Apoptosis is deregulated in most, if not all, cancers, including hematological malignancies. Smac mimetics that antagonize Inhibitor of Apoptosis (IAP) proteins have so far largely been investigated in acute myeloid leukemia (AML) cell lines; however, little is yet known on the therapeutic potential of Smac mimetics in primary AML samples. In this study, we therefore investigated the antileukemic activity of the Smac mimetic BV6 in diagnostic samples of 67 adult AML patients and correlated the response to clinical, cytogenetic and molecular markers and gene expression profiles. Treatment with cytarabine (ara-C) was used as a standard chemotherapeutic agent. Interestingly, about half (51%) of primary AML samples are sensitive to BV6 and 21% intermediate responsive, while 28% are resistant. Notably, 69% of ara-C-resistant samples show a good to fair response to BV6. Furthermore, combination treatment with ara-C and BV6 exerts additive effects in most samples. Whole-genome gene expression profiling identifies cell death, TNFR1 and NF-κB signaling among the top pathways that are activated by BV6 in BV6-sensitive, but not in BV6-resistant cases. Furthermore, sensitivity of primary AML blasts to BV6 correlates with significantly elevated expression levels of TNF and lower levels of XIAP in diagnostic samples, as well as with NPM1 mutation. In a large set of primary AML samples, these data provide novel insights into factors regulating Smac mimetic response in AML and have important implications for the development of Smac mimetic-based therapies and related diagnostics in AML.

Ubiquitin ligases are critical components of the ubiquitination process that determine substrate specificity and, in collaboration with E2 ubiquitin-conjugating enzymes, regulate the nature of polyubiquitin chains assembled on their substrates. Cellular inhibitor of apoptosis (c-IAP1 and c-IAP2) proteins are recruited to TNFR1-associated signalling complexes where they regulate receptor-stimulated NF-κB activation through their RING domain ubiquitin ligase activity. Using a directed yeast two-hybrid screen, we found several novel and previously identified E2 partners of IAP RING domains. Among these, the UbcH5 family of E2 enzymes are critical regulators of the stability of c-IAP1 protein following destabilizing stimuli such as TWEAK or CD40 signalling or IAP antagonists. We demonstrate that c-IAP1 and UbcH5 family promote K11-linked polyubiquitination of receptor-interacting protein 1 (RIP1) in vitro and in vivo. We further show that TNFα-stimulated NF-κB activation involves endogenous K11-linked ubiquitination of RIP1 within the TNFR1 signalling complex that is c-IAP1 and UbcH5 dependent. Lastly, NF-κB essential modifier efficiently binds K11-linked ubiquitin chains, suggesting that this ubiquitin linkage may have a signalling role in the activation of proliferative cellular pathways.

Inflammatory responses mediated by NOD2 rely on RIP2 kinase and ubiquitin ligase XIAP for the activation of nuclear factor κB (NF-κB), mitogen-activated protein kinases (MAPKs), and cytokine production. Herein, we demonstrate that selective XIAP antagonism blocks NOD2-mediated inflammatory signaling and cytokine production by interfering with XIAP-RIP2 binding, which removes XIAP from its ubiquitination substrate RIP2. We also establish that the kinase activity of RIP2 is dispensable for NOD2 signaling. Rather, the conformation of the RIP2 kinase domain functions to regulate binding to the XIAP-BIR2 domain. Effective RIP2 kinase inhibitors block NOD2 signaling by disrupting RIP2-XIAP interaction. Finally, we identify NOD2 signaling and XIAP-dependent ubiquitination sites on RIP2 and show that mutating these lysine residues adversely affects NOD2 pathway signaling. Overall, these results reveal a critical role for the XIAP-RIP2 interaction in NOD2 inflammatory signaling and provide a molecular basis for the design of innovative therapeutic strategies based on XIAP antagonists and RIP2 kinase inhibitors.

Inflammasomes activate caspase-1 for processing and secretion of the cytokines interleukin-1beta (IL-1beta) and IL-18. Cryopyrin/NALP3/NLRP3 is an essential component of inflammasomes triggered by microbial ligands, danger-associated molecular patterns (DAMPs), and crystals. Inappropriate Cryopyrin activity has been incriminated in the pathogenesis of gouty arthritis, Alzheimer's, and silicosis. Therefore, inhibitors of the Nalp3 inflammasome offer considerable therapeutic promise. In this study, we show that the type 2 diabetes drug glyburide prevented activation of the Cryopyrin inflammasome. Glyburide's cyclohexylurea group, which binds to adenosine triphosphatase (ATP)-sensitive K(+) (K(ATP)) channels for insulin secretion, is dispensable for inflammasome inhibition. Macrophages lacking K(ATP) subunits or ATP-binding cassette transporters also activate the Cryopyrin inflammasome normally. Glyburide analogues inhibit ATP- but not hypothermia-induced IL-1beta secretion from human monocytes expressing familial cold-associated autoinflammatory syndrome-associated Cryopyrin mutations, thus suggesting that inhibition occurs upstream of Cryopyrin. Concurrent with the role of Cryopyrin in endotoxemia, glyburide significantly delays lipopolysaccharide-induced lethality in mice. Therefore, glyburide is the first identified compound to prevent Cryopyrin activation and microbial ligand-, DAMP-, and crystal-induced IL-1beta secretion.

The cellular inhibitor of apoptosis (c-IAP) proteins are E3 ubiquitin ligases that are critical regulators of tumour necrosis factor (TNF) receptor (TNFR)-mediated signalling. Through their E3 ligase activity c-IAP proteins promote ubiquitination of receptor-interaction protein 1 (RIP1), NF-κB-inducing kinase (NIK) and themselves, and regulate the assembly of TNFR signalling complexes. Consequently, in the absence of c-IAP proteins, TNFR-mediated activation of NF-κB and MAPK pathways and the induction of gene expression are severely reduced. Here, we describe the identification of OTUB1 as a c-IAP-associated deubiquitinating enzyme that regulates c-IAP1 stability. OTUB1 disassembles K48-linked polyubiquitin chains from c-IAP1 in vitro and in vivo within the TWEAK receptor-signalling complex. Downregulation of OTUB1 promotes TWEAK- and IAP antagonist-stimulated caspase activation and cell death, and enhances c-IAP1 degradation. Furthermore, knockdown of OTUB1 reduces TWEAK-induced activation of canonical NF-κB and MAPK signalling pathways and modulates TWEAK-induced gene expression. Finally, suppression of OTUB1 expression in zebrafish destabilizes c-IAP (Birc2) protein levels and disrupts fish vasculature. These results suggest that OTUB1 regulates NF-κB and MAPK signalling pathways and TNF-dependent cell death by modulating c-IAP1 stability.

Context-independent anti-hypusine antibodies that bind to the post-translational modification (PTM), hypusine, with minimal dependence on flanking amino acid sequences, were identified. The antibodies bind to both hypusine and deoxyhypusine or selectively to hypusine but not to deoxyhypusine. Phage display was used to further enhance the affinity of the antibodies. Affinity maturation of these anti-hypusine antibodies improved their performance in affinity capture of the only currently known hypusinated protein, eukaryotic translation initiation factor 5A. These anti-hypusine antibodies may have utility in the identification of novel hypusinated proteins. Crystal structures of the corresponding Fab fragments were determined in complex with hypusine- or deoxyhypusine-containing peptides. The hypusine or deoxyhypusine moiety was found to reside in a deep pocket formed between VH and VL domains of the Fab fragments. Interaction between the antibodies and hypusine includes an extensive hydrogen bond network. These are, to our knowledge, the first reported structures of context-independent anti-PTM antibodies in complex with the corresponding PTM.

Inhibitor of apoptosis (IAP) proteins are antiapoptotic regulators that block cell death in response to diverse stimuli. They are expressed at elevated levels in human malignancies and are attractive targets for the development of novel cancer therapeutics. Herein, we demonstrate that small-molecule IAP antagonists bind to select baculovirus IAP repeat (BIR) domains resulting in dramatic induction of auto-ubiquitination activity and rapid proteasomal degradation of c-IAPs. The IAP antagonists also induce cell death that is dependent on TNF signaling and de novo protein biosynthesis. Additionally, the c-IAP proteins were found to function as regulators of NF-kappaB signaling. Through their ubiquitin E3 ligase activities c-IAP1 and c-IAP2 promote proteasomal degradation of NIK, the central ser/thr kinase in the noncanonical NF-kappaB pathway.