James B. Jaquith

cIAP1 and cIAP2 Facilitate Cancer Cell Survival by Functioning as E3 Ligases that Promote RIP1 Ubiquitination

The inhibitor of apoptosis (IAP) family of proteins enhances cell survival through mechanisms that remain uncertain. In this report, we show that cIAP1 and cIAP2 promote cancer cell survival by functioning as E3 ubiquitin ligases that maintain constitutive ubiquitination of the RIP1 adaptor protein. We demonstrate that AEG40730, a compound modeled on BIR-binding tetrapeptides, binds to cIAP1 and cIAP2, facilitates their autoubiquitination and proteosomal degradation, and causes a dramatic reduction in RIP1 ubiquitination. We show that cIAP1 and cIAP2 directly ubiquitinate RIP1 and induce constitutive RIP1 ubiquitination in cancer cells and demonstrate that constitutively ubiquitinated RIP1 associates with the prosurvival kinase TAK1. When deubiquitinated by AEG40730 treatment, RIP1 binds caspase-8 and induces apoptosis. These findings provide insights into the function of the IAPs and provide new therapeutic opportunities in the treatment of cancer.

The sonic hedgehog factor GLI1 imparts drug resistance through inducible glucuronidation

Drug resistance is a major hurdle in oncology. Responses of acute myeloid leukaemia (AML) patients to cytarabine (Ara-C)-based therapies are often short lived with a median overall survival of months. Therapies are under development to improve outcomes and include targeting the eukaryotic translation initiation factor (eIF4E) with its inhibitor ribavirin. In a Phase II clinical trial in poor prognosis AML, ribavirin monotherapy yielded promising responses including remissions; however, all patients relapsed. Here we identify a novel form of drug resistance to ribavirin and Ara-C. We observe that the sonic hedgehog transcription factor glioma-associated protein 1 (GLI1) and the UDP glucuronosyltransferase (UGT1A) family of enzymes are elevated in resistant cells. UGT1As add glucuronic acid to many drugs, modifying their activity in diverse tissues. GLI1 alone is sufficient to drive UGT1A-dependent glucuronidation of ribavirin and Ara-C, and thus drug resistance. Resistance is overcome by genetic or pharmacological inhibition of GLI1, revealing a potential strategy to overcome drug resistance in some patients.

Cytoprotective effects of IAPs revealed by a small molecule antagonist.

Deregulated expression of members of the IAP (inhibitor of apoptosis) family has been identified in a wide variety of neoplastic cells, and synthetic IAP antagonists represent a promising novel class of chemotherapeutic agents. Early work focused on the ability of these compounds to block the caspase-inhibitory function of XIAP (X-linked IAP). However, recent studies have shown that IAP antagonists, although primarily designed to target XIAP, trigger ubiquitin-mediated degradation of two related proteins, c-IAP (cellular IAP) 1 and c-IAP2, and through this process potentiates the death of tumour cells via autocrine cellular-signalling pathways. In this context, the relative contribution of XIAP as a target of this class of compounds is unclear. In the present study, we examine the involvement of XIAP using a recently described synthetic IAP antagonist, AEG40730, and through comparison of a human XIAP-depleted tumour cell line with its isogenic wild-type control line. Treatment with nanomolar concentrations of AEG40730 resulted in the loss of both XIAP and c-IAP1 proteins, albeit with different kinetics. Although XIAP-deficient HCT116 cells retained some sensitivity to external apoptotic stimuli, the results suggest that IAP antagonists, such as AEG40730, exert their apoptosis-enhancing effects through XIAP in addition to the c-IAPs. These results indicate that IAP antagonists can target multiple IAPs to augment distinct pro-apoptotic signalling pathways, thereby revealing the potential for these compounds in cancer therapy and underscoring the promise of IAP-targeted therapies.

Abstract 139: The identification of potent inhibitor of apoptosis protein (IAP) inhibitors for clinical development

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The inhibitor of apoptosis proteins (IAPs) are important regulators of cell death. Over-expression of IAPs, including XIAP, cIAP1 and cIAP2, predicts poor patient outcome in several types of cancer. XIAP binds to and inhibits caspases 3, 7 and 9. Inhibition of IAP function can enhance cell death in tumor cells. Inhibition of IAPs is being explored clinically with XIAP anti-sense ([AEG35156][1]) and small molecule inhibitor (HGS1029) strategies. We describe small molecule inhibitors of the IAPs that have been designed to bind to the homologous BIR3 domains on the IAPs, which is the site of binding of second mitochondria-derived activator of caspase (SMAC). Structure-activity trends of IAP inhibitors were evaluated for binding potency and selectivity towards IAP BIR domains and analyzed for their ability to sensitize cancer cells to death alone or in conjunction with the agonistic TRAIL receptor 1 monoclonal antibody, mapatumumab, or chemotherapeutic agents. Bridging selected compounds at various sites led to compounds with binding affinities for IAPs in the picomolar range. In cellular assays, IAP inhibitor compounds caused rapid and robust loss of cIAP1, consistent with stimulation of E3 ligase activity. In vitro cytotoxicity assays demonstrated sensitization of tumor cell lines to co-treatment with mapatumumab or chemotherapy at low nanomolar concentrations. In vitro ADME studies were conducted in multiple species to select stable molecules which were negative in CYP and hERG binding assays. Selected compounds had high protein binding in plasma from multiple species. IV administration in mice resulted in Cmax values in excess of the EC50 required for cancer cell death in vitro. Efficacy of lead compounds was demonstrated against several xenograft tumor models as single agents and in combination with either mapatumumab or conventional chemotherapeutics. This led to the identification of a highly potent compound series from which a clinical development candidate, HGS1029, was selected. Solid tumor and lymphoid malignancies Phase 1 cancer trials with HGS1029 have been initiated. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 139. [1]: /lookup/external-ref?link_type=GENPEPT&access_num=AEG35156&atom=%2Fcanres%2F70%2F8_Supplement%2F139.atom