Inhibitor Of Apoptosis Proteins Antagonist Research Articles

179: IFNγ and Smac mimetics induce synergistic killing via necroptosis and apoptosis

Inhibitor of Apoptosis proteins (IAPs) are a group of E3 ligases involved in tumor necrosis factor (TNF) signalling. Upon TNF stimulation IAPs are needed for proper activation of NF-κB signalling, thereby protecting cells from cytotoxic activity of TNF. IAPs have emerged as attractive targets for anti-cancer therapeutics since it has been shown that overexpression of the IAP antagonist Smac sensitises tumour cells to TNF driven apoptosis as do the synthetic counterparts, Smac mimetics. We demonstrated that cell death induced by inhibition of IAPs through Smac mimetics is not strictly TNF dependent. The combined treatment of Interferon γ (IFNγ), a potent pro-inflammatory cytokine, and Smac mimetics shows potent synergistic killing of a wide range of tumour cell lines as well as primary and transformed cells. Further investigation showed that IFNγ and Smac mimetic induced killing requires NF-κB as well as Jak/STAT signalling and displayed classic apoptotic hallmarkers. Whereas killing of several cell lines could be blocked by the inhibition of caspases, other cell lines like HT29, KATOIII, mouse dermal fibroblasts (MDFs) and keratinocytes remained sensitive. In order to elucidate the involvement of the necroptotic pathway in this apoptosis independent IFNγ and Smac mimetic combined killing we performed knock down experiments and analysed primary and transformed knock out MDFs and keratinocytes lacking members of the necroptotic pathway like RIPK3 −/− and MLKL −/− . The results suggest that IFNγ and Smac mimetic combined treatment can activate both apoptotic and necroptotic death pathways, involving members of the necroptotic pathway. This may provide new insights into yet unknown cell death mechanisms and extend the clinical use of Smac mimetics.

Clinical pharmacokinetic (PK)/pharmacodynamic (PD) model for Debio 1143, a novel antagonist of IAPs in cancer treatment.

2585 Background: Inhibitors of apoptosis proteins (IAPs) modulate multiple processes, including caspase activation and NF-kB signaling. Expression and/or overexpression of IAPs have been reported for a variety of tumor types and are correlated with tumor growth and resistance to apoptosis induced by standard chemo and radiation therapies. The small molecule Debio 1143 is an orally-active IAP antagonist able to promote apoptosis in tumor cells. Methods: In a first-in-human Phase I study in patients with advanced cancer, Debio 1143 was given orally once daily on days 1-5 every 2 or 3 weeks. Ten dose levels ranging from 5 mg to 900 mg were explored. Blood samples were taken on days 1-5 for PK (Debio 1143) and PD assessments (IAPs levels, blood markers of apoptosis and inflammation) with rich sampling at day 1 and day 5. A population PK model was built within NONMEM 7.2. Further to a graphical PK vs PD check, population PK/PD models were explored. Results: The PK of Debio 1143 was well described by a two-compartment model with two absorption pathways. Debio 1143 bioavailability appeared to decrease after repeated administrations, suggesting a possible time-dependent PK. Graphically, markers of target engagement (cIAP1 PBMCs levels) and apoptosis (caspase-3 generated cytokeratine-18 fragments plasma levels [CK18-M30]) showed changes with Debio 1143 PK. cIAP1 levels could be fitted using an indirect response model where the elimination of cIAP1 was stimulated by Debio 1143. The CK18-M30 data was best fitted by a model with a delayed effect, onsetting after the first day. Simulations of d14q21 treatment predicted a clear decrease in cIAP1, with small differences between 100 mg and 200 mg dosing. A CK18-M30 increase ranging from 20 to 200% is predicted at day 14 after 200 mg qd. Conclusions: Debio 1143PK is compatible with oral administration q5d21. A clear relationship between PK and markers of target engagement or apoptosis was evidenced over the dose range explored. Findings from the q14d21 simulations indicate a promising PK/PD relationship for such schedule. Results from ongoing studies including PK/safety and PK/efficacy exploration will also feed the model to support Debio 1143 treatment optimization.

Smac mimetic promotes glioblastoma cancer stem-like cell differentiation by activating NF-κB

Recently, a broader role of inhibitor of apoptosis (IAP) proteins besides their antiapoptotic functions has been described. Therefore, we investigated the effect of non-toxic concentrations of the small-molecule Smac mimetic BV6, which antagonizes IAP proteins, on differentiation of cancer stem-like cells (CSLCs) derived from primary glioblastoma (GBM) specimens. Here, we identify a novel function of BV6 in regulating differentiation of GBM CSLCs by activating NF-κB. BV6 at non-lethal doses stimulates morphological changes associated with the differentiation of GBM CSLCs. BV6 increases transcriptional activity, mRNA and protein levels of the astrocytic marker GFAP without altering expression of the neuronal marker β-III-tubulin, indicating that BV6 induces astrocytic differentiation of GBM CSLCs. Molecular studies reveal that BV6 triggers processing of the NF-κB subunit p100 to p52, nuclear translocation of p52 and p50 and increased NF-κB DNA-binding. Intriguingly, inhibition of NF-κB by overexpression of dominant-negative IκBα super-repressor (IκBα-SR) blocks the BV6-stimulated increase in GFAP and differentiation. Interestingly, this BV6-stimulated differentiation is associated with reduced expression of stemness markers such as CD133, Nanog and Sox2 in GBM CSLCs. In contrast, BV6 does not alter cell morphology, differentiation and expression of stemness markers in non-malignant neural stem cells. Importantly, BV6 treatment reduces clonogenicity of GBM CSLCs in vitro and in vivo, suppresses their tumorigenicity in orthotopic and subcutaneous mouse models and significantly increases the survival of mice. By identifying a novel role of BV6 in promoting differentiation of GBM CSLCs, these findings provide new insights into Smac mimetic-regulated non-apoptotic functions with important implications for targeting GBM CSLCs.

Small-Molecule IAP Antagonists Sensitize Cancer Cells to TRAIL-Induced Apoptosis: Roles of XIAP and cIAPs

TNF-related apoptosis-inducing ligand (TRAIL) is a promising anticancer agent because it shows apoptosis-inducing activity in transformed, but not in normal, cells. As with most anticancer agents, however, its clinical use is restricted by either inherent or acquired resistance by cancer cells. We demonstrate here that small-molecule SMAC mimetics that antagonize the inhibitor of apoptosis proteins (IAP) potently sensitize previously resistant human cancer cell lines, but not normal cells, to TRAIL-induced apoptosis, and that they do so in a caspase-8-dependent manner. We further show that the compounds have no cytotoxicity as single agents. Also, we demonstrate that several IAP family members likely participate in the modulation of cellular sensitivity to TRAIL. Finally, we note that the compounds that sensitize cancer cells to TRAIL are the most efficacious in binding to X-linked IAP, and in inducing cellular-IAP (cIAP)-1 and cIAP-2 degradation. Our studies thus describe valuable compounds that allow elucidation of the signaling events occurring in TRAIL resistance, and demonstrate that these agents act as potent TRAIL-sensitizing agents in a variety of cancer cell lines.

Abstract OT1-4-04: A phase II randomized, open-label, neoadjuvant study of LCL161, an oral antagonist of inhibitor of apoptosis proteins, in combination with paclitaxel in patients with triple-negative breast cancer

Abstract Background: Inhibitor of apoptosis proteins (IAPs) negatively regulate cell death through a variety of mechanisms. LCL161 is an oral small-molecule antagonist of IAPs that has demonstrated single-agent activity and synergy with paclitaxel in breast cancer tumor models. In preclinical studies, a gene expression signature has been shown to enrich for response to LCL161. The recommended dose of LCL161 1800 mg once weekly has demonstrated preliminary antitumor activity with paclitaxel in an ongoing Phase Ib study in patients with breast cancer. Trial design: This is a Phase II, randomized, open-label study of neoadjuvant paclitaxel with or without LCL161 in women with operable, newly diagnosed triple-negative breast cancer (NCT01617668). Key inclusion criteria include women with histologically confirmed diagnosis of triple-negative breast cancer; clinical stages T2, N0–N2, M0; candidates for mastectomy or breast-conserving surgery; ECOG performance status ≤1; known status of the LCL161-predictive gene expression signature (positive and negative gene signature is a stratification factor); and adequate bone marrow and organ function. Key exclusion criteria are: bilateral or inflammatory breast cancer; locally recurrent breast cancer; patients currently receiving systemic therapy for any other malignancy, or having received systemic therapy for a malignancy in the preceding 3 months; impaired gastrointestinal function that may affect the absorption of LCL161; or uncontrolled cardiac disease. Patients are randomized 1:1 to receive paclitaxel IV (80 mg/m2 weekly) with or without oral LCL161 (1800 mg once weekly) for 12 weeks (corresponding to 4 treatment cycles). Each treatment arm is stratified 1:1 based on gene expression signature status (positive or negative). Endpoints: The primary endpoint is pathologic complete response (pCR), defined as the absence of invasive disease in the breast after 12 weeks of therapy, analyzed separately in the gene expression signature positive and negative groups. The key secondary endpoint is the pCR rate following treatment with LCL161 and paclitaxel in gene expression signature-positive or -negative tumors. Other secondary endpoints include: pCR rate in breast after 12 weeks of therapy in the full study population, and in patients with gene expression signature-positive and -negative tumors treated with paclitaxel alone; pCR rate in breast, regional nodes and axilla; biomarker evaluation including caspase 3 activation in tumor; safety; and pharmacokinetics of LCL161. Statistical methods: pCR analysis will be performed according to treatment group and gene expression signature status. An absolute increase of at least 7.5% in pCR rate of the experimental arm over the control arm will be considered as evidence of clinically relevant efficacy. Target accrual: Approximately 200 patients will be randomized into this study. Recruitment is ongoing across America, Europe, and Asia. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr OT1-4-04.

Discovery of a Novel Class of Dimeric Smac Mimetics as Potent IAP Antagonists Resulting in a Clinical Candidate for the Treatment of Cancer (AZD5582)

A series of dimeric compounds based on the AVPI motif of Smac were designed and prepared as antagonists of the inhibitor of apoptosis proteins (IAPs). Optimization of cellular potency, physical properties, and pharmacokinetic parameters led to the identification of compound 14 (AZD5582), which binds potently to the BIR3 domains of cIAP1, cIAP2, and XIAP (IC50 = 15, 21, and 15 nM, respectively). This compound causes cIAP1 degradation and induces apoptosis in the MDA-MB-231 breast cancer cell line at subnanomolar concentrations in vitro. When administered intravenously to MDA-MB-231 xenograft-bearing mice, 14 results in cIAP1 degradation and caspase-3 cleavage within tumor cells and causes substantial tumor regressions following two weekly doses of 3.0 mg/kg. Antiproliferative effects are observed with 14 in only a small subset of the over 200 cancer cell lines examined, consistent with other published IAP inhibitors. As a result of its in vitro and in vivo profile, 14 was nominated as a candidate for clinical development.

Inhibitors of apoptosis proteins (IAPs) are required for effective T-cell expansion/survival during antiviral immunity in mice

Inhibitors of apoptosis proteins (IAPs) were originally described as regulating apoptosis by direct binding to caspases. More recently, IAPs have been identified as important modulators of canonical and noncanonical nuclear factor κB signaling via their ubiquitin-E3 ligase activity. IAPs are therefore, not only gatekeepers of cell death, but are probably also involved in the regulation of inflammation, as well as innate and adaptive immunity. In this study, we analyzed the role of IAPs in T-cell immunity during lymphocytic choriomeningitis virus (LCMV) infection by pharmacological targeting with an IAP antagonist/second mitochondria-derived activator of caspase-mimetic. Expansion of virus-specific CD8 T cells was drastically reduced in LCMV-infected mice exposed to IAP antagonists. Accordingly, virus control was substantially impaired, indicated by high virus titres in the spleen and the spread of LCMV to peripheral organs. The profound negative effect of IAP antagonists on T-cell immunity was partially linked to tumor necrosis factor-mediated cell death of activated T cells and required inhibition of X-linked inhibitor of apoptosis, as well as cellular IAP-1. Thus, IAPs play an important role in T-cell expansion and survival in the context of a highly inflammatory environment such as a virus infection, indicating that IAP antagonists may interfere with immune responses.

Abstract A55: Potent, dual cIAP1/XIAP antagonists induce apoptosis in a melanoma stem cell population.

Abstract The inhibitor of apoptosis proteins (IAP) are key regulators of anti-apoptotic and pro-survival signaling pathways. Overexpression of IAPs occurs in various cancers and has been associated with tumor progression and resistance to treatment. IAP antagonists activate the E3 ligase function of cIAP1 and stimulate rapid autoubiquitylation and proteosomal degradation of both cIAP1 and cIAP2. Elimination of these proteins leads to a switch in TNFα signalling from being pro-survival to being pro-apoptotic. However, a strong pro-apoptotic effect from cIAP loss cannot be achieved without sustained antagonism of XIAP-mediated caspase inhibition. Therefore, a best in class profile for IAP antagonists requires potent dual antagonism of cIAP1 and XIAP. Astex has used fragment based-drug discovery to develop a second generation of IAP antagonists, which are non-peptidomimetic and do not contain an alanine as a warhead. This series has the ability not only to efficiently degrade cIAP1 but also to potently antagonize XIAP, delivering a dual cIAP1/XIAP inhibitory profile which is not apparent in the first generation of IAP antagonists based on an alanine warhead. Here, we report the structural understanding of the unique molecular profile of the series together with the enhanced activity of these compounds in melanoma cancer stem cells (CSC). CSC populations are more resistant to apoptosis than the bulk cell population and they have been associated with resistance to cancer therapy, relapse and cancer progressions. Blockade of the apoptotic pathway by up-regulation of anti-apoptotic factors has been implicated in conferring resistance in CSC fractions and increased XIAP expression has also been reported in these cells. We have analysed the CD133+ population of three melanoma cell lines (SK-MEL-2, SK-MEL-5 and SK-MEL-28) and measured activation of caspase-3 (NucView™ cell staining) after treatment with IAP antagonists in presence of TNFα. Our potent dual cIAP1/XIAP antagonists (XIAP EC50 <= 5 nM in cells) have a significantly enhanced apoptosis-inducing capacity (p<0.05), on the CSC fraction in all three cell lines, compared to alanine-containing cIAP1 selective antagonists, with reduced XIAP potency (XIAP EC50 > 35 nM in cells). The enhanced XIAP potency of our compounds is overriding the resistance in CSC subpopulations, highlighting the importance of dual antagonism in promoting efficient induction of apoptosis. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A55. Citation Format: Gianni Chessari, Maria Ahn, Keisha Hearn, Christopher N. Johnson, Jon Lewis, Neil Thompson, George Ward, Pamela Williams. Potent, dual cIAP1/XIAP antagonists induce apoptosis in a melanoma stem cell population. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A55.

Abstract C20: In vitro effects of Debio 1143, a novel oral IAP inhibitor, in human SCCHN cell lines and tumor specimens.

Abstract Background: Resistance to radiotherapy and chemotherapy-induced apoptosis is a hallmark of cancer. Inhibitors of apoptosis proteins (IAPs) are negative modulators of apoptosis frequently expressed in various cancers, and, as such, attractive targets to overcome resistance to cancer therapy. The oral SMAC mimetic Debio 1143 (D1143, a.k.a AT-406), an antagonist of multiple IAPs (cIAP1/2 and XIAP), is currently investigated in a Phase I oncology clinical trial. This study aimed at evaluating D1143 activity as a single agent and in combination with TNF-α, TRAIL, cisplatin or carboplatin, in various SCCHN models. Materials and Methods: The antiproliferative effects of D1143 were evaluated in a panel of 5 human SCCHN cell lines by MTT assay. Baseline and phosphorylated protein levels were detected by Western blot analysis. Tumor samples from SCCHN patients were surgically resected and cut into 300 µm thick slices using a tissue slicer (TIPCAN®). Each slice was exposed to 10 µM D1143 and/or 1 µM of platinum-based drug for 48 hours. Tumor samples were analyzed by immunohistochemistry (IHC) or immunofluorescence to visualize the effects of the treatment on various biomarkers of cell apoptosis, proliferation, and drug target engagement. Results: A panel of 5 SCCHN cell lines was characterized for the expression of c-IAP1/2, XIAP, Bcl2, LRIG1, and other proteins implicated in resistance to cell death. D1143 alone displayed limited antiproliferative activity in only one cell line (Detroit 562). However, two SCCHN cell lines were sensitive to D1143 and TRAIL combined (SQ20B and SCC15), three were sensitive to D1143 and TNF-α combined (SQ20B, SCC61, and Detroit 562), and one was resistant to both combinations (HEP2). In Detroit 562 sensitive cells, 10 µM D1143 induced sustained cIAP1/2 degradation after 15 minutes of exposure. In contrast, in the HEP2 insensitive cell line, D1143 induced slight and transient inhibition of cIAP1/2. IHC analyses revealed that ex vivo exposure to D1143 of tumor explants freshly resected from SCCHN patients decreased cIAP1 staining. Treatment of tumor samples with D1143 combined with cisplatin or carboplatin augmented the cleavage of caspase 3 compared to controls suggesting induction of apoptosis. Conclusion: In 4 out of 5 SCCHN cell lines, D1143 induced cIAP1/2 degradation and potentiated TNF-α or TRAIL-induced antiproliferative effects. D1143 combined with carboplatin and cisplatin in SCCHN patient samples induced caspase 3-dependent apoptosis. D1143 in combination with conventional chemotherapies may be considered as a potential treatment for SCCHN patients. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C20. Citation Format: Marie Serova, Annemilaï Tijeras-Raballand, Sebastien Albert, Sandrine Faivre, Eric Raymond, Anne Vaslin, Claudio Zanna, Gregoire Vuagniaux, Armand de Gramont. In vitro effects of Debio 1143, a novel oral IAP inhibitor, in human SCCHN cell lines and tumor specimens. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C20.

Abstract CN04-04: Targeting death receptors and SMAC mimetics.

Abstract Evasion of apoptosis is a hallmark of human cancers and contributes to tumor formation and progression as well as resistance to current cancer therapies, which largely rely on intact cell death pathways in cancer cells. Apoptosis resistance can be caused by aberrant expression of antiapoptotic proteins, for example Inhibitor of Apoptosis (IAP) proteins, which are expressed at high levels in many human cancers. Since IAP proteins block apoptosis at the core of the apoptotic machinery by inhibiting effector caspases, they present promising therapeutic targets. Smac mimetics are small-molecule inhibitors that mimick the IAP-binding motif of the endogenous IAP antagonist Smac and are among the most widely used strategies to antagonize IAP proteins. In addition to their use as single agents, Smac mimetics have been used in combination with various cytotoxic stimuli to trigger cell death in cancer cells, in particular together with death receptor ligands such as soluble TRAIL or TRAIL receptor agonists. For example in pancreatic cancer_ one of the most lethal types of cancer_ IAP antagonists were shown to synergize with TRAIL receptor agonists to induce apoptosis and suppress tumor growth in preclinical in vitro and in vivo models. Furthermore, Smac mimetics and TRAIL acted in concert to trigger apoptosis in CLL, even in patients with poor prognostic features such chemoresistant disease, p53 mutation or chromosome 17p deletion. Thus, Smac mimetics may open new perspectives to overcome resistance to death receptor-mediated apoptosis in various forms of human cancers. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):CN04-04. Citation Format: Simone Fulda. Targeting death receptors and SMAC mimetics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr CN04-04.

Cellular inhibitor of apoptosis-2 is a critical regulator of apoptosis in airway epithelial cells treated with asthma-related inflammatory cytokines

Aberrant apoptosis of airway epithelial cells (AECs) is a disease contributing feature in the airways of asthmatics. The proinflammatory cytokines tumor necrosis factor α (TNFα) and interferon γ (IFNγ) are increased in asthma and have been shown to contribute to apoptosis at the airways. In the present study, we investigated the role of the inhibitor of apoptosis protein (IAP) family in primary AECs exposed to TNFα and IFNγ. IAPs are potent regulators of caspase activity elicited by the intrinsic and extrinsic apoptosis pathways. However, while caspase-mediated apoptosis was observed in AECs exposed to doxorubicin, it was not observed after cytokine treatment. Instead, AECs exhibited proapoptotic changes evidenced by an increased Bax:Bcl2 transcript ratio and partial processing of procaspase-3. Examination by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and Western analysis showed that proapoptotic changes were associated with a time- and dose-dependent induction of cellular IAP-2 (cIAP2), potentiated primarily by IFNγ. The abundance of the IAP antagonists X-linked IAP-associated factor 1 (XAF1) and second mitochondria-derived activator of caspases did not change, although a moderate nuclear redistribution was observed for XAF1, which was also observed for cIAP2. Small interfering RNA (siRNA)-mediated depletion of cIAP2 from AECs leads to caspase-3 activation and poly (ADP-ribose) polymerase cleavage, but this required extended cytokine exposure to produce a concomitant decrease in cIAP1 and Bcl2. These results indicate that AECs possess endogenous mechanisms making them highly resistant to apoptosis due to asthma-related inflammatory cytokines, and the activity of cIAP2 plays an important role in this protection.

Learning and Confirming with Preclinical Studies: Modeling and Simulation in the Discovery of GDC-0917, an Inhibitor of Apoptosis Proteins Antagonist

The application of modeling and simulation techniques is increasingly common in the preclinical stages of the drug development process. GDC-0917 [(S)-1-((S)-2-cyclohexyl-2-((S)-2-(methylamino)propanamido)acetyl)-N-(2-(oxazol-2-yl)-4-phenylthiazol-5-yl)pyrrolidine-2-carboxamide] is a potent second-generation antagonist of inhibitor of apoptosis (IAP) proteins that is being developed for the treatment of various cancers. GDC-0917 has low to moderate clearance in the mouse (12.0 ml/min/kg), rat (27.0 ml/min/kg), and dog (15.3 ml/min/kg), and high clearance in the monkey (67.6 ml/min/kg). Accordingly, oral bioavailability was lowest in monkeys compared with other species. Based on our experience with a prototype molecule with similar structure, in vitro-in vivo extrapolation was used to predict a moderate clearance (11.5 ml/min/kg) in humans. The predicted human volume of distribution was estimated using simple allometry at 6.69 l/kg. Translational pharmacokinetic-pharmacodynamic (PK-PD) analysis using results from MDA-MB-231-X1.1 breast cancer xenograft studies and predicted human pharmacokinetics suggests that ED50 and ED90 targets can be achieved in humans using acceptable doses (72 mg and 660 mg, respectively) and under an acceptable time frame. The relationship between GDC-0917 concentrations and pharmacodynamic response (cIAP1 degradation) was characterized using an in vitro peripheral blood mononuclear cell immunoassay. Simulations of human GDC-0917 plasma concentration-time profile and cIAP1 degradation at the 5-mg starting dose in the phase 1 clinical trial agreed well with observations. This work shows the importance of leveraging information from prototype molecules and illustrates how modeling and simulation can be used to add value to preclinical studies in the early stages of the drug development process.

Design, stereoselective synthesis, and biological evaluation of novel tri-cyclic compounds as inhibitor of apoptosis proteins (IAP) antagonists

We recently reported the discovery of octahydropyrrolo[1,2-a]pyrazine A as a lead compound for an inhibitor of apoptosis proteins (IAP) antagonist. To develop IAP antagonists with favorable PK profiles, we designed novel tri-cyclic compounds, octahydro-1H-cyclopropa[4,5]pyrrolo[1,2-a]pyrazines 1 and 2 based on co-crystal structural analysis of A with cellular IAP-1 (cIAP-1). The additional cyclopropane moiety was used to block the predicted metabolic site of compound A without detriment to the binding affinity for cIAP. Compounds 1 and 2 were stereoselectively synthesized via intermediates 4a and 5b′, which were obtained by Simmons−Smith cyclopropanation of ethylester 3a and silyl ether 3b′. Compounds 1 and 2 showed strong growth inhibition in MDA-MB-231 breast cancer cells and improved metabolic stability in comparison to A. Compound 2 exhibited significant in vivo PD effects to increase tumor necrosis factor-alpha mRNA in a dose dependent manner.

Induction of the DNA damage response by IAP inhibition triggers natural immunity via upregulation of NKG2D ligands in Hodgkin lymphoma in vitro

Evasion of apoptosis is a hallmark of cancer cells. Inhibitor of apoptosis proteins (IAPs) act as endogenous inhibitors of programmed cell death and are overexpressed in several tumors including Hodgkin lymphoma (HL). Preclinical studies indicate antitumor activity of IAP antagonists and clinical studies in hematological malignancies are underway. Here, we investigate the impact of the small molecule IAP antagonist LCL161 on HL cell lines. Although the antagonist caused rapid degradation of cIAP1 leading to TNFα secretion, LCL161 did not promote apoptosis significantly. However, LCL161 induced expression of MICA and MICB, ligands for the activating immune receptor NKG2D, and enhanced the susceptibility of HL cells to NKG2D-dependent lysis by NK cells. MICA/B upregulation was dependent on activation of the DNA damage response upon LCL161 treatment. Taken together, we demonstrate a novel link between IAP inhibition, DNA damage and immune recognition.

Phase I study of safety and pharmacokinetics (PK) of GDC-0917, an antagonist of inhibitor of apoptosis (IAP) proteins in patients (Pts) with refractory solid tumors or lymphoma.

2503 Background: GDCE0917 is a small molecule that triggers tumor cell apoptosis by selectively antagonizing IAP proteins. Preclinical studies demonstrated antitumor efficacy of GDC-0917 alone or in combination with chemotherapeutic agents. Methods: Oral GDC-0917 was given on Day (d) 1 followed by 2d off and a 2-week (w) on/ 1w off treatment (tx) schedule (21d cycle) starting d4. A modified continual reassessment method was used for dose escalation. Dose-limiting toxicity (DLT, assessed d1-24), PK, adverse events (AEs), pharmacodynamics (PD), and clinical activity were evaluated. Results: 42 pts of age 36-86 (median 60.5) were enrolled in 11 cohorts (5-600 mg) and received 1-15 cycles (median 2) of GDC-0917. One DLT, Grade (G) 3 fatigue, was observed at 450 mg. The maximum tolerated dose was not determined although plasma concentrations of preclinically defined IC90 were reached. The most frequent AEs were diarrhea, fatigue and nausea (26.2% each), vomiting (23.8%), and constipation (19%). The most frequent AEs reported as tx-related were mostly G1-2 and included fatigue and nausea (21.4% each), vomiting (14.3%), rash (11.9%) and pruritus (9.5%). AEs reported as tx-related that were ≥ G3 in > 1 pt were elevated AST and ALT (2 pts, at 450 and 600 mg). AEs reported as tx-related that resulted in tx discontinuation were G3 fatigue, G2 QTc prolongation, G2 drug hypersensitivity, G2 pneumonitis (1 pt each), and G3 pruritus/G2 rash (same pt). GDC-0917 peak concentrations were observed 2-3h post dosing. Exposure was dose-proportional with a mean plasma elimination t1/2of 4-8h and no apparent accumulation at steady state. Rapid down-modulation of cIAP1 was observed in PBMCs at all dose levels. Evaluation of tumor biopsies demonstrated decreases in cIAP1 (2 pts total, at 40 and 200 mg) and increases in activated caspase-3 and cPARP (1 pt at 200 mg). Two pts (4.8%) had a complete response (both unconfirmed, ovarian Ca and MALT lymphoma [PET]); 4 pts (9.5%) had stable disease for ≥ 3 months. Conclusions: GDC-0917 had a favorable safety, PK and PD profile in pts with advanced malignancies. These encouraging results support further clinical evaluation of this agent. Clinical trial information: NCT01226277.

Design, synthesis and evaluation of inhibitor of apoptosis protein (IAP) antagonists that are highly selective for the BIR2 domain of XIAP

We recently reported the systematic ligand-based rational design and synthesis of monovalent Smac mimetics that bind preferentially to the BIR2 domain of the anti-apoptotic protein XIAP. Expanded structure–activity relationship (SAR) studies around these peptidomimetics led to compounds with significantly improved selectivity (>60-fold) for the BIR2 domain versus the BIR3 domain of XIAP. The potent and highly selective IAP antagonist 8q (ML183) sensitized TRAIL-resistant prostate cancer cells to apoptotic cell death, highlighting the merit of this probe compound as a valuable tool to investigate the biology of XIAP.

Abstract 837: Identification of non-canonical NF-κB signaling as a critical mediator of Smac mimetic-stimulated migration and invasion of glioblastoma cells.

Abstract Since inhibitor of apoptosis (IAP) proteins can regulate additional signaling pathways beyond apoptosis, we investigated the effect of the Smac mimetic BV6, which antagonizes IAP proteins, on non-apoptotic functions in glioblastoma (GBM). Here, we identify non-canonical NF-κB signaling and a TNFα/TNFR1 autocrine/paracrine loop as critical mediators of BV6-stimulated migration and invasion of GBM cells. In addition to GBM cell lines, BV6 triggers cell elongation, migration and invasion in primary, patient-derived GBM cells at non-toxic concentrations, which do not affect cell viability or proliferation, and also increases infiltrative tumor growth in vivo underscoring the relevance of these findings. Molecular studies reveal that BV6 causes rapid degradation of cIAP proteins, accumulation of NIK, processing of p100 to p52, translocation of p52 into the nucleus, increased NF-κB DNA binding and enhanced NF-κB transcriptional activity. EMSA supershift shows that the NF-κB DNA binding subunits consist of p50, p52 and RelB further confirming activation of the non-canonical NF-κB pathway. BV6-stimulated NF-κB activation leads to elevated mRNA levels of TNFα and additional NF-κB target genes involved in migration (i.e. IL-8, MCP-1, CXCR4) and invasion (i.e. MMP9). Importantly, inhibition of NF-κB by overexpression of dominant-negative IκBα superrepressor prevents the BV6-stimulated cell elongation, migration and invasion. Similarly, specific inhibition of non-canonical NF-κB signaling by RNA interference-mediated silencing of NIK suppresses the BV6-induced cell elongation, migration and invasion as well as upregulation of NF-κB target genes. Intriguingly, pharmacological or genetic inhibition of the BV6-stimulated TNFα autocrine/paracrine loop by the TNFα-blocking antibody Enbrel or by knockdown of TNFR1 abrogates the BV6-induced cell elongation, migration and invasion. By demonstrating that the Smac mimetic BV6 at non-toxic concentrations promotes migration and invasion of GBM cells via non-canonical NF-κB signaling, our findings have Citation Format: Aurelie Tchoghandjian, Claudia Jennewein, Ines Eckhardt, Krishna Rajalingam, Simone Fulda. Identification of non-canonical NF-κB signaling as a critical mediator of Smac mimetic-stimulated migration and invasion of glioblastoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 837. doi:10.1158/1538-7445.AM2013-837

Abstract 2944: AT-IAP, a dual cIAP1 and XIAP antagonist with oral antitumor activity in melanoma models.

Abstract Melanoma is a highly aggressive malignancy with an exceptional ability to develop resistance and no curative therapy is available for patients with metastatic disease. Inhibitor of apoptosis proteins (IAP) play a key role in preventing cell death by apoptosis. In normal cell, IAPs are highly regulated by endogenous antagonists (e.g. SMAC) but in melanoma cell lines and in patient samples expression levels of IAPs are generally high and depleting IAPs by siRNA tended to reduce cell viability, with XIAP reduction being the most efficient [1]. Small molecule IAP antagonists have the ability to switch IAP-controlled pro-survival pathways towards apoptosis and cell death. Recent evidence suggests that a true dual antagonist of both cIAP1 and XIAP will promote an effective apoptotic response through generation of death-inducing ripoptosome complexes, with resultant caspase activation [2, 3]. We have used our fragment-based drug discovery technology PyramidTM to derive a non-peptidomimetic IAP antagonist, AT-IAP, which does not have an alanine warhead and has nanomolar cellular potency for both XIAP and cIAP1. Initial pharmacokinetic and pharmacodynamic modeling of AT-IAP in mice bearing the MDA-MB-231 cell line indicated that daily oral dosing of AT-IAP at 30 mg/kg ensures high concentrations of compound in tumor and plasma over a 24 h period with resultant inhibition of both XIAP and cIAP1 and induction of apoptosis markers (cleaved PARP and cleaved caspase-3). In this paper, we describe the characterization of AT-IAP in melanoma models. An in vitro cell line proliferation screen demonstrated that 36% of melanoma cell lines exhibited enhanced sensitivity to AT-IAP, which was improved on addition of exogenous 1 ng/ml TNF-α (92% of cell lines were sensitive to AT-IAP + TNF-α). Sensitivity of melanoma cells to AT-IAP has also been confirmed in a panel of 20 primary melanoma tumors in colony formation assays set up in the presence and absence of added TNF-α. Finally, a set of biomarkers has been identified and used to predict single agent activity of AT-IAP in a range of melanoma cell line and patient derived xenograft models. [1] Engesaeter et al., Cancer Biology & Therapy, 2011, 12 (1), 47 [2] Ndubaku et al., ACS Chem Biol., 2009, 4 (7), 557 [3] Meier, P., Nat Rev. Cancer, 2010, 10 (8), 561 Citation Format: Gianni Chessari, Ahn Maria, Ildiko Buck, Elisabetta Chiarparin, Joe Coyle, James Day, Martyn Frederickson, Charlotte Griffiths-Jones, Keisha Hearn, Steven Howard, Tom Heightman, Petra Hillmann, Aman Iqbal, Christopher N. Johnson, Jon Lewis, Vanessa Martins, Joanne Munck, Mike Reader, Lee Page, Anna Hopkins, Alessia Millemaggi, Caroline Richardson, Gordon Saxty, Tomoko Smyth, Emiliano Tamanini, Neil Thompson, George Ward, Glyn Williams, Pamela Williams, Nicola Wilsher, Alison Woolford. AT-IAP, a dual cIAP1 and XIAP antagonist with oral antitumor activity in melanoma models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2944. doi:10.1158/1538-7445.AM2013-2944

Time‐Dependent Inhibition and Induction of Human Cytochrome P4503A4/5 by an Oral IAP Antagonist, LCL161, In Vitro and In Vivo in Healthy Subjects

Tumor cells can evade programmed cell death via up-regulation of inhibitor of apoptosis proteins (IAPs). LCL161 is a small molecule oral IAP antagonist in development for use in combination with cytotoxic agents. The effect of LCL161 on CYP3A4/5 (CYP3A) activity was investigated in vitro and in a clinical study. Results in human liver microsomes indicated LCL161 inhibited CYP3A in a concentration- and time-dependent manner (KI of 0.797 µM and kinact of 0.0803 min(-1) ). LCL161 activated human PXR in a reporter gene assay and induced CYP3A4 mRNA up to ∼5-fold in human hepatocytes. In healthy subjects, the dual inhibitor and inductive effects of a single dose of LCL161 were characterized using single midazolam doses, given before and at three time points after the LCL161 dose. Midazolam Cmax increased 3.22-fold and AUC(0-inf) increased 9.32-fold when administered four hours after LCL161. Three days later, midazolam Cmax decreased by 27% and AUC(0-inf) decreased by 30%. No drug interaction remained one week later. The strong CYP3A inhibition by LCL161 was accurately predicted using dynamic physiologically-based pharmacokinetic (PBPK) modeling approaches in Simcyp. However, the observed induction effect after the LCL161 dose could not be modeled; suggesting direct enzyme induction by LCL161 was not the underlying mechanism.

Abstract 3325: Role of smac-mimetic in restoring apoptosis in chronic lymphocytic leukemia .

Abstract Defective apoptosis is the fundamental hallmark feature of CLL biology. Inhibitors of apoptosis proteins (IAPs) inhibit apoptosis by neutralizing the activities of caspases, a family of proteases that act in concert to execute apoptosis signals. SMAC (Second Mitochondria Derived Activator of Caspases) released from mitochondria in response to apoptotic stimuli is an endogenous antagonist of IAP that promote apoptosis by neutralizing the anti-apoptotic properties of IAPs. The IAP-binding tetrapeptide motifs from SMAC (AVPI; Ala-Val-Pro-Ile) and from caspase-9 (ATPF; Ala-Thr-Pro-Phe) both bind to the same conserved surface groove on the BIR3 domain of XIAP, allowing SMAC to remove the XIAP mediated inhibition of caspase-9. This provided rationale to use SMAC mimetics to reverse the IAP-mediated inhibition of caspases. Monomeric smac mimetic (smac066) demonstrated low nM potency for MDA-MB231 cell line and low μM potency for HL-60 and PC-3 cells (Seneci et al., 2009). Using CLL primary cells, we show that IAPs (XIAP, cIAP1 and cIAP2) are expressed at high levels in CLL lymphocytes (n=9) compared to normal lymphocytes (n=7), while SMAC is present at similar levels suggesting that though SMAC is present, it is important that the caspases are unbound from IAPs to execute apoptosis. CLL lymphocytes treated with smac066 (10 μM; 24 hr) demonstrated a significant activation of apoptosis (n=37; p=0.0001; median 22%; range 1-74%) with IC50 values of 8 μM, 6 μM, and 6 μM at 24, 48 and 72 hr respectively. The correlative analysis between the sensitization and respective prognostic markers demonstrate that samples with poor prognostic markers such as 17p deletion (n=3) (tumor suppressor protein p53 locus), 11q deletion (n=3) (DNA damage response gene, ATM locus), unmutated IGVH gene status (n=7), trisomy12 (n=6) and number of prior treatments (n=4) required higher concentrations of drug compared to patients with no cytogenetic abnormalities. Alternatively, samples that had 13q deletion (n=6) (locus of miRs 15a and 16 that regulate the Mcl-1 and Bcl-2 expression) were significantly sensitive to smac-mediated apoptosis. The mechanism involved in restoring the apoptosis by smac mimetic in CLL cells was associated with decrease in IAPs (5 μM; n=16; 24 hr; p=0.003) such as XIAP and cIAP2, but not cIAP1. PARP cleavage (a substrate of caspases), cleavages of procaspase 8 and 9 (initiator caspases) and procaspase 3 (executioner caspase) suggested that both extrinsic and intrinsic pathways are activated by smac066. The immuno-precipitation studies showed that in untreated cells, XIAP and caspase-3 are in association with each other. However, upon treatment with smac mimetic caspase-3 dissociated significantly from the XIAP. The level of SMAC bound to XIAP increased with smac mimetic. In summary, smac066 disrupts the IAP-mediated inhibition of apoptosis in CLL primary cells; thereby should be combined and evaluated with other chemotherapeutic agents. Citation Format: Kumudha Balakrishnan, Min Fu, Francesco Onida, William G. Wierda, Michael Keating, Varsha Gandhi. Role of smac-mimetic in restoring apoptosis in chronic lymphocytic leukemia . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3325. doi:10.1158/1538-7445.AM2013-3325