Inhibitor Of Apoptosis Protein Research Articles

Epigenetic priming by hypomethylation enhances the immunogenic potential of tolinapant in T-cell lymphoma.

Programmed cell death mechanisms are important for the regulation of tumor development and progression. Evasion of and resistance to apoptosis are significant factors in tumorigenesis and drug resistance. Bypassing apoptotic pathways and eliciting another form of regulated cell death, namely necroptosis, an immunogenic cell death (ICD), may override apoptotic resistance. Here, we present the mechanistic rationale for combining tolinapant, an antagonist of the inhibitor of apoptosis proteins (IAP), with decitabine, a hypomethylating agent (HMA), in T-cell lymphoma (TCL). Tolinapant treatment alone of TCL cells in vitro and in syngeneic in vivo models demonstrated that ICD markers can be upregulated, and we have shown that epigenetic priming with decitabine further enhances this effect. The clinical relevance of ICD markers was confirmed by the direct measurement of plasma proteins from peripheral TCL patients treated with tolinapant. We showed increased levels of necroptosis in TCL lines, along with the expression of cancer-specific antigens (such as cancer testis antigens) and increases in genes involved in interferon signaling induced by HMA treatment, together deliver a strong adaptive immune response to the tumor. These results highlight the potential of a decitabine and tolinapant combination for TCL and could lead to clinical evaluation.

Deoxyshikonin triggers apoptosis in cervical cancer cells through p38 MAPK-mediated caspase activation.

Deoxyshikonin (DSK) is a biological component derived from Lithospermum erythrorhizon. Although DSK possesses potential anticancer activities, whether DSK exerts anticancer effects on cervical cancer cells is incompletely explored. This study was aimed to investigate the anticancer activity of DSK against cervical cancer cells and its molecular mechanisms. Cell viability was evaluated by MTT assay. Level of phosphorylation and protein was determined using Western blot. Involvement of signaling kinases was assessed by specific inhibitors. Our results revealed that DSK reduced viability of human cervical cell in a dose-dependent fashion. Meanwhile, DSK significantly elicited apoptosis of HeLa and SiHa cells. Apoptosis microarray was used to elucidate the involved pathways, and the results showed that DSK dose-dependently diminished cellular inhibitor of apoptosis protein 1 (cIAP1), cIAP2, and XIAP, and induced cleavage of poly(ADP-ribose) polymerase (PARP) and caspase-8/9/3. Furthermore, we observed that DSK significantly triggered activation of ERK, JNK, and p38 MAPK (p38), and only inhibition of p38 diminished the DSK-mediated pro-caspases cleavage. Taken together, our results demonstrate that DSK has anti-cervical cancer effects via the apoptotic cascade elicited by downregulation of IAPs and p38-mediated caspase activation. This suggests that DSK could act as an adjuvant to facilitate cervical cancer management.

Downregulation of miR-4284 can inhibit the apoptosis of Human Arterial Smooth Muscle Cells (HASMCs) in arteriosclerosis obliterans (ASO).

Introduction The disease arteriosclerosis obliterans (ASO) affects the lower extremities. ASO's mechanism involves the proliferation and migration of vascular smooth muscle cells (VSMCs). The miR-4284 is involved in several biological processes of the cardiovascular system, including VSMC proliferation, migration, and death. However, it is unknown if the miR-4284 gene is involved in the control of ASO. Furthermore, the molecular processes behind the contribution of human arterial smooth muscle cells (HASMCs), one of the most significant components of the arterial wall, to arteriosclerosis obliterans (ASO) pathogenesis remain unknown. Previously, we explored the alterations of miRNAs in the blood of ASO patients, and now we wanted to test further whether these changes also take place in the HASMCs that are responsible for the pathogenesis of ASO. Methods The expression levels of miR-29a in arterial walls were analyzed via a real-time polymerase chain reaction. An ASO cell model was established to investigate the expression of miR-4284 on HASMCs. The Transwell system and CCK-8 detection were used to assess the migration and proliferation of HASMCs. The proportion of apoptotic cells as well as the concentrations of apoptotic signal protein production were assessed using flow cytometry. A Western blot technique was used to identify B cell lymphoma-2 (Bcl2), Bcl2-associated X protein (BAX), as well as X-linked inhibitors of apoptosis protein (XIAP). Results The results showed that PCR confirmed that the qualified production or expression of miR-4284 was significantly reduced in HASMCs after they were cultured without FBS and in an atmosphere of 1% O2 + 5% CO2 + 94% N2 and that glucose had no effect on its expression. MiR-4284 has no effect on migration and proliferation, but downregulation of miR-4284 can decrease the apoptotic rate of HASMCs, as revealed by flow cytometry. Furthermore, western blot experiments showed that the expression of BAX was low, while the expression of the other two proteins, viz., Bcl2 and XIAP, was over-expressed. Conclusion We found that miR-4284 downregulation enhanced Bcl2, as well as XIAP, and decreased Bax. This shows that downregulated miR-4284 regulates apoptosis-related protein expression in HASMCs. The mechanism is not clear, and we need further study to confirm it.

(2,6-Dimethylphenyl)arsonic Acid Induces Apoptosis through the Mitochondrial Pathway, Downregulates XIAP, and Overcomes Multidrug Resistance to Cytostatic Drugs in Leukemia and Lymphoma Cells In Vitro.

Cancer treatment is greatly challenged by drug resistance, highlighting the need for novel drug discoveries. Here, we investigated novel organoarsenic compounds regarding their resistance-breaking and apoptosis-inducing properties in leukemia and lymphoma. Notably, the compound (2,6-dimethylphenyl)arsonic acid (As2) demonstrated significant inhibition of cell proliferation and induction of apoptosis in leukemia and lymphoma cells while sparing healthy leukocytes. As2 reached half of its maximum activity (AC50) against leukemia cells at around 6.3 µM. Further experiments showed that As2 overcomes multidrug resistance and sensitizes drug-resistant leukemia and lymphoma cell lines to treatments with the common cytostatic drugs vincristine, daunorubicin, and cytarabine at low micromolar concentrations. Mechanistic investigations of As2-mediated apoptosis involving FADD (FAS-associated death domain)-deficient or Smac (second mitochondria-derived activator of caspases)/DIABLO (direct IAP binding protein with low pI)-overexpressing cell lines, western blot analysis of caspase-9 cleavage, and measurements of mitochondrial membrane integrity identified the mitochondrial apoptosis pathway as the main mode of action. Downregulation of XIAP (x-linked inhibitor of apoptosis protein) and apoptosis induction independent of Bcl-2 (B-cell lymphoma 2) and caspase-3 expression levels suggest the activation of additional apoptosis-promoting mechanisms. Due to the selective apoptosis induction, the synergistic effects with common anti-cancer drugs, and the ability to overcome multidrug resistance in vitro, As2 represents a promising candidate for further preclinical investigations with respect to refractory malignancies.

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.

Repurposing the diuretic benzamil as an anti-osteosarcoma agent that acts by suppressing integrin/FAK/STAT3 signalling and compromising mitochondrial function.

Osteosarcoma is the most common primary bone malignancy among children and adolescents. We investigated whether benzamil, an amiloride analogue and sodium-calcium exchange blocker, may exhibit therapeutic potential for osteosarcoma in vitro. MG63 and U2OS cells were treated with benzamil for 24 hours. Cell viability was evaluated with the MTS/PMS assay, colony formation assay, and flow cytometry (forward/side scatter). Chromosome condensation, the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, cleavage of poly-ADP ribose polymerase (PARP) and caspase-7, and FITC annexin V/PI double staining were monitored as indicators of apoptosis. Intracellular calcium was detected by flow cytometry with Fluo-4 AM. The phosphorylation and activation of focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) were measured by western blot. The expression levels of X-linked inhibitor of apoptosis protein (XIAP), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), SOD1, and SOD2 were also assessed by western blot. Mitochondrial status was assessed with tetramethylrhodamine, ethyl ester (TMRE), and intracellular adenosine triphosphate (ATP) was measured with BioTracker ATP-Red Live Cell Dye. Total cellular integrin levels were evaluated by western blot, and the expression of cell surface integrins was assessed using fluorescent-labelled antibodies and flow cytometry. Benzamil suppressed growth of osteosarcoma cells by inducing apoptosis. Benzamil reduced the expression of cell surface integrins α5, αV, and β1 in MG63 cells, while it only reduced the expression of αV in U2OS cells. Benzamil suppressed the phosphorylation and activation of FAK and STAT3. In addition, mitochondrial function and ATP production were compromised by benzamil. The levels of anti-apoptotic proteins XIAP, Bcl-2, and Bcl-xL were reduced by benzamil. Correspondingly, benzamil potentiated cisplatin- and methotrexate-induced apoptosis in osteosarcoma cells. Benzamil exerts anti-osteosarcoma activity by inducing apoptosis. In terms of mechanism, benzamil appears to inhibit integrin/FAK/STAT3 signalling, which triggers mitochondrial dysfunction and ATP depletion.

Xevinapant Combined with Pembrolizumab in Patients with Advanced, Pretreated, Colorectal and Pancreatic Cancer: Results of the Phase Ib/II CATRIPCA Trial.

Xevinapant is an orally available inhibitor of apoptosis proteins (IAP) inhibitor. Preclinical data suggest that IAP antagonism may synergize with immune checkpoint blockers by modulating the NFκB pathway in immune cells. Adult patients with non-high microsatellite instability advanced/metastatic pancreatic ductal adenocarcinoma (PDAC) or colorectal cancer were enrolled in this phase Ib/II study and received pembrolizumab 200 mg every 3 weeks intravenously, and ascending doses of oral xevinapant (100, 150, and 200 mg daily for 14 days on/7 days off). Dose escalation followed a 3+3 design with a 21-day dose-limiting toxicity (DLT) evaluation period. Following the determination of the recommended phase II dose (RP2D), 14 patients with PDAC and 14 patients with colorectal cancer were enrolled in expansion cohorts to assess preliminary efficacy. Forty-one patients (26 males) with a median age of 64 years were enrolled: 13 in the dose escalation and 28 in the two expansion cohorts. No DLT was observed during dose escalation. The RP2D was identified as xevinapant 200 mg/day + pembrolizumab 200 mg every 3 weeks. The most common adverse events (AE) were fatigue (37%), gastrointestinal AE (decreased appetite in 37%, nausea in 24%, stomatitis in 12%, and diarrhea and vomiting in 10% each), and cutaneous AE (pruritus, dry skin, and rash seen in 20%, 15%, and 15% of patients, respectively). The best overall response according to RECIST1.1 was partial response (confirmed) in 1 (3%), stable disease in 4 (10%), and progressive disease in 35 (88%). Xevinapant combined with pembrolizumab was well tolerated with no unexpected AEs. However, antitumor activity was low.

Sterigmatocystin induces autophagic and apoptotic cell death of liver cancer cells via downregulation of XIAP

XIAP, or the X-linked Inhibitor of Apoptosis Protein, is the most extensively studied member within the IAP gene family. It possesses the capability to impede apoptosis through direct inhibition of caspase activity. Various kinds of cancers overexpress XIAP to enable cancer cells to avoid apoptosis. Consequently, the inhibition of XIAP holds significant clinical implications for the development of anti-tumor medications and the treatment of cancer. In this study, sterigmatocystin, a natural compound obtained from the genus asperigillus, was demonstrated to be able to induce apoptotic and autophagic cell death in liver cancer cells. Mechanistically, sterigmatocystin induces apoptosis by downregulation of XIAP expression. Additionally, sterigmatocystin treatment induces cell cycle arrest, blocks cell proliferation, and slows down colony formation in liver cancer cells. Importantly, sterigmatocystin exhibits a remarkable therapeutic effect in a nude mice model. Our findings revealed a novel mechanism through which sterigmatocystin induces apoptotic and autophagic cell death of liver cancer cells by suppressing XIAP expression, this offers a promising therapeutic approach for treating liver cancer patients.

Exploring the role of histone deacetylase inhibitors in cancer development and therapeutic potential.

The process of acetylation and deacetylation of histones within the nucleus operates within a dynamic equilibrium. Histone acetyltransferases (HATs) and histone deacetylases (HDACs) collaboratively and precisely regulate normal gene transcription and expression. Any disorder in the activity of HATs/HDACs can lead to uncontrolled gene expression, consequently resulting in tumorigenesis. Histone deacetylase inhibitors (HDACIs) have the capacity to block the cell cycle, thereby restraining tumor cell proliferation and tumor growth. Also, HDACIs exhibit a significant capability to diminish the expression of apoptosis protein inhibitors such as Bcl-2 and B-cell lymphoma-extra-large (Bcl-xL), while concurrently up-regulating pro-apoptotic proteins such as Bax, Bad, and Bim. Also, HDACIs demonstrate the ability to inhibit tumor cell angiogenesis. Representing a new category of targeted anti-cancer therapeutics, HDACIs possess the capability to restore the expression of tumor suppressor genes, induce apoptosis, and stimulate cell differentiation. Additionally, they exert anti-cancer effects through diverse pathways both in vivo and in vitro, thereby presenting promising prospects in tumor therapy. This review delves into the involvement of HDACs in cancer pathology and the therapeutic potential of HDACIs as emerging drugs in cancer treatment.

Birinapant Reshapes the Tumor Immunopeptidome and Enhances Antigen Presentation.

Birinapant, an antagonist of the inhibitor of apoptosis proteins, upregulates MHCs in tumor cells and displays a better tumoricidal effect when used in combination with immune checkpoint inhibitors, indicating that Birinapant may affect the antigen presentation pathway; however, the mechanism remains elusive. Based on high-resolution mass spectrometry and in vitro and in vivo models, we adopted integrated genomics, proteomics, and immunopeptidomics strategies to study the mechanism underlying the regulation of tumor immunity by Birinapant from the perspective of antigen presentation. Firstly, in HT29 and MCF7 cells, Birinapant increased the number and abundance of immunopeptides and source proteins. Secondly, a greater number of cancer/testis antigen peptides with increased abundance and more neoantigens were identified following Birinapant treatment. Moreover, we demonstrate the existence and immunogenicity of a neoantigen derived from insertion/deletion mutation. Thirdly, in HT29 cell-derived xenograft models, Birinapant administration also reshaped the immunopeptidome, and the tumor exhibited better immunogenicity. These data suggest that Birinapant can reshape the tumor immunopeptidome with respect to quality and quantity, which improves the presentation of CTA peptides and neoantigens, thus enhancing the immunogenicity of tumor cells. Such changes may be vital to the effectiveness of combination therapy, which can be further transferred to the clinic or aid in the development of new immunotherapeutic strategies to improve the anti-tumor immune response.

Research Progress of Apoptosis Inhibitory Protein Survivin in Gastric Cancer

Survivin is an important member of the apoptosis inhibitory protein family, which plays an important role in suppressing apoptosis, promoting proliferation and vascular formation. The occurrence and development of tumors are related to apoptosis and imbalance of proliferation. Gastric cancer is the most common malignant tumor in the digestive system. Due to the inadequate symptoms of early symptoms, most of them have entered the middle and late stages during diagnosis. It is easy to relapse and metastasize. The mortality rate is high, which seriously endangers human health. Research have found that Survivin expresses high in gastric cancer tissue, participating in the proliferation of tumor cells, inhibiting apoptosis and the formation of blood vessels, and is of great significance to the diagnosis, judgment transfer and prognosis evaluation of gastric cancer, especially the value of combined with a number of indicators is greater. Therefore, finding an early diagnosis and judging the ideal tumor logo combination of the early diagnosis and judgment of the prognosis is important. This article reviews the biological characteristics of Survivin and its research progress in gastric cancer, so as to provide theoretical basis for the diagnosis of gastric cancer and new ideas for the successful prevention and treatment of tumors.

Abstract 2921: Extended treatment with IAP inhibitor xevinapant post radiotherapy improves therapeutic efficacy and promotes antitumor immunity in preclinical models

Abstract Background: Xevinapant is a first-in-class, oral IAP (inhibitor of apoptosis protein) inhibitor designed to restore cancer cell sensitivity to apoptosis induced by radiotherapy (RT) and chemotherapy. In a randomized phase 2 study of patients with unresected locally advanced squamous cell carcinoma of the head and neck, xevinapant + chemoradiotherapy (CRT) significantly improved locoregional control at 18 months, 3-year progression-free survival and nearly doubled 5-year overall survival vs. placebo + CRT (53% vs. 28%). Two phase 3 trials, in which patients receive 6 cycles of xevinapant or placebo monotherapy (3 cycles in combination with standard-of-care (C)RT followed by 3 cycles of monotherapy), are ongoing. Based on the roles of IAPs in apoptosis and tumor immunity, continual dosing of xevinapant post RT may deliver additional therapeutic benefit through modulation of multiple tumor microenvironment (TME) compartments. Methods: The impact on antitumor efficacy of xevinapant dosing duration was evaluated in A1419 (head and neck), LLC (lung) and MC38 (colorectal) syngeneic tumor-bearing mice treated with vehicle control, RT alone (3.6Gy, QD, 5 days), or RT + xevinapant (100 mg/kg, QD, 1, 2, or 4 weeks). To assess treatment-mediated TME modulation in MC38 tumors, RNAseq and FACS-based immune profiling was performed, followed by functional validation with ex vivo ELISpot assays, in vitro immunogenic cell death assays, T cell activation assays, and macrophage polarization and viability assays. To evaluate the effect of xevinapant on RT-induced cancer associated fibroblast (CAF) activation and CAF-modulated immune phenotype, activation assay and cytokine profiling was performed. Results: In the tumor models tested, RT in combination with extended xevinapant dosing markedly improved efficacy and prolonged survival compared to treatment arms of RT with shorter xevinapant dosing durations, RT alone, and vehicle control. RNAseq and FACS analyses suggested a TME with enhanced antitumor immunity, while ELISpot and in vitro T cell assays confirmed that extended xevinapant dosing promoted antigen-specific T cell response and T cell activation. Furthermore, macrophage polarization and viability assays suggested that xevinapant reduced the viability of M2 macrophages which may have contributed to the increased M1:M2 macrophage ratio and the improved antitumor immune response observed in vivo. In addition, via the suppression of RT-mediated CAF activation, xevinapant may promote M1 polarization of macrophages and recruitment of T cells by upregulating GM-CSF and CXCL10 in CAFs. Conclusions: Extended dosing of xevinapant post concurrent RT improved antitumor efficacy and prolonged survival of tumor-bearing mice. Mechanistic investigation suggested that such benefit may be, in part, modulated by xevinapant enhanced antitumor immunity. Citation Format: Tsz-Lun Yeung, Feng Jiang, Hui Huang, Huakui Yu, Li-Ya Chiu, Mathilde Bonnemaison, Bo Marelli, Roberta Ferretti, Ralph Lindemann, Christina Esdar. Extended treatment with IAP inhibitor xevinapant post radiotherapy improves therapeutic efficacy and promotes antitumor immunity in preclinical models [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 2921.

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.

Abstract 3964: A cancer survival program against activated T cell attack

Abstract The immune-cancer interactions downstream of immune checkpoint inhibition present the next targetable interface of cancer immunity. Despite evidence for cytotoxic T lymphocyte (CTL) infiltration and cytolytic activity within immunotherapy-responsive tumors, partial responses and acquired resistance are common, indicating additional barriers for complete tumor elimination. We report a survival program engaged within residual cancer cells exposed to weeks of activated CTLs. Contrary to immune-evasion mechanisms, we observe a residual antigenic cancer cell subpopulation which activates CTLs, survives within IFNγ-high environments, and withstands CTL-induced apoptosis. This cancer survival program, termed immune-induced persistence, is reversibly-driven (non-genetic) and prevents the complete CTL-mediated elimination of cancer cell populations by supporting the survival of quiescent cancer ‘persister’ cells against activated CTL attack. Persister cells display sublethal caspase activity and caspase-dependent DNA damage, consistent with CTL-induced genomic instability, and depend on the inhibitors of apoptosis proteins for survival. Notably, immune-induced persistence presents a markedly distinct survival program than drug-induced persistence, including distinctive apoptotic profiles, stress response pathways, and vulnerabilities. Thus, cancer cell populations can transiently adopt non-redundant survival programs in response to various cytotoxic environments. Features of immune-induced persistence are observed within CTL-treated human cancer cell lines, immune-treated human tumor slice cultures, and intratumor transcriptional heterogeneity programs. Under continued CTL exposure, persistent cancer populations give rise to genetically distinct cancer clones, including stably resistant immune escape variants, modeling acquired resistance to cancer immunotherapies. We propose sublethal interactions between effector immune cells and cancer persister cells as a critical and targetable variable at the interface between activated immunity and cancer. Citation Format: Michael X. Wang, Brandon E. Mauch, August F. Williams, Tania Barazande-Pour, Filipe Araujo Hoffmann, Sophie H. Harris, Cooper P. Lathrop, Claire E. Turkal, Michelle H. Paw, David A. Gervasio, Tiffany Tran, Theresa Guo, Gregory A. Daniels, Soo J. Park, Matthew J. Hangauer. A cancer survival program against activated T cell attack [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 3964.

Abstract 7599: Evaluate the treatment efficacy of magnolol combined with cisplatin in non-small cell lung cancer

Abstract Lung cancer is one of the five most common cancers in the world. Statistics indicate that approximately 80% to 90% of lung cancer cases fall under the classification of non-small cell lung cancer (NSCLC). For several decades, non-small cell lung cancer (NSCLC) has often been treated with chemotherapy drugs. However, lung cancer has high incidence and recurrence rates, mainly due to drug resistance and side effects during chemotherapy and radiotherapy. This is the hinder problem in the treatment of NSCLC, which highlight the necessity of developing new drugs or new treatments for NSCLC. Cisplatin is a chemotherapy drug that can treat about 50% of solid tumors, but the cisplatin-induced cytoprotective autophagy is the resistance to clinical treatment. It is urgent to elucidate the underlying molecular mechanisms regulating cisplatin resistance to improve its therapeutic value in lung cancer. Magnolol is a phenolic component isolated from the roots and bark of Magnolia officinalis and is used in traditional Chinese medicine. With a wide range of pharmacological activities, various studies have focused on the anti-cancer properties of magnolol in recent years. The effect of the drug is to keep cells in the G2/M phase, induce mitochondrial dysfunction and ROS production, affect the supply of cellular energy, and then lead to cell apoptosis. In this study, we investigated the ability of magnolol enhance cisplatin-induced cytotoxicity, showing dose dependence in NSCLC cell by MTT assay. We utilized the immunofluorescence staining and western blotting assay to confirm the downgrade expression of LC3B and the upregulate expression of p62 that define magnolol is able to suppress cisplatin-induced cytoprotective autophagy in NSCLC. We also detected the extrinsic/intrinsic apoptosis induction ability of magnolol combined with cisplatin in NSCLC. In the result, the cisplatin combined with magnolol have more significantly triggered the loss of mitochondrial membrane potential, and activate more protein cleaved caspase-3, -8 and -9 than monotherapy. We also used western blotting assay to confirm the expression of apoptosis inhibitory protein Bcl-2 and pro-apoptotic protein Bax to measure the degree of apoptosis. The migration and invasion effects of magnolol and cisplatin were identified by transwell invasion and wound healing on NSCLC. The magnolol combined with cisplatin extended the mouse survival rate and can inhibit tumor progression. These results suggest that combinational therapy of cisplatin and magnolol could be a promising treatment strategy for NSCLC. The potential regulation of magnolol on NSCLC is associated with triggering cisplatin-induced apoptosis and diminishing cisplatin-induce cytoprotective autophagy. Citation Format: Rong-Er Lu, Bo-Xun Chen, Fei-Ting Hsu, I-Tsang Chiang. Evaluate the treatment efficacy of magnolol combined with cisplatin in 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 7599.

The novel drug candidate S2/IAPinh improves survival in models of pancreatic and ovarian cancer

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.

Curcumin Analog L48H37 Induces Apoptosis in Human Oral Cancer Cells by Activating Caspase Cascades and Downregulating the Inhibitor of Apoptosis Proteins through JNK/p38 Signaling.

L48H37 is a synthetic curcumin analog that has anticancer potentials. Here, we further explored the anticancer effect of L48H37 on oral cancer cells and its mechanistic acts. Cell cycle distribution was assessed using flow cytometric analysis. Apoptosis was elucidated by staining with PI/Annexin V and activation of the caspase cascade. Cellular signaling was explored using apoptotic protein profiling, Western blotting, and specific inhibitors. Our findings showed that L48H37 significantly reduced the cell viability of SCC-9 and HSC-3 cells, resulting in sub-G1 phase accumulation and increased apoptotic cells. Apoptotic protein profiling revealed that L48H37 increased cleaved caspase-3, and downregulated cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP) in SCC-9 cells, and the downregulated cIAP1 and XIAP in both oral cancer cells were also demonstrated by Western blotting. Meanwhile, L48H37 triggered the activation of caspases and mitogen-activated protein kinases (MAPKs). The involvement of c-Jun N-terminal kinase (JNK) and p38 MAPK (p38) in the L48H37-triggered apoptotic cascade in oral cancer cells was also elucidated by specific inhibitors. Collectively, these findings indicate that L48H37 has potent anticancer activity against oral cancer cells, which may be attributed to JNK/p38-mediated caspase activation and the resulting apoptosis. This suggests a potential benefit for L48H37 for the treatment of oral cancer.

Targeting inhibitor of apoptosis proteins (IAPs) enhances susceptibility of oral squamous carcinoma cells to cisplatin

PurposeOral 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. MethodsCisplatin-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. ResultsWe 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. ConclusionThese findings provide pre-clinical evidence that IAP inhibition may be a valuable therapeutic option in OSCC.

Cancer cell-specific and pro-apoptotic SMAC peptide-doxorubicin conjugated prodrug encapsulated aposomes for synergistic cancer immunotherapy

BackgroundImmunogenic cell death (ICD) is a crucial approach to turn immunosuppressive tumor microenvironment (ITM) into immune-responsive milieu and improve the response rate of immune checkpoint blockade (ICB) therapy. However, cancer cells show resistance to ICD-inducing chemotherapeutic drugs, and non-specific toxicity of those drugs against immune cells reduce the immunotherapy efficiency.MethodsHerein, we propose cancer cell-specific and pro-apoptotic liposomes (Aposomes) encapsulating second mitochondria-derived activator of caspases mimetic peptide (SMAC-P)-doxorubicin (DOX) conjugated prodrug to potentiate combinational ICB therapy with ICD. The SMAC-P (AVPIAQ) with cathepsin B-cleavable peptide (FRRG) was directly conjugated to DOX, and the resulting SMAC-P-FRRG-DOX prodrug was encapsulated into PEGylated liposomes.ResultsThe SMAC-P-FRRG-DOX encapsulated PEGylated liposomes (Aposomes) form a stable nanostructure with an average diameter of 109.1 ± 5.14 nm and promote the apoptotic cell death mainly in cathepsin B-overexpressed cancer cells. Therefore, Aposomes induce a potent ICD in targeted cancer cells in synergy of SMAC-P with DOX in cultured cells. In colon tumor models, Aposomes efficiently accumulate in targeted tumor tissues via enhanced permeability and retention (EPR) effect and release the encapsulated prodrug of SMAC-P-FRRG-DOX, which is subsequently cleaved to SMAC-P and DOX in cancer cells. Importantly, the synergistic activity of inhibitors of apoptosis proteins (IAPs)-inhibitory SMAC-P sensitizing the effects of DOX induces a potent ICD in the cancer cells to promote dendritic cell (DC) maturation and stimulate T cell proliferation and activation, turning ITM into immune-responsive milieu.ConclusionsEventually, the combination of Aposomes with anti-PD-L1 antibody results in a high rate of complete tumor regression (CR: 80%) and also prevent the tumor recurrence by immunological memory established during treatments.Graphical

Systematic analysis of PANoptosis-related genes identifies XIAP as a functional oncogene in breast cancer

BackgroundBreast cancer (BC) is the most prevalent malignant disease affecting women globally. PANoptosis, a novel form of cell death combining features of pyroptosis, apoptosis, and necroptosis, has recently gained attention. However, its precise function in BC and the predictive values of PANoptosis-related genes remain unclear. MethodsWe used the expression data and clinical information of BC tissues or normal breast tissues from public databases, and then successfully developed and verified a BC PANoptosis-related risk model through a combination of univariate Cox regression, least absolute shrinkage and selection operator (LASSO) regression, and Kaplan-Meier (KM) analysis. A nomogram was constructed to estimate survival probability, and its accuracy was assessed using calibration curves. ResultsAmong 37 PANoptosis-related genes, we identified 4 differentially expressed genes related to overall survival (OS). Next, a risk model incorporating these four PANoptosis-related genes was established. Patients were stratified into low/high-risk groups based on the median risk score, with the low-risk group showing better prognoses and higher levels of immune infiltration. Utilizing the risk score and clinical features, we developed a nomogram to predict 1-, 3- and 5-year survival probability. X-linked inhibitor of apoptosis protein (XIAP) emerged as a potentially risky factor with the highest hazard ratio. In vitro experiments demonstrated that XIAP inhibition enhances the antitumor effect of doxorubicin through the PANoptosis pathway. ConclusionPANoptosis holds an important role in BC prognosis and treatment.