Inhibitor Of Apoptosis Antagonists Research Articles

Heterobifunctional Ligase Recruiters Enable pan-Degradation of Inhibitor of Apoptosis Proteins.

Proteolysis targeting chimeras (PROTACs) represent a new pharmacological modality to inactivate disease-causing proteins. PROTACs operate via recruiting E3 ubiquitin ligases, which enable the transfer of ubiquitin tags onto their target proteins, leading to proteasomal degradation. However, several E3 ligases are validated pharmacological targets themselves, of which inhibitor of apoptosis (IAP) proteins are considered druggable in cancer. Here, we report three series of heterobifunctional PROTACs, which consist of an IAP antagonist linked to either von Hippel-Lindau- or cereblon-recruiting ligands. Hijacking E3 ligases against each other led to potent, rapid, and preferential depletion of cellular IAPs. In addition, these compounds caused complete X-chromosome-linked IAP knockdown, which was rarely observed for monovalent and homobivalent IAP antagonists. In cellular assays, hit degrader 9 outperformed antagonists and showed potent inhibition of cancer cell viability. The hetero-PROTACs disclosed herein are valuable tools to facilitate studies of the biological roles of IAPs and will stimulate further efforts toward E3-targeting therapies.

Efficacy of birinapant in combination with carboplatin in targeting platinum‑resistant epithelial ovarian cancers.

Patients diagnosed with epithelial ovarian cancers (EOCs) often suffer from disease relapse associated with the emergence of resistance to standard platinum-based chemotherapy. Treatment of patients with chemo-resistant disease remains a clinical challenge. One mechanism of chemoresistance includes overexpression of pro-survival proteins called inhibitors of apoptosis (IAP) which enable cancer cells to evade apoptosis. Due to their anti-apoptotic activity, association with poor prognosis, and correlation with therapy resistance in multiple malignancies, IAP proteins have become an attractive target for development of anticancer therapeutics. Second mitochondrial activator of caspase (SMAC) mimetics are the most widely used IAP antagonists currently being tested in clinical trials as a monotherapy and in combination with different chemotherapeutic drugs to target different types of cancer. In the present study, the antitumor efficacy of combination therapy with birinapant, a bivalent SMAC mimetic compound, and carboplatin to target platinum-resistant EOC cells was investigated. A 3D organoid bioassay was utilized to test the efficacy of the combination therapy in a panel of 7 EOC cell lines and 10 platinum-resistant primary patient tumor samples. Findings from the in vitro studies demonstrated that the birinapant and carboplatin combination was effective in targeting a subset of ovarian cancer cell lines and platinum-resistant primary patient tumor samples. This combination therapy was also effective in vitro and in vivo in targeting a platinum-resistant patient-derived xenograft (PDX) model established from one of the patient tumors tested. Overall, our study demonstrated that birinapant and carboplatin combination could target a subset of platinum-resistant ovarian cancers and also highlights the potential of the 3D organoid bioassay as a preclinical tool to assess the response to chemotherapy or targeted therapies in ovarian cancer.

Combinatorial Treatment of Birinapant and Zosuquidar Enhances Effective Control of HBV Replication In Vivo.

Chronic hepatitis B virus (HBV) infection remains a global health threat and affects hundreds of millions worldwide. Small molecule compounds that mimic natural antagonists of inhibitor of apoptosis (IAP) proteins, known as Smac-mimetics (second mitochondria-derived activator of caspases-mimetics), can promote the death of HBV-replicating liver cells and promote clearance of infection in preclinical models of HBV infection. The Smac-mimetic birinapant is a substrate of the multidrug resistance protein 1 (MDR1) efflux pump, and therefore inhibitors of MDR1 increase intracellular concentration of birinapant in MDR1 expressing cells. Liver cells are known to express MDR1 and other drug pump proteins. In this study, we investigated whether combining the clinical drugs, birinapant and the MDR1 inhibitor zosuquidar, increases the efficacy of birinapant in killing HBV expressing liver cells. We showed that this combination treatment is well tolerated and, compared to birinapant single agent, was more efficient at inducing death of HBV-positive liver cells and improving HBV-DNA and HBV surface antigen (HBsAg) control kinetics in an immunocompetent mouse model of HBV infection. Thus, this study identifies a novel and safe combinatorial treatment strategy to potentiate substantial reduction of HBV replication using an IAP antagonist.

Abstract 2405: Inhibitor of apoptosis protein antagonists as novel targeted chemotherapeutic agents for acute myeloid leukemia

Abstract Acute myeloid leukemia (AML) is a diverse collection of hematological cancers characterized by the bone marrow's excessive production of certain immature myeloid blood cells. The American Cancer Society estimated that there was ~11,000 deaths and ~21,500 new incidences of AML in the U.S. in 2019. While the various AML subtypes have differing prognoses, more than 30% of AML patients fail to enter complete remission upon standard chemotherapy regimens. Furthermore, most patients that do initially respond will relapse within 5 years in the absence of bone marrow transplantation (BMT). As such, the need for novel targets and drugs to treat this disease is a significant unmet medical need. This is especially true in patient populations unable to tolerate aggressive chemotherapy or where BMT is impossible or not recommended. The current standard of care (SOC) for AML has been the same for decades - various combinations of anthracyclines and nucleoside analogues are used as front line agents with debilitating toxicities. Recent evidence showing efficacy of Bcl2 family inhibitors in some leukemias prompted us to investigate whether antagonism of the inhibitor of apoptosis (IAP) proteins may be effective against AML. IAP proteins are involved in many processes including cell death, immunity, inflammation, cell cycle and migration, and are therefore promising targets for the development of cancer therapeutics. As the name suggests, IAP proteins play an important role in the control of apoptotic pathways and have been implicated in AML development and progression. It has been shown that IAPs are over-expressed in AML patient cells and high expression levels are linked to poor prognosis. Although Bcl2 family members only regulate mitochondrial apoptosis, IAPs modulate apoptotic signaling at multiple, and more distal, points suggesting a potential improvement in activity for drugs targeting them. We recently described the characterization and preliminary optimization of monovalent small molecule IAP antagonists. Here we show the design and synthesis of novel second generation bivalent IAP antagonists that kill AML cells much more potently than drugs currently undergoing clinical evaluation, while sparing healthy cells. Our data in leukemia cell lines and patient-derived AML samples using these novel, highly potent, IAP antagonists suggest that these small molecules may be effective as therapeutic agents. Citation Format: Darren Finlay, Nicole Bata, Allison Limpert, Dominik Heimann, Peter Teriete, Carol Burian, James Mason, Nicholas D. Cosford, Kristiina Vuori. Inhibitor of apoptosis protein antagonists as novel targeted chemotherapeutic agents for acute myeloid leukemia [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2405.

MON-LB77 Inhibitors of XIAP as Novel Therapeutic Agents in Thyroid Cancer

Treatment options for radioiodine-refractory epithelial thyroid cancer (ETC) are limited to tyrosine kinase inhibitors (TKIs), which are associated with significant adverse effects and are not curative. The inhibitors of apoptosis (IAP) family have oncogenic properties and overexpression of X-linked IAP (XIAP) in papillary thyroid carcinoma (PTC) is associated with a poor prognosis. Our group has previously reported a synergistic interaction between SMAC mimetics (SM) (IAP antagonists with XIAP and cIAP1 specificity), and PPARgamma agonists in granulosa cell tumors (1). Such an approach has not been explored in ETC.We hypothesize that SM may be efficacious alone or in combination with a secondary agent to inhibit proliferation, induce cell death and/or promote differentiation to resensitize cells to radioiodine.Four ETC-derived cell lines (K1, Nthy-ori 3-1, TPC-1 and SW-1736) were examined for cIAP1, cIAP2 and XIAP expression by RT-PCR. The K-1 and TPC-1 cell lines (PTC origin, BRAFV600E and PI3KCA mutation positive) were chosen to investigate the effects of an SM in combination with either a PPARgamma agonist (rosiglitazone) or a broad-spectrum TKI (sorafenib). Cell proliferation was examined using xCELLigence Real-Time Cell Analysis. Viability was assessed using total cell counts at 24 and 48 hours.In the four cell lines, we found abundant cIAP1 and XIAP expression and low cIAP2 expression. When an SM was used in combination with either rosiglitazone or sorafenib, we observed significant impairment of cell proliferation and viability with a clear morphological response. These effects appear SM-dependent, as SM treatment alone also showed significant effects on proliferation. However, these effects were enhanced when combined with sorafenib, which was ineffective alone. Markers of differentiation are currently being examined.Our findings suggest a novel role for SM in treating or redifferentiating radioiodine-refractory ETC. Clinically, this may involve lower doses of individual agents than when used alone, thereby reducing adverse effects.

Future Therapeutic Directions for Smac-Mimetics.

It is well accepted that the ability of cancer cells to circumvent the cell death program that untransformed cells are subject to helps promote tumor growth. Strategies designed to reinstate the cell death program in cancer cells have therefore been investigated for decades. Overexpression of members of the Inhibitor of APoptosis (IAP) protein family is one possible mechanism hindering the death of cancer cells. To promote cell death, drugs that mimic natural IAP antagonists, such as second mitochondria-derived activator of caspases (Smac/DIABLO) were developed. Smac-Mimetics (SMs) have entered clinical trials for hematological and solid cancers, unfortunately with variable and limited results so far. This review explores the use of SMs for the treatment of cancer, their potential to synergize with up-coming treatments and, finally, discusses the challenges and optimism facing this strategy.

Abstract 724: Preclinical evaluation of TQB3728, a potent orally active IAP antagonist

Abstract Objectives: Evasion of apoptosis and avoidance of immune surveillance represent two key hallmarks of cancer. Members of the inhibitor of apoptosis (IAP) gene play important interconnecting roles in both of these characteristic pathways of tumorigenesis, providing a critical nexus in targeting of cancer. Small-molecule antagonists of the IAPs, known as Smac mimetic compounds (SMCs), are in quick clinical development for cancer therapy. We disclose here a novel IAP antagonist TQB3728 and its preclinical evaluation on tumor therapy. Methods: The biochemical activities of TQB3728 for cIAP1 BIR3, cIAP2 BIR3 and XIAP BIR3 were measured by the corresponding biochemical assays. The in vitro anti-proliferative activity was evaluated on MDA-MB-231 and EMT6 cell lines. The in vivo antitumor activity of TQB3728 was evaluated in two cell-derived xenograft (CDX) models of TNBC (MDA-MB-231) and lymphoma (Karpas 422), and one syngeneic model of TNBC (EMT6). PK/PD was assessed by western blot analysis of cIAP1 expression and cleaved caspase 3 level in the treated tumor tissues. Results: TQB3728 displayed potent biochemical activities for cIAP1 BIR3, cIAP2 BIR3 and XIAP BIR3 with IC50 of 0.7, 11.8 and 22.5 nM, respectively. The expected anti-proliferative activities were also observed in both tumor cell lines, with a IC50 of 14.3 Nm for MDA-MB-231, and IC50 of 149.5 nM for EMT6 in the presence of exogenous TNFα (1 ng/ml). TQB3728 showed in vivo antitumor activity in the MDA-MB-231 CDX model with 89.8% TGI @30 mpk QD, which is better than LCL-161 (71.9% TGI @30 mpk, QD). TQB3728 also showed good antitumor activity in vivo in the Karpas 422 lymphoma CDX model with 112.2% TGI @100 mpk Q3D. In EMT6 tumor-bearing mice, the combination of TQB3728 and anti-PD1 antibody significantly regressed the tumor growth (TGI = 106.3%), whereas monotherapies only displayed moderate activities ( 60.6% and 0.4% TGI for anti-PD1 and TQB3728, respectively). Western blot analysis of the tumor samples showed that TQB3728 promoted caspase 3 cleavage, indicating that the tumor growth inhibition could be mediated by cell apoptosis signaling. Conclusions: We have discovered a potent orally active IAP antagonist, TQB3728, which shows activity of cIAP1 inhibition and strong antitumor activity in both in vitro and in vivo preclinical tumor models. These results are highly promising and warrant moving the compound forward to clinical investigation. Citation Format: Yingchun Liu, Xiquan Zhang, Ling Yang, Xin Tian, Tiantian Dong, Charles Z. Ding, Lihong Hu, Zhaobing Xu, Yuanfeng Xia, Haijuan Tong, Wenxi Li, Lijuan Hou, Chichung Chan, Shuhui Chen. Preclinical evaluation of TQB3728, a potent orally active IAP antagonist [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 724.

Paclitaxel With Inhibitor of Apoptosis Antagonist, LCL161, for Localized Triple-Negative Breast Cancer, Prospectively Stratified by Gene Signature in a Biomarker-Driven Neoadjuvant Trial.

There are currently no targeted therapies approved for triple-negative breast cancer (TNBC). A tumor necrosis factor α ( TNFα)-based gene expression signature (GS) predictive of sensitivity to LCL161, inhibitor of apoptosis antagonist, was translated into a clinical assay and evaluated in a neoadjuvant trial. Women with localized TNBC (T2/N0-2/M0) were prospectively stratified by GS status and randomly assigned (1:1) to receive oral LCL161 (1,800 mg once per week) and intravenous paclitaxel (80 mg/m2 once per week; combination arm) or paclitaxel alone (control arm) for 12 weeks, followed by surgery. The primary objective was to determine whether neoadjuvant LCL161 enhances efficacy of paclitaxel, defined by > 7.5% increase in the pathologic complete response (pCR, breast) rate, stratified by GS. Of 209 patients enrolled (207 with valid GS scores), 30.4% had GS-positive TNBC. In the GS-positive group, pCR was higher in the combination versus the control arm (38.2% v 17.2%), with 88.8% posterior probability of > 7.5% increase in pCR. However, in the GS-negative group, the pCR was lower in the combination group (5.6% v 16.4%), with 0% posterior probability of > 7.5% increase in pCR. A higher incidence of grade 3 or 4 adverse events in the combination arm included neutropenia (24.5%) and diarrhea (5.7%). Overall, 19 patients (18.1%) in the combination arm discontinued treatment because of adverse events, including pyrexia (n = 5), pneumonia (n = 4), and pneumonitis (n = 4), versus five patients (4.9%) in the control arm. This neoadjuvant trial provides evidence supporting a biomarker-driven targeted therapy approach for selected patients with GS-positive TNBC and demonstrates the utility of a neoadjuvant trial for biomarker validation and drug development, but also highlights toxicity risk. Future neoadjuvant clinical trials should carefully weigh these considerations for targeted therapy development in biomarker-defined TNBC.

Manipulation of the Alternative NF-κB Pathway in Mice Has Sexually Dimorphic Effects on Bone.

ABSTRACTAlternative NF‐κB signaling promotes osteoclastogenesis and pathological bone loss, but the effect of sex on phenotype has not been explored. We disrupted alternative NF‐κB signaling by deletion of upstream kinase NF‐κB‐inducing kinase (NIK) or NF‐κB subunit RelB and found that both NIK‐deficient and RelB‐deficient female mice possessed more than twofold higher trabecular bone mass compared to controls, whereas no differences were observed in males. In vitro, RelB‐deficient precursors from female mice showed a more severe osteoclast (OC) differentiation defect than male, while WT had no sex bias. Next, we asked whether pharmacologic activation of alternative NF‐κB by inhibitor of apoptosis (IAP) antagonist BV6 has sex‐dependent effects on bone. Unlike male mice that lost bone, female mice on BV6 for 4 weeks showed no changes in either trabecular bone mass or OC number. Because estrogen generally suppresses NF‐κB, we hypothesized that estrogen protects bone from BV6 effects in vivo. Thus, we performed ovariectomy or sham surgery in female mice, then treated with BV6 or vehicle for 4 weeks. Although ovariectomy caused bone loss, BV6 did not have any additional impact, suggesting that direct estrogen effects do not cause resistance to BV6 in vivo. The osteopenic effects of IAP antagonists in males may have implications for their use in cancer therapy. © 2018 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Regulation of innate and adaptive antitumor immunity by IAP antagonists.

Inhibition of the T-cell co-inhibitory checkpoint receptors or their ligands CTLA-4, PD-1 and PD-L1 using monoclonal antibodies has proven to be highly effective against many cancers. Yet many cancers remain resistant to checkpoint blockade, and durable remissions occur in only a minority of patients. Novel approaches to enhancing antitumor responses are thus necessary in order to expand the reach of these treatments. The inhibitor of apoptosis (IAP) protein family comprises a diverse group of proteins, many of which have immunoregulatory roles. Small molecule IAP antagonists have been developed and are undergoing early phase clinical testing. These drugs were initially developed to promote tumor cell apoptosis; however, a considerable body of work now indicates that IAP antagonists induce antitumor activity through modulation of innate and adaptive immunity. Primarily through inhibition of cellular (c)-IAP1 and c-IAP2, IAP antagonists can activate alternative NF-κB signaling, promoting B-cell survival, activation of dendritic cells and delivering a broad co-stimulatory signal to T cells. At the same time, IAP antagonists can promote tumor cell intrinsic sensitization to innate immune signals, and enhance tumor cell killing by inflammatory cytokines and phagocytic macrophages. These drugs thus represent an attractive investigational approach to immunotherapy, providing a positive signaling counterpart to the relief of signal inhibition conferred by checkpoint blockade.

Inhibitor of apoptosis proteins, NAIP, cIAP1 and cIAP2 expression during macrophage differentiation and M1/M2 polarization.

Monocytes and macrophages constitute the first line of defense of the immune system against external pathogens. Macrophages have a highly plastic phenotype depending on environmental conditions; the extremes of this phenotypic spectrum are a pro-inflammatory defensive role (M1 phenotype) and an anti-inflammatory tissue-repair one (M2 phenotype). The Inhibitor of Apoptosis (IAP) proteins have important roles in the regulation of several cellular processes, including innate and adaptive immunity. In this study we have analyzed the differential expression of the IAPs, NAIP, cIAP1 and cIAP2, during macrophage differentiation and polarization into M1 or M2. In polarized THP-1 cells and primary human macrophages, NAIP is abundantly expressed in M2 macrophages, while cIAP1 and cIAP2 show an inverse pattern of expression in polarized macrophages, with elevated expression levels of cIAP1 in M2 and cIAP2 preferentially expressed in M1. Interestingly, treatment with the IAP antagonist SMC-LCL161, induced the upregulation of NAIP in M2, the downregulation of cIAP1 in M1 and M2 and an induction of cIAP2 in M1 macrophages.

Inhibition of IAP (inhibitor of apoptosis) proteins represses inflammatory status via nuclear factor-kappa B pathway in murine endometriosis lesions.

How is the role of inhibitor of apoptosis proteins (IAPs) in the development of murine endometriosis lesions? BALB/c female mice (n=36) were used for the murine endometriosis model. Endometriotic lesions were surgically induced in mice by transplanting mouse uterine tissue. After 4weeks of IAP antagonist (BV6) treatment, the expression of inflammatory factors in the implants was evaluated using real-time RT-PCR. Inflammatory state, angiogenic activity, and nuclear factor-kappa B (NF-κB) activation were assessed by immunohistochemical staining. The number, size, and level of inflammatory cytokines (Vegf, Il-6, Ccl-2, Lif) gene expression in the murine endometriosis-like lesions were reduced by BV6 treatment. BV6 repressed the intensity and rate of positive cells of CD3, F4/80, and PECAM immunostaining; in addition, the expression of NF-κB p65 and phospho-NF-κB p65 was also attenuated. Inhibitor of apoptosis proteins antagonist represses the inflammation status of murine endometriosis-like lesions viaNF-κB pathway. IAPs may be a novel therapeutic target for endometriosis.

Bivalent IAP antagonists, but not monovalent IAP antagonists, inhibit TNF-mediated NF-\u03baB signaling by degrading TRAF2-associated cIAP1 in cancer cells

The inhibitor of apoptosis (IAP) proteins have pivotal roles in cell proliferation and differentiation, and antagonizing IAPs in certain cancer cell lines results in induction of cell death. A variety of IAP antagonist compounds targeting the baculovirus IAP protein repeat 3 (BIR3) domain of cIAP1have advanced into clinical trials. Here we sought to compare and contrast the biochemical activities of selected monovalent and bivalent IAP antagonists with the intent of identifying functional differences between these two classes of IAP antagonist drug candidates. The anti-cellular IAP1 (cIAP1) and pro-apoptotic activities of monovalent IAP antagonists were increased by using a single covalent bond to combine the monovalent moieties at the P4 position. In addition, regardless of drug concentration, treatment with monovalent compounds resulted in consistently higher levels of residual cIAP1 compared with that seen following bivalent compound treatment. We found that the remaining residual cIAP1 following monovalent compound treatment was predominantly tumor necrosis factor (TNF) receptor-associated factor 2 (TRAF2)-associated cIAP1. As a consequence, bivalent compounds were more effective at inhibiting TNF-induced activation of p65/NF-κB compared with monovalent compounds. Moreover, extension of the linker chain at the P4 position of bivalent compounds resulted in a decreased ability to degrade TRAF2-associated cIAP1 in a manner similar to monovalent compounds. This result implied that specific bivalent IAP antagonists but not monovalent compounds were capable of inducing formation of a cIAP1 E3 ubiquitin ligase complex with the capacity to effectively degrade TRAF2-associated cIAP1. These results further suggested that only certain bivalent IAP antagonists are preferred for the targeting of TNF-dependent signaling for the treatment of cancer or infectious diseases.

Identification of mTOR as a primary resistance factor of the IAP antagonist AT406 in hepatocellular carcinoma cells.

Dysregulation of inhibitor of apoptosis (IAP) proteins (IAPs) in hepatocellular carcinoma (HCC) is often associated with poor prognosis. Here we showed that AT406, an IAP antagonist, was cytotoxic and pro-apoptotic to both established (HepG2, SMMC-7721 lines) and primary HCC cells. Activation of mTOR could be a key resistance factor of AT406 in HCC cells. mTOR inhibition (by OSI-027), kinase-dead mutation or knockdown remarkably enhanced AT406-induced lethality in HCC cells. Reversely, forced-activation of mTOR by adding SC79 or exogenous expressing a constitutively active S6K1 (T389E) attenuated AT406-induced cytotoxicity against HCC cells. We showed that AT406 induced degradation of IAPs (cIAP-1 and XIAP), but didn't affect another anti-apoptosis protein Mcl-1. Co-treatment of OSI-027 caused simultaneous Mcl-1 downregulation to overcome AT406's resistance. Significantly, shRNA knockdown of Mcl-1 remarkably facilitated AT406-induced apoptosis in HCC cells. In vivo, AT406 oral administration suppressed HepG2 tumor growth in nude mice. Its activity was potentiated with co-administration of OSI-027. We conclude that mTOR could be a key resistance factor of AT406 in HCC cells.

Oncolytic virus synergizes with Smac mimetic compounds to induce rhabdomyosarcoma cell death in a syngeneic murine model

Rhabdomyosarcoma (RMS), a neoplasm characterized by undifferentiated myoblasts, is the most common soft tissue tumour in children. Therapeutic resistance is common in RMS and is often caused by acquired defects in the cellular apoptotic program. Smac mimetic compounds (SMCs) are a novel class of inhibitor of apoptosis (IAP) antagonists that are currently under clinical development as cancer therapeutics. We previously reported that cIAP1 is overexpressed in human primary RMS tumours and in patient-derived RMS cell lines where it drives resistance to apoptosis. In this study, we investigated whether inflammatory cytokine production triggered by activators of innate immunity synergizes with LCL161 to induce bystander killing of RMS cells in vitro and in vivo. Indeed, we show that innate immune stimuli (oncolytic virus (VSVΔ51-GFP), interferon γ (IFNγ), and tumour necrosis factor-like weak inducer of apoptosis (TWEAK)) combine with SMCs in vitro to reduce cell viability in the Kym-1 RMS cancer cell line. Other human RMS cell lines (RH36, RH41, RD, RH18, RH28, and RH30) and the murine RMS cell line 76-9 are resistant to treatment with LCL161 alone or in combination with immune stimulants in in vitro cell viability assays. In contrast, we report that the combination of LCL161 and VSVΔ51-GFP reduces tumour volume and prolongs survival in a 76-9 syngeneic murine model. Our results support further exploration of the combined use of IAP antagonists and innate immune stimuli as a therapeutic approach for RMS cancers.

IAPs: Mediators of Oncogenesis and Targets for Anticancer Therapy.

The inhibitor of apoptosis (IAP) family members are potent regulators of cell homeostasis able to regulate several fundamental cellular processes that include cell death, cell proliferation, cell differentiation, and inflammation. Regarding this broad spectrum of activity, it is now becoming clear that some members of the family possess oncogenic properties. Analysis of genomic database from tumor sequencing studies has revealed a number of genetic alterations affecting some IAP genes and resulting in gain or loss of function. In this review, we discuss the importance of IAP alterations in cell transformation and their link with key oncogenic pathways, focusing on nuclear factor-kappa B (NF-κB)-activating signaling pathways. Then we highlight the therapeutic potential of IAP antagonists and nitric oxide (NO) donors as inhibitors of NF-κB in anticancer therapy.

A Phase 1b Study of Birinapant in Combination with 5-Azacitadine in Patients with Myelodysplastic Syndrome Who Are Naïve, Refractory or Have Relapsed to 5-Azacitadine

Background: Although 5-azacitidine (5-AZA) is the standard of care for patients with higher-risk patients with Myelodysplastic Syndrome (MDS) almost all patients will fail 5-AZA therapy with the majority of patients progressing within 2 years. Two-year survival for MDS patients refractory to 5-AZA is only 15%. There is a need for more effective therapies for these patients with higher-risk MDS. The Inhibitors of Apoptosis (IAP) Proteins are a family of molecules that suppress apoptotic cell death. Expression of IAPs is dysregulated in MDS, supporting potential as therapeutic targets. SMAC (second mitochondrial activator of caspases) is an IAP antagonist, resulting in caspase activation, inhibition of the pro-survival transcription factor NF-kB and increased apoptosis leading to tumor cell death. Birinapant is a potent bivalent SMAC mimetic in clinical development for the treatment of several cancers. Pre-clinical data support its investigation in myeloid malignancies, specifically MDS. Methods: This Phase 1b study was designed to determine the MTD and recommended Phase 2 dose and schedule of birinapant in combination with 5-AZA in patients with higher-risk MDS who are naïve to or have relapsed or are refractory to 5-AZA. Secondary objectives included determining the clinical activity of the combination of birinapant plus 5-AZA, and assessing the pharmacokinetics (PK) and exploratory biomarkers. Results: Twenty-one patients were enrolled, of whom 15 had high/very high risk MDS, with median IPSS score of 3 (range, 1.5-8). Median age (range) was 73 years (48-84 years). Eight patients were therapy-naïve. All patients received 5-AZA, administered either intravenous (IV; 13 patients) or subcutaneous (SC; 8 of whom 4 later switched to IV), at 75 mg/m2 for 7 days of a 28-day cycle in combination with birinapant. Birinapant was administered IV twice weekly (Days 1 and 4) at 13 mg/m2 for either 3 of 4 weeks (15 patients; Cohort 1 and Expansion Cohort) or 4 of 4 weeks (6 patients; Cohort 2) in 28-day cycles. No cycle 1 dose-limiting toxicities were observed. Adverse events ≥Grade 3 observed in two or more patients were thrombocytopenia (9 patients); pneumonia (including fungal pneumonia; 8); anemia (8); leukopenia (7); febrile neutropenia (7) neutropenia (5); fatigue (4); respiratory failure (3); cellulitis or soft tissue necrosis (3); and nausea, vomiting, or hypoxia (2 patients each). SAEs seen in more than one patient included pneumonia (6 patients, including fungal pneumonia); febrile neutropenia (4); sepsis (3) and cellulitis or soft tissue necrosis (3). Grade 3 abdominal soft tissue necrosis occurred in the area of subcutaneous injections of 5-AZA, and two events of Grade 3 cellulitis were judged related to treatment. These local injection site reactions were more severe than expected with SC 5-AZA, and were not observed with subsequent IV administration. These events were not considered DLTs. Treatment-related suppression of NF-k B activity was evident in circulating blast cells from patients in Cohort 1 suggesting pharmacodynamic activity of birinapant at this dose and schedule. Based on this, the Phase 2 dosing regimen has been recommended as birinapant administered at 13 mg/m2 IV twice weekly for 3 weeks of a 28-day cycle in combination with 5-AZA at 75 mg/m2 IV daily for 7 days of a 28-day cycle. Although not a primary endpoint, evidence of clinical activity, as demonstrated by significant reduction in marrow blast percentages, was seen, including 3 patients considered relapsed or refractory to prior therapy. Nine patients of 20 evaluable (45%) demonstrated either a ≥50% decrease in marrow blast count, or a blast count of ≤5% at the end of Cycle 1 or 2. Two additional patients (10%) demonstrated hematological improvement in one or more peripheral cell lineages without transfusions. In addition, 2 patients, one of whom had previously received 5-AZA, could be bridged to stem cell transplant subsequent to response with birinapant plus 5-AZA. Several patients remain in active follow-up. Conclusion: This Phase 1b trial confirms the acceptable safety profile of the combination of birinapant administered with IV 5-AZA. Evidence of clinical activity in both 5-AZA naïve and refractory patients provide the rationale for an ongoing global randomized, blinded Phase 2 study comparing birinapant in combination with 5-AZA vs. 5-AZA alone in patients with higher-risk MDS in the front-line setting. Disclosures Wang: Immunogen: Research Funding. Rakkar:TetraLogic Pharmaceuticals: Research Funding. Minderman:TetraLogic Pharmaceuticals: Research Funding. Burns:TetraLogic Pharmaceuticals: Employment. Tibes:TetraLogic Pharmaceuticals: Research Funding.

A Phase 2 Study of Birinapant in Combination with 5-Azacitadine in Patients with Myelodysplastic Syndrome Who Are Naïve to 5-Azacitadine: A Preliminary Analysis of Phase 2a

Background: The Inhibitors of Apoptosis (IAP) proteins are a family of molecules that suppress apoptotic cell death. Expression of IAPs is dysregulated in Myelodysplastic Syndrome (MDS) and is a potential path for therapeutic intervention. SMAC (second mitochondrial-derived activator of caspases) is an IAP antagonist, resulting in caspase activation, inhibition of NF-k B and increased apoptosis. Birinapant is a potent bivalent SMAC mimetic under clinical investigation for several cancer types. Pre-clinical data support its investigation in myeloid malignancies, including MDS, and a previous phase 1b study demonstrated promising clinical activity in patients with higher-risk MDS who were naïve or had relapsed or were refractory to prior therapy. In this trial birinapant is combined with 5-azacitidine (5-AZA) in high-risk MDS patients who have not yet been treated with a hypomethylating agent.Methods: This phase 2 ongoing study of birinapant plus 5-AZA in patients with MDS or CMMoL recently completed enrollment to the unblinded portion, in which 9 patients were dosed with birinapant 13 mg/m2 IV twice weekly for 3 weeks of a 28-day cycle plus 5-AZA 75 mg/m2 by IV infusion daily for 7 days of a 28-day cycle. The primary objective of this study is to determine the objective response rate by International Working Group criteria of birinapant plus 5-AZA in patients with high-risk MDS or CMMoL who are naïve to hypomethylating agents. Secondary objectives include assessing the tolerability of this regimen, and determining the pharmacokinetics and exploratory pharmacodynamics of the combination.Results: Of the 9 patients enrolled, 6 patients had high or very high risk MDS; no patients had CMMoL. 8 patients were age ≥60 years. All patients were naïve to 5-AZA. Of the 9 patients enrolled, 6 completed up to four cycles of chemotherapy and were considered evaluable for response; three discontinued therapy prior to Cycle 4 (patient or investigator decision, or occurrence of adverse events (AEs)). Of the 6 evaluable patients, 3 had a complete response (CR), one had a marrow CR, and one had a partial response (PR). A sixth patient had a best response of stable disease at Cycle 4. Of these six patients evaluable, one (PR) was able to proceed to stem cell transplantation. The median duration of response (range) in these patients has not yet been assessed. Adverse events were consistent with the disease under investigation or the known profile of birinapant or 5-AZA. The most common ≥Grade 3 AEs seen in ≥2 patients were neutropenia and thrombocytopenia (5 patients each); fatigue, anemia, increased lipase and syncope (2 patients each). One patient developed a Grade 1 VIth cranial nerve palsy. No serious adverse events were reported in more than one patient, and one fatal event (sepsis) was reported. No patients developed infusion site reaction. Data showing inhibition of NF-k B, a downstream pro-survival molecule activated by IAPs, in circulating blast cells, demonstrated evidence of a potential biomarker in this population.Conclusion: This ongoing phase 2 study provide data for an acceptable safety profile of the combination of birinapant administered with IV 5-AZA. Preliminary evidence of promising clinical activity in this non-randomized, open-label phase was observed. These results provide rationale for the continuation to the next phase of this global randomized, blinded phase 2 study comparing birinapant in combination with 5-AZA vs. 5-AZA alone in patients with high-risk MDS or CMMoL in the front-line setting. DisclosuresRitchie:Celgene: Speakers Bureau; Incyte: Speakers Bureau. Skolnik:TetraLogic Pharmaceuticals: Employment, Equity Ownership.

Baculovirus Inhibitor-of-Apoptosis Op-IAP3 Blocks Apoptosis by Interaction with and Stabilization of a Host Insect Cellular IAP.

Baculovirus-encoded inhibitor of apoptosis (IAP) proteins likely evolved from their host cell IAP homologs, which function as critical regulators of cell death. Despite their striking relatedness to cellular IAPs, including the conservation of two baculovirus IAP repeat (BIR) domains and a C-terminal RING, viral IAPs use an unresolved mechanism to suppress apoptosis in insects. To define this mechanism, we investigated Op-IAP3, the prototypical IAP from baculovirus OpMNPV. We found that Op-IAP3 forms a stable complex with SfIAP, the native, short-lived IAP of host insect Spodoptera frugiperda. Long-lived Op-IAP3 prevented virus-induced SfIAP degradation, which normally causes caspase activation and apoptosis. In uninfected cells, Op-IAP3 also increased SfIAP steady-state levels and extended SfIAP's half-life. Conversely, SfIAP stabilization was lost or reversed in the presence of mutated Op-IAP3 that was engineered for reduced stability. Thus, Op-IAP3 stabilizes SfIAP and preserves its antiapoptotic function. In contrast to SfIAP, Op-IAP3 failed to bind or inhibit native Spodoptera caspases. Furthermore, BIR mutations that abrogate binding of well-conserved IAP antagonists did not affect Op-IAP3's capacity to prevent virus-induced apoptosis. Remarkably, Op-IAP3 also failed to prevent apoptosis when endogenous SfIAP was ablated by RNA silencing. Thus, Op-IAP3 requires SfIAP as a cofactor. Our findings suggest a new model wherein Op-IAP3 interacts directly with SfIAP to maintain its intracellular level, thereby suppressing virus-induced apoptosis indirectly. Consistent with this model, Op-IAP3 has evolved an intrinsic stability that may serve to repress signal-induced turnover and autoubiquitination when bound to its targeted cellular IAP. The IAPs were first discovered in baculoviruses because of their potency for preventing apoptosis. However, the antiapoptotic mechanism of viral IAPs in host insects has been elusive. We show here that the prototypical viral IAP, Op-IAP3, blocks apoptosis indirectly by associating with unstable, autoubiquitinating host IAP in such a way that cellular IAP levels and antiapoptotic activities are maintained. This mechanism explains Op-IAP3's requirement for native cellular IAP as a cofactor and the dispensability of caspase inhibition. Viral IAP-mediated preservation of the host IAP homolog capitalizes on normal IAP-IAP interactions and is likely the result of viral IAP evolution in which degron-mediated destabilization and ubiquitination potential have been reduced. This mechanism illustrates another novel means by which DNA viruses incorporate host death regulators that are modified for resistance to host regulatory controls for the purpose of suppressing host cell apoptosis and acquiring replication advantages.

Pharmacology of smac mimetics; chemotype differentiation based on physical association with caspase regulators and cellular transport

Cellular levels of inhibitor of apoptosis (IAP) proteins are elevated in multiple human cancers and their activities often play a part in promoting cancer cell survival by blocking apoptotic pathways, controlling signal transduction pathways and contributing to resistance. These proteins function through interactions of their BIR (baculoviral IAP repeat) protein domains with pathway components and these interactions are endogenously antagonized by Smac/Diablo (second mitochondrial activator of caspases/direct IAP binding protein with low isoelectric point). This report describes development of synthetic smac mimetics (SM) and compares their binding, antiproliferative and anti-tumor activities. All dimeric antagonists inhibit in vitro smac tetrapeptide binding to recombinant IAP proteins, rescue IAP-bound caspase-3 activity and show anti-proliferative activity against human A875 melanoma cells. One heterodimeric SM, SM3, binds tightly to IAP proteins in vitro and slowly dissociates (greater than two hours) from these protein complexes compared to the other antagonists. In addition, in vitro SM anti-proliferation potency is influenced by ABCB1 transporter (ATP-binding cassette, sub-family B; MDR1, P-gp) activities and one antagonist, SM5, does not appear to be an ABCB1 efflux pump substrate. All dimeric smac mimetics inhibit the growth of human melanoma A875 tumors implanted in athymic mice at well-tolerated doses. One antagonist, SM4, shows broad spectrum in vivo anti-tumor activity and modulates known pharmacodynamic markers of IAP antagonism. These data taken together demonstrate the range of diverse dimeric IAP antagonist activities and supports their potential as anticancer agents.