SMAC Mimetic/IAP Inhibitor Birinapant Enhances Radiosensitivity of Glioblastoma Multiforme.

Birinapant is a novel SMAC peptidomimetic molecule in clinical development. It suppresses the inhibitor of apoptosis proteins (IAPs) and promotes cytochrome-C/Apaf-1/caspase-9 activation to induce effective apoptosis. Because IAP inhibition has been shown to enhance the sensitivity of cancer cells to radiation, we investigated the role of birinapant in radiosensitization of glioblastoma cells in vitro and in vivo. Two glioblastoma cell lines, U-251 and U-87, were used to analyze radiosensitization in vitro with 7-AAD cell death/apoptosis and clonogenic assays. Subcutaneous flank (U-251 and U-87) and intracranial orthotopic (U-251) xenografts in nude mice were used to evaluate radiosensitization in vivo. TNF-α levels in media and serum were measured using electrochemiluminescence. Radiosensitization in vitro was more prominent for U-251 cells than for U-87 cells. In vivo, in both tumor models, significant tumor growth delay was observed with combination treatment compared to radiation alone. There was a survival benefit with combination treatment in the orthotopic U-251 model. TNF-α levels in media correlated directly with radiation dose in vitro. These findings show that birinapant can enhance the radiosensitivity of glioblastoma cell lines in cell-based assays and tumor models via radiation-induced TNF-α. Further study into the use of birinapant with radiation therapy is warranted.

RIPK3 promotes cell death and NLRP3 inflammasome activation in the absence of MLKL.

RIPK3 and its substrate MLKL are essential for necroptosis, a lytic cell death proposed to cause inflammation via the release of intracellular molecules. Whether and how RIPK3 might drive inflammation in a manner independent of MLKL and cell lysis remains unclear. Here we show that following LPS treatment, or LPS-induced necroptosis, the TLR adaptor protein TRIF and inhibitor of apoptosis proteins (IAPs: X-linked IAP, cellular IAP1 and IAP2) regulate RIPK3 and MLKL ubiquitylation. Hence, when IAPs are absent, LPS triggers RIPK3 to activate caspase-8, promoting apoptosis and NLRP3-caspase-1 activation, independent of RIPK3 kinase activity and MLKL. In contrast, in the absence of both IAPs and caspase-8, RIPK3 kinase activity and MLKL are essential for TLR-induced NLRP3 activation. Consistent with in vitro experiments, interleukin-1 (IL-1)-dependent autoantibody-mediated arthritis is exacerbated in mice lacking IAPs, and is reduced by deletion of RIPK3, but not MLKL. Therefore RIPK3 can promote NLRP3 inflammasome and IL-1ß inflammatory responses independent of MLKL and necroptotic cell death.

Birinapant, a smac-mimetic with improved tolerability for the treatment of solid tumors and hematological malignancies.

Birinapant (1) is a second-generation bivalent antagonist of IAP proteins that is currently undergoing clinical development for the treatment of cancer. Using a range of assays that evaluated cIAP1 stability and oligomeric state, we demonstrated that 1 stabilized the cIAP1-BUCR (BIR3-UBA-CARD-RING) dimer and promoted autoubiquitylation of cIAP1 in vitro. Smac-mimetic 1-induced loss of cIAPs correlated with inhibition of TNF-mediated NF-κB activation, caspase activation, and tumor cell killing. Many first-generation Smac-mimetics such as compound A (2) were poorly tolerated. Notably, animals that lack functional cIAP1, cIAP2, and XIAP are not viable, and 2 mimicked features of triple IAP knockout cells in vitro. The improved tolerability of 1 was associated with (i) decreased potency against cIAP2 and affinity for XIAP BIR3 and (ii) decreased ability to inhibit XIAP-dependent signaling pathways. The P2' position of 1 was critical to this differential activity, and this improved tolerability has allowed 1 to proceed into clinical studies.

Birinapant (TL32711), a bivalent SMAC mimetic, targets TRAF2-associated cIAPs, abrogates TNF-induced NF-κB activation, and is active in patient-derived xenograft models.

The acquisition of apoptosis resistance is a fundamental event in cancer development. Among the mechanisms used by cancer cells to evade apoptosis is the dysregulation of inhibitor of apoptosis (IAP) proteins. The activity of the IAPs is regulated by endogenous IAP antagonists such as SMAC (also termed DIABLO). Antagonism of IAP proteins by SMAC occurs via binding of the N-terminal tetrapeptide (AVPI) of SMAC to selected BIR domains of the IAPs. Small molecule compounds that mimic the AVPI motif of SMAC have been designed to overcome IAP-mediated apoptosis resistance of cancer cells. Here, we report the preclinical characterization of birinapant (TL32711), a bivalent SMAC-mimetic compound currently in clinical trials for the treatment of cancer. Birinapant bound to the BIR3 domains of cIAP1, cIAP2, XIAP, and the BIR domain of ML-IAP in vitro and induced the autoubiquitylation and proteasomal degradation of cIAP1 and cIAP2 in intact cells, which resulted in formation of a RIPK1:caspase-8 complex, caspase-8 activation, and induction of tumor cell death. Birinapant preferentially targeted the TRAF2-associated cIAP1 and cIAP2 with subsequent inhibition of TNF-induced NF-κB activation. The activity of a variety of chemotherapeutic cancer drugs was potentiated by birinapant both in a TNF-dependent or TNF-independent manner. Tumor growth in multiple primary patient-derived xenotransplant models was inhibited by birinapant at well-tolerated doses. These results support the therapeutic combination of birinapant with multiple chemotherapies, in particular, those therapies that can induce TNF secretion.

The novel SMAC mimetic birinapant exhibits potent activity against human melanoma cells.

PURPOSE: Inhibitor of apoptosis proteins (IAP) promote cancer cell survival and confer resistance to therapy. We report on the ability of second mitochondria-derived activator of caspases mimetic, birinapant, which acts as antagonist to cIAP1 and cIAP2, to restore the sensitivity to apoptotic stimuli such as TNF-α in melanomas. EXPERIMENTAL DESIGN: Seventeen melanoma cell lines, representing five major genetic subgroups of cutaneous melanoma, were treated with birinapant as a single agent or in combination with TNF-α. Effects on cell viability, target inhibition, and initiation of apoptosis were assessed and findings were validated in 2-dimensional (2D), 3D spheroid, and in vivo xenograft models. RESULTS: When birinapant was combined with TNF-α, strong combination activity, that is, neither compound was effective individually but the combination was highly effective, was observed in 12 of 18 cell lines. This response was conserved in spheroid models, whereas in vivo birinapant inhibited tumor growth without adding TNF-α in in vitro resistant cell lines. Birinapant combined with TNF-α inhibited the growth of a melanoma cell line with acquired resistance to BRAF inhibition to the same extent as in the parental cell line. CONCLUSIONS: Birinapant in combination with TNF-α exhibits a strong antimelanoma effect in vitro. Birinapant as a single agent shows in vivo antitumor activity, even if cells are resistant to single agent therapy in vitro. Birinapant in combination with TNF-α is effective in a melanoma cell line with acquired resistance to BRAF inhibitors.

Frequent activation of AKT in non-small cell lung carcinomas and preneoplastic bronchial lesions.

AKT is frequently activated in various cancers, but its involvement in lung tumor development and progression is not well established. We examined AKT activity by immunohistochemistry in 110 non-small cell lung carcinomas (NSCLCs) using tissue microarrays. AKT activation was observed in 56 (51%) tumors. To further validate activation of the AKT pathway in this series, we examined the phosphorylation status of the mammalian target of rapamycin (mTOR) and forkhead (FKHR), two downstream targets of AKT. Positive staining for phospho-mTOR and phospho-FKHR were detected in 74% and 68% of tumors, respectively, and was significantly associated with activation of AKT. Tumors positive for phosphorylated (active) AKT were present with a similar frequency in low stage (I/II) and high stage (III/IV) tumors, raising the possibility that AKT activation occurs early in tumor progression. We therefore examined AKT activity in 25 bronchial epithelial lesions from 12 patients at high risk of lung cancer. Metaplastic/dysplastic areas showed AKT activity, whereas normal and hyperplastic bronchial epithelia exhibited little or no activity. Since some bronchial epithelial lesions may develop into invasive cancers, we examined the effect of AKT on invasiveness of lung cancer cells, using an in vitro cell invasion assay. Transfection of NSCLC cells with wild-type AKT increased invasiveness in response to hepatocyte growth factor, whereas transfection with dominant negative AKT abrogated this effect. Collectively, these data suggest that AKT activation is a frequent and early event in lung tumorigenesis, which may enhance risk of progression to malignancy. Thus, AKT represents a potentially important target for chemoprevention in individuals at high risk of NSCLC.

Growth inhibition and induction of apoptosis in mesothelioma cells by selenium and dependence on selenoprotein SEP15 genotype.

Malignant mesotheliomas (MMs) are aggressive tumors derived from mesothelial cells lining the lungs, pericardium and peritoneum, and are often associated with occupational asbestos exposure. Suppression subtractive hybridization was used to identify genes differentially expressed in MM cells compared to normal mesothelial cells. A gene, SEP15, encoding a 15-kDa selenium-containing protein was isolated using this approach and was subsequently shown to be downregulated in approximately 60% of MM cell lines and tumor specimens. A SEP15 polymorphic variant, 1125A, resides in the SECIS recognition element in the 3'-UTR and may influence the efficiency of Sec incorporation into the protein during translation. Since previous studies have implicated a potential role of the trace element selenium as a chemopreventive agent in animal models and in several types of human cancer, we investigated the effect of selenium on MM cells and its dependence on SEP15 genotype. Selenium was shown to inhibit cell growth and induce apoptosis in a dose-dependent manner in MM cells but had minimal effect on normal mesothelial cells. However, MM cells with downregulated SEP15 or the 1125A variant were somewhat less responsive to the growth inhibitory and apoptotic effects of selenium than MM cells expressing wild-type protein. RNAi-based knockdown studies demonstrated that SEP15 inhibition makes sensitive MM cells more resistant to selenium. These data imply that selenium may be useful as a chemopreventive agent in individuals at high risk of MM due to asbestos exposure, although those with the 1125A polymorphism may be less responsive to the protective benefits of dietary selenium supplementation.

Protein kinase C promotes apoptosis in LNCaP prostate cancer cells through activation of p38 MAPK and inhibition of the Akt survival pathway.

Activation of protein kinase C (PKC) by phorbol esters or diacylglycerol mimetics induces apoptosis in androgen-dependent prostate cancer cells, an effect that involves both the activation of the classic PKC alpha and the novel PKC delta isozymes (Fujii, T., García-Bermejo, M. L., Bernabó, J. L., Caamaño, J., Ohba, M., Kuroki, T., Li, L., Yuspa, S. H., and Kazanietz, M. G. (2000) J. Biol. Chem. 275, 7574-7582 and Garcia-Bermejo, M. L., Leskow, F. C., Fujii, T., Wang, Q., Blumberg, P. M., Ohba, M., Kuroki, T., Han, K. C., Lee, J., Marquez, V. E., and Kazanietz, M. G. (2002) J. Biol. Chem. 277, 645-655). In the present study we explored the signaling events involved in this PKC-mediated effect, using the androgen-dependent LNCaP cell line as a model. Stimulation of PKC by phorbol 12-myristate 13-acetate (PMA) leads to the activation of ERK1/2, p38 MAPK, and JNK in LNCaP cells. Here we present evidence that p38 MAPK, but not JNK, mediates PKC-induced apoptosis. Because LNCaP cells have hyperactivated Akt function due to PTEN inactivation, we examined whether this survival pathway could be affected by PKC activation. Interestingly, activation of PKC leads to a rapid and reversible dephosphorylation of Akt, an effect that was prevented by the pan-PKC inhibitor GF109302X and the cPKC inhibitor Gö6976. In addition, the diacylglycerol mimetic agent HK654, which selectively stimulates PKC alpha in LNCaP cells, also induced the dephosphorylation of Akt in LNCaP cells. Inactivation of Akt function by PKC does not involve the inhibition of PI3K, and it is prevented by okadaic acid, suggesting the involvement of a phosphatase 2A in PMA-induced Akt dephosphorylation. Finally, we show that, when an activated form of Akt is delivered into LNCaP cells by either transient transfection or adenoviral infection, the apoptotic effect of PMA is significantly reduced. Our results highlight a complex array of signaling pathways regulated by PKC isozymes in LNCaP prostate cancer cells and suggest that both p38 MAPK and Akt play critical roles as downstream effectors of PKC isozymes in this cellular model.

Cytogenetics and molecular genetics of lung cancer.

The development and progression of lung cancer is a multistep process characterized by the accumulation of numerous genetic and epigenetic alterations, some of which occur early in the course of disease. In this review, we summarize cytogenetic imbalances and molecular genetic/epigenetic changes seen in human small-cell and non-small-cell lung cancer. Alterations of tumor suppressor genes and oncogenes leading to perturbations of key cell-regulatory and growth-control pathways are highlighted. The translational implications of molecular biomarkers for risk assessment, early detection, and monitoring of chemoprevention trials are discussed.