition by the dimeric IAPi AZD5582 is promising. This study provides a rationale of AZD5582 as a new treatment strategy in monotherapy and in combination therapy., Competing Interests: Declaration of competing interest The authors declare they have no conflict of interest to disclose., (Copyright © 2023 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Survivin, a homodimeric protein and a member of the IAP family, plays a vital function in cell survival and cycle progression by interacting with various proteins and complexes. Its expression is upregulated in cancers but not detectable in normal tissues. Thus, it has been regarded and validated as an ideal cancer target. However, survivin is "undruggable" due to its lack of enzymatic activities or active sites for small molecules to bind/inhibit. Academic and industrial laboratories have explored different strategies to overcome this hurdle over the past two decades, with some compounds advanced into clinical testing. These strategies include inhibiting survivin expression, its interaction with binding partners and homodimerization. Here, we provide comprehensive analyses of these strategies and perspective on different small molecule survivin inhibitors to help drug discovery targeting "undruggable" proteins in general and survivin specifically with a true survivin inhibitor that will prevail in the foreseeable future.

Survivin is a member of the family of inhibitors of apoptosis proteins (IAPs). It is involved in the normal mitotic process and acts as an anti-apoptotic molecule. While terminally differentiated normal tissues lack survivin, several human malignancies have significant protein levels. Resistance to chemotherapy and radiation in tumor cells is associated with survivin expression. Decreased tumor development, apoptosis, and increased sensitivity to chemotherapy and radiation are all effects of downregulating survivin expression or activity. As a prospective cancer treatment, small molecules targeting the transcription and translation of survivin and molecules that can directly bind with the survivin are being explored both in pre-clinical and clinics. Pre-clinical investigations have found and demonstrated the effectiveness of several small-molecule survivin inhibitors. Unfortunately, these inhibitors have also been shown to have off-target effects, which could limit their clinical utility. In addition to small molecules, several survivin peptide vaccines are currently under development. These vaccines are designed to elicit a cytotoxic T-cell response against survivin, which could lead to the destruction of tumor cells expressing survivin. Some survivin-based vaccines are advancing through Phase II clinical studies. Overall, survivin is a promising cancer drug target. However, challenges still need to be addressed before the survivin targeted therapies can be widely used in the clinics., Competing Interests: Declaration of competing interest The authors declare that no conflicts of interests., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Multidisciplinary therapy centered on antitumor drugs is indicated in patients with unresectable pancreatic neuroendocrine tumors (PanNET). However, the criteria for selection of optimal therapeutic agents is controversial. The aim of this study was to assess the malignancy of PanNET for optimal therapeutic drug selection., Methods: Forty-seven patients with PanNET who underwent surgery were reviewed retrospectively, and immunohistochemical characteristics, including expression of GLUT1, SSTR2a, SSTR5, Survivin, X-chromosome-linked inhibitor of apoptosis protein (XIAP), and Caspase3 in the resected specimens, were investigated. Relapse-free survival (RFS) and overall survival (OS) were evaluated with regard to the characteristics using the Kaplan-Meier method and compared with the log-rank test., Results: GLUT1 expression showed significant correlation with sex (p = 0.036) and mitotic rate (p = 0.048). Survivin and XIAP expression showed significant correlation with T-stage (p = 0.014 and 0.009), p-Stage (p = 0.028 and 0.045), and mitotic rate (p = 0.023 and 0.007). XIAP expression also significantly influenced OS (p = 0.044)., Conclusions: Survivin and XIAP correlated with grade of malignancy, and expression of XIAP in particular was associated with a poor prognosis. Expression of these proteins may be a useful indicator to select optimal therapeutic agents in PanNET., (© 2023. The Author(s).)

Purpose: Chemoresistance is a primary factor for treatment failure and tumor recurrence in non-small cell lung cancer (NSCLC) patients. The oncoprotein survivin is commonly upregulated in human malignancies and is associated with poor prognosis, but its effect on carcinogenesis and chemoresistance in NSCLC is not yet evident, and to explore an effective inhibitor targeting survivin expression is urgently needed., Methods: The protumor characteristics of survivin and antitumor activities of bergenin in NSCLC cells were examined by MTS, colony formation assays, immunoblot, immunohistochemistry, and in vivo xenograft development., Results: Survivin was upregulated in non-small cell lung cancer (NSCLC) tissues, while its depletion inhibited NSCLC tumorigenesis. The current study focused on bergenin, identifying its effective antitumor effect on NSCLC cells both in vivo and in vitro. The results showed that bergenin could inhibit cell proliferation and induce the intrinsic pathway of apoptosis via downregulating survivin. Mechanistically, bergenin reduced the phosphorylation of survivin via inhibiting the Akt/Wee1/CDK1 signaling pathway, thus resulting in enhanced interaction between survivin and E3 ligase Fbxl7 to promote survivin ubiquitination and degradation. Furthermore, bergenin promoted chemoresistance in NSCLC cells re-sensitized to pemetrexed treatment., Conclusions: Survivin overexpression is required for maintaining multiple malignant phenotypes of NSCLC cells. Bergenin exerts a potent antitumor effect on NSCLC via targeting survivin, rendering it a promising agent for the treatment of NSCLC., (© 2023. Springer Nature Switzerland AG.)

Immunotoxins (ITs) target cancer cells via antibody binding to surface antigens followed by internalization and toxin-mediated inhibition of protein synthesis. The fate of cells responding to IT treatment depends on the amount and stability of specific pro-apoptotic and pro-survival proteins. When treated with a pseudomonas exotoxin-based immunotoxin (HB21PE40), the triple-negative breast cancer (TNBC) cell line MDA-MB-468 displayed a notable resistance to toxin-mediated killing compared to the epidermoid carcinoma cell line, A431, despite succumbing to the same level of protein synthesis inhibition. In a combination screen of ~1912 clinically relevant and mechanistically annotated compounds, we identified several agents that greatly enhanced IT-mediated killing of MDA-MB-468 cells while exhibiting only a modest enhancement for A431 cells. Of interest, two Smac mimetics, birinapant and SM164, exhibited this kind of differential enhancement. To investigate the basis for this, we probed cells for the presence of inhibitor of apoptosis (IAP) proteins and monitored their stability after the addition of immunotoxin. We found that high levels of IAPs inhibited immunotoxin-mediated cell death. Further, TNFα levels were not relevant for the combination's efficacy. In tumor xenograft studies, combinations of immunotoxin and birinapant caused complete regressions in MDA-MB-468tumor-bearing mice but not in mice with A431 tumors. We propose that IAPs constitute a barrier to immunotoxin efficacy which can be overcome with combination treatments that include Smac mimetics., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA.)

XIAP, the most potent mammalian inhibitor of apoptosis protein (IAP), critically restricts developmental culling of sympathetic neuronal progenitors, and is correspondingly overexpressed in most MYCN-amplified neuroblastoma tumors. Because apoptosis-related protein in the TGFβ signaling pathway (ARTS) is the only XIAP antagonist that directly binds and degrades XIAP, we evaluated the preclinical effectiveness and tolerability of XIAP antagonism as a novel targeting strategy for neuroblastoma. We found that antagonism of XIAP, but not other IAPs, triggered apoptotic death in neuroblastoma cells. XIAP silencing induced apoptosis while overexpression conferred protection from drug-induced apoptosis. From a screen of IAP inhibitors, first-in-class ARTS mimetic A4 was most effective against high-risk and high XIAP-expressing neuroblastoma cells, and least toxic toward normal liver- and bone marrow-derived cells, compared with pan-IAP antagonists. On target engagement assays and nuclear magnetic resonance spectroscopy, A4 was observed to degrade rather than inhibit XIAP, catalyzing rapid degradation of XIAP through the ubiquitin-proteasome pathway. In MYCN-amplified neuroblastoma patient-derived xenografts, A4 significantly prolonged survival as a single agent, and demonstrated synergism with standard-of-care agents to reduce their effective required doses 3- to 6-fold. Engagement and degradation of XIAP by ARTS mimetics is a novel targeting strategy for neuroblastoma that may be especially effective against MYCN-amplified disease with intrinsically high XIAP expression. First-in-class ARTS mimetic A4 demonstrates preclinical efficacy and warrants further development and study., Significance: XIAP degradation is sufficient to kill MYCN-amplified neuroblastoma which overexpresses and relies on XIAP as a brake against cell death, without affecting normal cells., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Cellular inhibitor of apoptosis proteins (cIAPs) are RING-containing E3 ubiquitin ligases that ubiquitylate receptor-interacting protein kinase 1 (RIPK1) to regulate TNF signalling. Here, we established mice simultaneously expressing enzymatically inactive cIAP1/2 variants, bearing mutations in the RING domains of cIAP1/2 (cIAP1/2 mutant RING, cIAP1/2 MutR ). cIap1/2 MutR/MutR mice died during embryonic development due to RIPK1-mediated apoptosis. While expression of kinase-inactive RIPK1 D138N rescued embryonic development, Ripk1 D138N/D138N /cIap1/2 MutR/MutR mice developed systemic inflammation and died postweaning. Cells expressing cIAP1/2 MutR and RIPK1 D138N were still susceptible to TNF-induced apoptosis and necroptosis, implying additional kinase-independent RIPK1 activities in regulating TNF signalling. Although further ablation of Ripk3 did not lead to any phenotypic improvement, Tnfr1 gene knock-out prevented early onset of systemic inflammation and premature mortality, indicating that cIAPs control TNFR1-mediated toxicity independent of RIPK1 and RIPK3. Beyond providing novel molecular insights into TNF-signalling, the mouse model established in this study can serve as a useful tool to further evaluate ongoing therapeutic protocols using inhibitors of TNF, cIAPs and RIPK1., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Bruton's tyrosine kinase inhibitors (BTKi) and CAR T-cell therapy have demonstrated tremendous clinical benefits in mantle cell lymphoma (MCL) patients, but intrinsic or acquired resistance inevitably develops. In this study, we assessed the efficacy of the highly potent and selective MCL-1 inhibitor AZD5991 in various therapy-resistant MCL cell models. AZD5991 markedly induced apoptosis in these cells. In addition to liberating BAK from the antiapoptotic MCL-1/BAK complex for the subsequent apoptosis cascade, AZD5991 downregulated inhibitor of apoptosis proteins (IAPs) through a BAK-dependent mechanism to amplify the apoptotic signal. The combination of AZD5991 with venetoclax enhanced apoptosis and reduced mitochondrial oxygen consumption capacity in MCL cell lines irrespective of their BTKi or venetoclax sensitivity. This combination also dramatically inhibited tumor growth and prolonged mouse survival in two aggressive MCL patient-derived xenograft models. Mechanistically, the augmented cell lethality was accompanied by the synergistic suppression of IAPs. Supporting this notion, the IAP antagonist BV6 induced dramatic apoptosis in resistant MCL cells and sensitized the resistant MCL cells to venetoclax. Our study uncovered another unique route for MCL-1 inhibitor to trigger apoptosis, implying that the pro-apoptotic combination of IAP antagonists and apoptosis inducers could be further exploited for MCL patients with multiple therapeutic resistance., (© 2023. The Author(s).)

Aim: Rheumatoid arthritis (RA) is a chronic inflammation mediated by an autoimmune response. Baculoviral IAP repeat-containing 2 (BIRC2) and tumor necrosis factor receptor 1-associated death domain protein (TRADD) have been reported to be highly expressed in RA, while their specific roles during RA progression remain unclear. This study aims to explore the specific regulation of BIRC2/TRADD during the progression of RA., Methods: C28/I2 cells were stimulated by lipopolysaccharide (LPS) to establish an in vitro RA cellular model. The expression level of BIRC2 and TRADD was examined by quantitative real-time polymerase chain reaction and western blot. Cell Counting Kit-8 and flow cytometry assays were performed to examine cell viability and necroptosis, respectively. The oxidative stress markers were detected using commercial kits, and the pro-inflammatory cytokines were measured by ELISA assay. The interaction between BIRC2 and TRADD was verified by co-immunoprecipitation assay., Results: BIRC2 and TRADD were discovered to be highly expressed in LPS-mediated C28/I2 cells. BIRC2 knockdown was demonstrated to inhibit LPS-induced cell viability loss, necroptosis, oxidative stress, and inflammation in C28/I2 cells. BIRC2 could interact with TRADD and positively regulate TRADD expression. In addition, the protective role of BIRC2 knockdown against LPS-mediated injuries in C28/I2 cells was partly weakened by TRADD overexpression., Conclusion: In summary, BIRC2 knockdown alleviated necroptosis, oxidative stress, and inflammation in LPS-mediated C28/I2 cells, which might correlate to the regulatory role of TRADD, indicating a novel target for the treatment of RA., (© 2023 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Drug tolerance and minimal residual disease (MRD) are likely to prelude acquired resistance to targeted therapy. Mechanisms that allow persister cells to survive in the presence of targeted therapy are being characterized but selective vulnerabilities for these subpopulations remain uncertain. We identified cellular inhibitor of apoptosis protein 2 (cIAP2) as being highly expressed in SOX10-deficient drug tolerant persister (DTP) melanoma cells. Here, we show that cIAP2 is sufficient to induce tolerance to MEK inhibitors, likely by decreasing the levels of cell death. Mechanistically, cIAP2 is upregulated at the transcript level in SOX10-deficient cells and the AP-1 complex protein, JUND, is required for its expression. Using a patient-derived xenograft model, we demonstrate that treatment with the cIAP1/2 inhibitor, birinapant, during the MRD phase delays the onset of resistance to BRAF inhibitor and MEK inhibitor combination therapy. Together, our data suggest that cIAP2 upregulation in SOX10-deficient subpopulations of melanoma cells induces drug tolerance to MAPK targeting agents and provides a rationale to test a novel therapeutical approach to target MRD., (©2023 American Association for Cancer Research.)

The inhibitor of apoptosis protein BIRC2 regulates fundamental cell death and survival signaling pathways. Here we show that BIRC2 accumulates in the nucleus via binding of its second and third BIR domains, BIRC2 BIR2 and BIRC2 BIR3 , to the histone H3 tail and report the structure of the BIRC2 BIR3 -H3 complex. RNA-seq analysis reveals that the genes involved in interferon and defense response signaling and cell-cycle regulation are most affected by depletion of BIRC2. Overexpression of BIRC2 delays DNA damage repair and recovery of the cell-cycle progression. We describe the structural mechanism for targeting of BIRC2 BIR3 by a potent but biochemically uncharacterized small molecule inhibitor LCL161 and demonstrate that LCL161 disrupts the association of endogenous BIRC2 with H3 and stimulates cell death in cancer cells. We further show that LCL161 mediates degradation of BIRC2 in human immunodeficiency virus type 1-infected human CD4 + T cells. Our findings provide mechanistic insights into the nuclear accumulation of and blocking BIRC2., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Survivin, a well-known member of the inhibitor of apoptosis protein family, is upregulated in many cancer cells, which is associated with resistance to chemotherapy. To circumvent this, inhibitors are currently being developed to interfere with the nuclear export of survivin by targeting its protein-protein interaction (PPI) with the export receptor CRM1. Here, we combine for the first time a supramolecular tweezer motif, sequence-defined macromolecular scaffolds, and ultrasmall Au nanoparticles (us-AuNPs) to tailor a high avidity inhibitor targeting the survivin-CRM1 interaction. A series of biophysical and biochemical experiments, including surface plasmon resonance measurements and their multivalent evaluation by EVILFIT, reveal that for divalent macromolecular constructs with increasing linker distance, the longest linkers show superior affinity, slower dissociation, as well as more efficient PPI inhibition. As a drawback, these macromolecular tweezer conjugates do not enter cells, a critical feature for potential applications. The problem is solved by immobilizing the tweezer conjugates onto us-AuNPs, which enables efficient transport into HeLa cells. On the nanoparticles, the tweezer valency rises from 2 to 16 and produces a 100-fold avidity increase. The hierarchical combination of different scaffolds and controlled multivalent presentation of supramolecular binders was the key to the development of highly efficient survivin-CRM1 competitors. This concept may also be useful for other PPIs.

Nanobodies are highly affine binders, often used to track disease-relevant proteins inside cells. However, they often fail to interfere with pathobiological functions, required for their clinical exploitation. Here, a nanobody targeting the disease-relevant apoptosis inhibitor and mitosis regulator Survivin (SuN) is utilized. Survivin's multifaceted functions are regulated by an interplay of dynamic cellular localization, dimerization, and protein-protein interactions. However, as Survivin harbors no classical "druggable" binding pocket, one must aim at blocking extended protein surface areas. Comprehensive experimental evidence demonstrates that intracellular expression of SuN allows to track Survivin at low nanomolar concentrations but failed to inhibit its biological functions. Small angle X-ray scattering of the Survivin-SuN complex locates the proposed interaction interface between the C-terminus and the globular domain, as such not blocking any pivotal interaction. By clicking multiple SuN to ultrasmall (2 nm) gold nanoparticles (SuN-N), not only intracellular uptake is enabled, but additionally, Survivin crosslinking and interference with mitotic progression in living cells are also enabled. In sum, it is demonstrated that coupling of nanobodies to nanosized scaffolds can be universally applicable to improve their function and therapeutic applicability., (© 2023 The Authors. Small published by Wiley-VCH GmbH.)

Survivin (BIRC5) is a tumor-associated antigen (TAA) overexpressed in various tumors but present at low to undetectable levels in normal tissue. Survivin is known to have a high expression in breast cancer (e.g., Ductal Carcinoma in situ (DCIS) and triple negative breast cancer). Previous studies have not compared survivin expression levels in DCIS tumor samples to levels in adjacent, normal breast tissue from the same patient. To ensure the effective use of survivin as a target for T cell immunotherapy of breast cancer, it is essential to ascertain the varying levels of survivin expression between DCIS tumor tissue samples and the adjacent normal breast tissue taken from the same patient simultaneously. Next-generation sequencing of RNA (RNA-seq) in normal breast tissue and tumor breast tissue from five women presenting with DCIS for lumpectomy was used to identify sequence variation and expression levels of survivin. The identity of both tumor and adjacent normal tissue samples were corroborated by histopathology. Survivin was overexpressed in human breast tissue tumor samples relative to the corresponding adjacent human normal breast tissue. Wild-type survivin transcripts were the predominant species identified in all tumor tissue sequenced. This study demonstrates upregulated expression of wild type survivin in DCIS tumor tissue versus normal breast tissue taken from the same patient at the same time, and provides evidence that developing selective cytotoxic T lymphocyte (CTL) immunotherapy for DCIS targeting survivin warrants further study.

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.

Roles for the baculoviral inhibitor of apoptosis repeat-containing (BIRC) genes, BIRC2 and BIRC3, may include signaling to the inflammatory transcription factor, nuclear factor-κB (NF-κB) and protection from cell death. However, distinct functions for each BIRC are not well-delineated. Given roles for the epithelium in barrier function and host defence, BIRC2 and BIRC3 expression was characterized in pulmonary epithelial cell lines and primary human bronchial epithelial cells (pHBECs) grown as undifferentiated cells in submersion culture (SC) or as highly differentiated cells at air-liquid interface (ALI). In A549 cells, interleukin-1β (IL1B) and tumor necrosis factor α (TNF) induced BIRC3 mRNA (~20-50-fold), with maximal protein expression from 6-24 h. Similar effects occurred in BEAS-2B and Calu-3 cells, as well as SC and ALI pHBECs. BIRC2 protein was readily detected in unstimulated cells, but was not markedly modulated by IL1B or TNF. Glucocorticoids (dexamethasone, budesonide) modestly increased BIRC3 mRNA and protein, but showed little effect on BIRC2 expression. In A549 cells, BIRC3 mRNA induced by IL1B was unchanged by glucocorticoids and showed supra-additivity with TNF-plus-glucocorticoid. Supra-additivity was also evident for IL1B-plus-budesonide induced-BIRC3 in SC and ALI pHBECs. Using A549 cells, IL1B- and TNF-induced BIRC3 expression, and to a lesser extent, BIRC2, was prevented by NF-κB inhibition. Glucocorticoid-induced BIRC3 expression was prevented by silencing and antagonism of the glucocorticoid receptor. Whereas TNF, but not IL1B, induced degradation of basal BIRC2 and BIRC3 protein, IL1B- and TNF-induced BIRC3 protein remained stable. Differential regulation by cytokines and glucocorticoids shows BIRC2 protein expression to be consistent with roles in rapid signaling events, whereas cytokine-induced BIRC3 may be more important in later effects. While TNF-induced degradation of both BIRCs may restrict their activity, cytokine-enhanced BIRC3 expression could prime for its function. Finally, shielding from glucocorticoid repression, or further enhancement by glucocorticoid, may indicate a key protective role for BIRC3., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Thorne et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Drug resistance is one of the most important obstacles in effective cancer therapy triggered through various mechanisms. One of these mechanisms is caused by the upregulation of Inhibitor of Apoptosis Proteins (IAPs). IAPs, inhibit apoptosis through direct and/or indirect caspase inhibition, which themselves are antagonized by an endogenous protein called Second Mitochondrial-derived Activator of Caspases, Smac/Diablo, mediated by the presence of a tetrapeptide IAP binding motif at its N-terminus. Accordingly, Smac-based peptides are under intense investigation as anti-cancer drugs and have reached Phase 2 clinical trials, although, Smac based peptides or mimetics alone have not been effective as anti-cancer agents. On the other hand, KLA peptide has shown major toxicity against cancer cells through the induction of apoptosis. Consequently, we designed an anti-cancer chimera by fusing an octa-peptide from the N-terminus of mature Smac protein to a modified proapoptotic KLA peptide (KLAKLCKKLAKLCK) to be called Smac-KLA. This chimera, therefore, possesses both proapoptotic and anti-IAP activities. In addition, we dimerized this chimera via intermolecular disulfide bonds in order to enhance their cellular permeability. Both the Smac-KLA monomeric and dimeric peptides exhibited cytotoxic activity against both MCF-7 and MDA-MB231 breast cancer cell lines at low micromolar concentrations. Importantly, the dimerization of the chimeras enhanced their potency 2-4- fold due to higher cellular uptake., Competing Interests: Declarations of competing interest None, (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: The genesis of SMAC mimetic drugs is founded on the observation that many cancers amplify IAP proteins to facilitate their survival, and therefore removal of these pathways would re-sensitize the cells towards apoptosis. It has become increasingly clear that SMAC mimetics also interface with the immune system in a modulatory manner. Suppression of IAP function by SMAC mimetics activates the non-canonical NF-κB pathway which can augment T cell function, opening the possibility of using SMAC mimetics to enhance immunotherapeutics., Methods: We have investigated the SMAC mimetic LCL161, which promotes degradation of cIAP-1 and cIAP-2, as an agent for delivering transient costimulation to engineered BMCA-specific human TAC T cells. In doing so we also sought to understand the cellular and molecular effects of LCL161 on T cell biology., Results: LCL161 activated the non-canonical NF-κB pathway and enhanced antigen-driven TAC T cell proliferation and survival. Transcriptional profiling from TAC T cells treated with LCL161 revealed differential expression of costimulatory and apoptosis-related proteins, namely CD30 and FAIM3. We hypothesized that regulation of these genes by LCL161 may influence the drug's effects on T cells. We reversed the differential expression through genetic engineering and observed impaired costimulation by LCL161, particularly when CD30 was deleted. While LCL161 can provide a costimulatory signal to TAC T cells following exposure to isolated antigen, we did not observe a similar pattern when TAC T cells were stimulated with myeloma cells expressing the target antigen. We questioned whether FasL expression by myeloma cells may antagonize the costimulatory effects of LCL161. Fas-KO TAC T cells displayed superior expansion following antigen stimulation in the presence of LCL161, suggesting a role for Fas-related T cell death in limiting the magnitude of the T cell response to antigen in the presence of LCL161., Conclusions: Our results demonstrate that LCL161 provides costimulation to TAC T cells exposed to antigen alone, however LCL161 did not enhance TAC T cell anti-tumor function when challenged with myeloma cells and may be limited due to sensitization of T cells towards Fas-mediated apoptosis., Competing Interests: JB is a co-founder and shareholder of Triumvira Immunologics and is a paid consultant for Triumvira Immunologics. JB is a co-inventor on patents related to the BCMA TAC described in this manuscript and licensed to Triumvira Immunologics. CB is paid through a sponsored research agreement with Triumvira Immunologics. EL is a co-founder, shareholder, and chief scientific officer of Protaxis Therapeutics. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Afsahi, Silvestri, Moore, Graham, Bacchiochi, St-Jean, Baker, Korneluk, Beug, LaCasse and Bramson.)

Proteolysis targeting chimeras (PROTACs) represent a new pharmacological modality to inactivate disease-causing proteins. PROTACs operate via recruiting E3 ubiquitin ligases, which enable the transfer of ubiquitin tags onto their target proteins, leading to proteasomal degradation. However, several E3 ligases are validated pharmacological targets themselves, of which inhibitor of apoptosis (IAP) proteins are considered druggable in cancer. Here, we report three series of heterobifunctional PROTACs, which consist of an IAP antagonist linked to either von Hippel-Lindau- or cereblon-recruiting ligands. Hijacking E3 ligases against each other led to potent, rapid, and preferential depletion of cellular IAPs. In addition, these compounds caused complete X-chromosome-linked IAP knockdown, which was rarely observed for monovalent and homobivalent IAP antagonists. In cellular assays, hit degrader 9 outperformed antagonists and showed potent inhibition of cancer cell viability. The hetero-PROTACs disclosed herein are valuable tools to facilitate studies of the biological roles of IAPs and will stimulate further efforts toward E3-targeting therapies.

Cross-presentation of tumor antigens by dendritic cells (DC) is crucial to prime, stimulate and restimulate CD8+ T cells. This process is important in initiating and maintaining an antitumor response. Here, we show that the presence of conventional type 1 DCs (cDC1), a DC subtype that excels in cross-presentation, in the tumor correlated with response to neoadjuvant immune checkpoint blockade (ICB) in melanoma. This led us to hypothesize that patients failing to respond to ICB could benefit from enhanced cross-presentation of tumor antigens. We therefore established a cross-presentation assay to screen over 5,500 compounds for enhancers of DC cross-presentation using induced T-cell proliferation as the readout. We identified 145 enhancers, including AZD5582, an antagonist of inhibitor of apoptosis proteins (IAP) cIAP1, cIAP2, and XIAP. AZD5582 treatment led to DC activation of the noncanonical NF-kB pathway, enhanced antigen import from endolysosomes into the cytosol, and increased expression of genes involved in cross-presentation. Furthermore, it upregulated expression of CD80, CD86, MHC class II, CD70 and secretion of TNF by DCs. This enhanced DC activation and maturation program was observed also in tumor-bearing mice upon AZD5582 treatment, culminating in an increased frequency of systemic tumor antigen-specific CD8+ T cells. Our results merit further exploration of AZD5582 to increase antigen cross-presentation for improving the clinical benefit of ICB in patients who are unlikely to respond to ICB., (©2023 American Association for Cancer Research.)

Structural analysis reveals how the decision to induce apoptotic cell death is regulated.