Smac Mimetic Compounds Research Articles

Astrocytes and the tumor microenvironment inflammatory state dictate the killing of glioblastoma cells by Smac mimetic compounds

Smac mimetic compounds (SMCs) are small molecule drugs that sensitize cancer cells to TNF-α-induced cell death and have multiple immunostimulatory effects through alterations in NF-κB signaling. The combination of SMCs with immunotherapies has been reported to result in durable cures of up to 40% in syngeneic, orthotopic murine glioblastoma (GBM) models. Herein, we find that SMC resistance is not due to a cell-intrinsic mechanism of resistance. We thus evaluated the contribution of GBM and brain stromal components to identify parameters leading to SMC efficacy and resistance. The common physiological features of GBM tumors, such as hypoxia, hyaluronic acid, and glucose deprivation were found not to play a significant role in SMC efficacy. SMCs induced the death of microglia and macrophages, which are the major immune infiltrates in the tumor microenvironment. This death of microglia and macrophages then enhances the ability of SMCs to induce GBM cell death. Conversely, astrocytes promoted GBM cell growth and abrogated the ability of SMCs to induce death of GBM cells. The astrocyte-mediated resistance can be overcome in the presence of exogenous TNF-α. Overall, our results highlight that SMCs can induce death of microglia and macrophages, which then provides a source of death ligands for GBM cells, and that the targeting of astrocytes is a potential mechanism for overcoming SMC resistance for the treatment of GBM.

Inhibition of Autophagy and the Cytoprotective Role of Smac Mimetic against ROS-Induced Cancer: A Potential Therapeutic Strategy in Relapse and Chemoresistance Cases in Breast Cancer.

With more than a million deaths each year, breast cancer is the top cause of death in women. Around 70% of breast cancers are hormonally responsive. Although several therapeutic options exist, cancer resistance and recurrence render them inefficient and insufficient. The major key reason behind this is the failure in the regulation of the cell death mechanism. In addition, ROS was also found to play a major role in this problem. The therapeutic benefits of Smac mimetic compound (SMC) BV6 on MCF7 were examined in the current study. Treatment with BV6 reduces viability and induces apoptosis in MCF7 breast cancer cells. BV6 suppresses autophagy and has demonstrated a defensive role in cancer cells against oxidative stress caused by H2O2. Overall, the present investigation shows that SMC has therapeutic and cytoprotective potential against oxidative stress in cancer cells. These Smac mimetic compounds may be used as anti-cancer drugs as well as antioxidants alone or in conjunction with other commonly used antioxidants.

SBP-0636457, a Novel Smac Mimetic, Cooperates with Doxorubicin to Induce Necroptosis in Breast Cancer Cells during Apoptosis Blockage.

Breast cancer (BC) is a common health concern worldwide. Doxorubicin (Dox) is a widely used chemotherapeutic agent to treat various cancers, including BC. However, drug resistance and severe side effects often hinder the clinical application of Dox. Combination therapy is an effective potent strategy to increase chemosensitivity and reduce the adverse effects. Smac is a proapoptotic protein that interacts with inhibitors of apoptosis proteins (IAPs) and thereby promotes cell death. Smac mimetic compounds can mimic its function and can be used to kill cancer cells. In this study, Dox and SBP-0636457, a novel Smac mimetic, were found to have cooperative effects in inducing BC cell death. Dox and SBP-0636457 cotreatment induced necroptosis instead of apoptosis in BC cells. Receptor-interacting serine/threonine-protein kinase 1 or mixed-lineage kinase domain-like silencing could attenuate cell death caused by Dox/SBP-0636457 in BC cells. In addition, this combined treatment caused synergistic induction of TNFα, and TNFα/TNFR signalling is essential for cell death induced by Dox/SBP-0636457 in BC cells. Moreover, both canonical and noncanonical nuclear factor kappa B pathways were found to contribute to TNFα upregulation induced by Dox/SBP-0636457. Therefore, the findings suggest that SBP-0636457 combined with Dox is an alternative strategy for treating BC.

Crotonate sensitizes IAPi in the disruption of latent HIV by modulating the ncNF‐κB signaling pathway at the step of p100 cleavage into p52

BackgroundAntiretroviral therapy (ART) effectively suppresses HIV‐1 (HIV) replication to an undetectable level in the periphery; however, the treatment is life‐long and HIV reservoirs cannot be eliminated by ART alone in people living with HIV (PLWH). The major obstacle that impedes HIV eradication in ART‐treated PLWH is the latent viral reservoir harbors in the resting CD4+ T cells. Recently, the activation of non‐canonical (nc) NF‐κB pathway via IAP inhibitor (IAPi) or the SMAC mimetic (SMACm) compounds has raised as an attractive mechanism to disrupt HIV from latency. IAPi/SMACm targets IAPs to activate NF‐κB inducing kinase (NIK) followed by phosphorylation of IkappaB kinase (IKK), leading to the cleavage of p100 into p52. The latter forms a complex with RelB to stimulate transcription of its target genes after translocation into the nucleus. Previously, we discovered that crotonylation stimulates HIV latency reversal that elicited by PKC/NF‐κB agonist. We hypothesize that there may exist a functional interaction between protein crotonylation and ncNF‐κB signaling, which may enhance IAPi‐induced latency reversal to optiomally disrupt HIV latency for a cure of HIV.MethodsMultiple HIV latency models were used, including Jurkat cell derived HIV latency models, the U1 promonocytic model of latency, the primary CD4+ T cell model of HIV latency and the resting CD4+ T cells isolated from ART‐suppressed PLWH. HIV expression was determined by flow cytometry and/or real‐time PCR. Protein expression and cleavage was determined by Western blot.ResultsWe found that IAPi/SMACm can disrupt latent HIV. However, its ability to reactivate latent HIV varied among different HIV latency models, with a lowest efficacy in the primary CD4+ T cell model of latency. Interestingly, IAPi/SMACm‐induced disruption of latent HIV was greatly enhanced in the presence of crotonate in all these in vitro and ex vivo models. This was possibly mediated by the enhancement of p100 cleavage into p52 in the ncNF‐κB signaling pathway.ConclusionsWe propose that a blockage of p100 cleavage prevents an efficient activation of ncNF‐κB signaling pathway in the latently infected CD4+ T cells in PLWH, which could be relieved by targeting IAP proteins to induce p100 cleavage for the activation of ncNF‐κB signaling. Crotonylation, which was recently defined by us as a new epigenetic mark for the active HIV transcription, potentiates the reactivation of HIV from latency by further releasing the blockage at the p100 cleavage step, thereby enhancing ncNF‐κB activation to disrupt HIV latency. These data indicate that crotonylation could be exploited to develop novel strategies for HIV cure or remission in the future.

Toward conditional control of Smac mimetic activity by RNA-templated reduction of azidopeptides on PNA or 2'-OMe-RNA.

Oligonucleotide templated reactions can be used to control the activity of functional molecules based on the presence of a specific trigger sequence. We report an RNA-controlled reaction system to conditionally restore the N-terminal amino group and thus binding affinity of azide-modified Smac mimetic compounds (SMCs) for their target protein X-linked Inhibitor of Apoptosis Protein (XIAP). Two templated reactions were compared: Staudinger reduction with phosphines and a photocatalytic reaction with Ru(bpy)2 (mcbpy). The latter proved faster and more efficient, especially for the activation of a bivalent SMC, which requires two consecutive reduction steps. The templated reaction proceeds with turnover when 2'-OMe-RNA probes are used, but is significantly more efficient with PNA, catalyzing a reaction in the presence of low, substoichiometric amounts (1%-3%, 10nM) of target RNA.

Biophysical characterization of the interaction between the full-length XIAP and Smac/DIABLO

XIAP is multi-functional protein which regulates apoptosis acting as a direct caspase inhibitor. It is overexpressed in cancer cells, where it antagonizes the pro-apoptotic action of chemotherapeutics, and therefore it has become an important target for the treatment of cancer. In cells undergoing programmed cell death, the pro-apoptotic protein Smac is released by the mitochondria and binds to XIAP, thereby blocking caspase inhibition. Thus, Smac is considered a master regulator of apoptosis in mammals. In this regard, several Smac mimetic compounds have been developed to inhibit XIAP activity in cancer tissues. These compounds have shown low efficacy, partly due to the lack of structural knowledge of the XIAP-Smac interaction. In this work, through SEC-MALS and circular dichroism, we provide the first biophysical characterization of the interaction between the full-length form of XIAP and Smac, determining the stoichiometry of the complex and providing important information to develop more effective XIAP inhibitors.

Smac mimetics can provoke lytic cell death that is neither apoptotic nor necroptotic.

Smac mimetics, or IAP antagonists, are a class of drugs currently being evaluated as anti-cancer therapeutics. These agents antagonize IAP proteins, including cIAP1/2 and XIAP, to induce cell death via apoptotic or, upon caspase-8 deficiency, necroptotic cell death pathways. Many cancer cells are unresponsive to Smac mimetic treatment as a single agent but can be sensitized to killing in the presence of the cytokine TNFα, provided either exogenously or via autocrine production. We found that high concentrations of a subset of Smac mimetics could provoke death in cells that did not produce TNFα, despite sensitization at lower concentrations by TNFα. The ability of these drugs to kill did not correlate with valency. These cells remained responsive to the lethal effects of Smac mimetics at high concentrations despite genetic or pharmacological impairments in apoptotic, necroptotic, pyroptotic, autophagic and ferroptotic cell death pathways. Analysis of dying cells revealed necrotic morphology, which was accompanied by the release of lactate dehydrogenase and cell membrane rupture without prior phosphatidylserine exposure implying cell lysis, which occurred over a several hours. Our study reveals that cells incapable of autocrine TNFα production are sensitive to some Smac mimetic compounds when used at high concentrations, and this exposure elicits a lytic cell death phenotype that occurs via a mechanism not requiring apoptotic caspases or necroptotic effectors RIPK3 or MLKL. These data reveal the possibility that non-canonical cell death pathways can be triggered by these drugs when applied at high concentrations.

Doxorubicin sensitizes cancer cells to Smac mimetic via synergistic activation of the CYLD/RIPK1/FADD/caspase-8-dependent apoptosis

Smac/Diablo is a pro-apoptotic protein via interaction with inhibitors of apoptosis proteins (IAPs) to relieve their inhibition of caspases. Smac mimetic compounds (also known as antagonists of IAPs) mimic the function of Smac/Diablo and sensitize cancer cells to TNF-induced apoptosis. However, the majority of cancer cells are resistant to Smac mimetic alone. Doxorubicin is a widely used chemotherapeutic drug and causes adverse effect of cardiotoxicity in many patients. Therefore, it is important to find strategies of combined chemotherapy to increase chemosensitivity and reduce the adverse effects. Here, we report that doxorubicin synergizes with Smac mimetic to trigger TNF-mediated apoptosis, which is mechanistically distinct from doxorubicin-induced cell death. Doxorubicin sensitizes cancer cells including human pancreatic and colorectal cancer cells to Smac mimetic treatment. The combined treatment leads to synergistic induction of TNFα to initiate apoptosis through activating NF-κB and c-Jun signaling pathways. Knockdown of caspase-8 or knockout of FADD significantly blocked apoptosis synergistically induced by Smac mimetic and doxorubicin, but had no effect on cell death caused by doxorubicin alone. Moreover, Smac mimetic and doxorubicin-induced apoptosis requires receptor-interacting protein kinase 1 (RIPK1) and its deubiquitinating enzyme cylindromatosis (CYLD), not A20. These in vitro findings demonstrate that combination of Smac mimetic and doxorubicin synergistically triggers apoptosis through the TNF/CYLD/RIPK1/FADD/caspase-8 signaling pathway. Importantly, the combined treatment induced in vivo synergistic anti-tumor effects in the xenograft tumor model. Thus, the combined therapy using Smac mimetic and doxorubicin presents a promising apoptosis-inducing strategy with great potential for the development of anti-cancer therapy.

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.

SP3 is key to Smac mimetic efficacy

Pharmacology Finding drugs that selectively target tumor cells and spare healthy tissue is challenging. Smac is a mitochondrial protein that is released into the cytosol during apoptosis. Smac mimetic compounds block antiapoptotic machinery and induce production of the cytokine tumor necrosis factor–α. Smac mimetics have emerged as promising cancer therapies. Beug et al. found that the transcription factor SP3 critically mediates the effects of Smac mimetics and is more abundantly expressed in tumors than in normal tissues from the same patients. Thus, SP3 may provide a biomarker for patients that will best respond to and tolerate these drugs. Sci. Signal. 12 , eaat9563 (2019).

Combination of IAP Antagonists and TNF-α-Armed Oncolytic Viruses Induce Tumor Vascular Shutdown and Tumor Regression

Smac mimetic compounds (SMCs) are anti-cancer drugs that antagonize Inhibitor of Apoptosis proteins, which consequently sensitize cancer cells to death in the presence of proinflammatory ligands such as tumor necrosis factor alpha (TNF-α). SMCs synergize with the attenuated oncolytic vesicular stomatitis virus (VSVΔ51) by eliciting an innate immune response, which is dependent on the endogenous production of TNF-α and type I interferon. To improve on this SMC-mediated synergistic response, we generated TNF-α-armed VSVΔ51 to produce elevated levels of this death ligand. Due to ectopic expression of TNF-α from infected cells, a lower viral dose of TNF-α-armed VSVΔ51 combined with treatment of the SMC LCL161 was sufficient to improve the survival rate compared to LCL161 and unarmed VSVΔ51 co-therapy. This improved response is attributed to a bystander effect whereby the spread of TNF-α from infected cells leads to the death of uninfected cells in the presence of LCL161. In addition, the treatments induced vascular collapse in solid tumors with a concomitant increase of tumor cell death, revealing another mechanism by which cytokine-armed VSVΔ51 in combination with LCL161 can kill tumor cells. Our studies demonstrate the potential for cytokine-engineered oncolytic virus and SMCs as a new combination immunotherapy for cancer treatment.

Apoptosis-sensitizing activity of birinapant in head and neck squamous cell carcinoma cell lines.

Inhibitor of apoptosis proteins, which are overexpressed in head and neck squamous cell carcinoma (HNSCC), may cause therapeutic resistance. Using SMAC mimetic compounds, including birinapant, to degrade and/or inhibit these proteins and sensitize apoptosis may enhance therapies in HNSCC. Fas expression was analyzed in nine HNSCC cell lines and one keratinocyte cell line via flow cytometry. These cell lines were treated with Fas ligand-Fc (FasL) and birinapant, a bivalent SMAC mimetic, in mono and combination therapies. Cytotoxicity was measured using a crystal violet assay. Annexin V assay was performed for detection of apoptosis. The treatment efficacy of mono and combination therapies was statistically analyzed. Nonlinear regression analysis was performed to determine the inhibitory concentration (IC10) of birinapant. Fas expression was detected in each cell line tested. Mono treatment with FasL revealed minor to no apoptotic effects in the majority of the cell lines. Crystal violet and Annexin V staining revealed increased apoptosis rates for all cell lines following incubation with birinapant in mono treatment. Combination treatment with FasL and birinapant (IC10) revealed additional and synergistic effects in eight out of the ten cell lines. To the best of our knowledge, the present study provided the first evidence of the apoptosis-sensitizing activity of combination treatment with FasL and birinapant in HNSCC cell lines.

Inhibitors of Apoptosis Protein Antagonists (Smac Mimetic Compounds) Control Polarization of Macrophages during Microbial Challenge and Sterile Inflammatory Responses.

Apoptosis is a physiological cell death process essential for development, tissue homeostasis, and for immune defense of multicellular animals. Inhibitors of apoptosis proteins (IAPs) regulate apoptosis in response to various cellular assaults. Using both genetic and pharmacological approaches we demonstrate here that the IAPs not only support opportunistic survival of intracellular human pathogens like Chlamydia pneumoniae but also control plasticity of iNOS+ M1 macrophage during the course of infection and render them refractory for immune stimulation. Treatment of Th1 primed macrophages with birinapant (IAP-specific antagonist) inhibited NO generation and relevant proteins involved in innate immune signaling. Accordingly, birinapant promoted hypoxia, angiogenesis, and tumor-induced M2 polarization of iNOS+ M1 macrophages. Interestingly, birinapant-driven changes in immune signaling were accompanied with changes in the expression of various proteins involved in the metabolism, and thus revealing the new role of IAPs in immune metabolic reprogramming in committed macrophages. Taken together, our study reveals the significance of IAP targeting approaches (Smac mimetic compounds) for the management of infectious and inflammatory diseases relying on macrophage plasticity.

Selective replication of oncolytic virus M1 results in a bystander killing effect that is potentiated by Smac mimetics

Oncolytic virotherapy is a treatment modality that uses native or genetically modified viruses that selectively replicate in and kill tumor cells. Viruses represent a type of pathogen-associated molecular pattern and thereby induce the up-regulation of dozens of cytokines via activating the host innate immune system. Second mitochondria-derived activator of caspases (Smac) mimetic compounds (SMCs), which antagonize the function of inhibitor of apoptosis proteins (IAPs) and induce apoptosis, sensitize tumor cells to multiple cytokines. Therefore, we sought to determine whether SMCs sensitize tumor cells to cytokines induced by the oncolytic M1 virus, thus enhancing a bystander killing effect. Here, we report that SMCs potentiate the oncolytic effect of M1 in vitro, in vivo, and ex vivo. This strengthened oncolytic efficacy resulted from the enhanced bystander killing effect caused by the M1 virus via cytokine induction. Through a microarray analysis and subsequent validation using recombinant cytokines, we identified IL-8, IL-1A, and TRAIL as the key cytokines in the bystander killing effect. Furthermore, SMCs increased the replication of M1, and the accumulation of virus protein induced irreversible endoplasmic reticulum stress- and c-Jun N-terminal kinase-mediated apoptosis. Nevertheless, the combined treatment with M1 and SMCs had little effect on normal and human primary cells. Because SMCs selectively and significantly enhance the bystander killing effect and the replication of oncolytic virus M1 specifically in cancer cells, this combined treatment may represent a promising therapeutic strategy.

Smac mimetics synergize with immune checkpoint inhibitors to promote tumour immunity against glioblastoma

Small-molecule inhibitor of apoptosis (IAP) antagonists, called Smac mimetic compounds (SMCs), sensitize tumours to TNF-α-induced killing while simultaneously blocking TNF-α growth-promoting activities. SMCs also regulate several immunomodulatory properties within immune cells. We report that SMCs synergize with innate immune stimulants and immune checkpoint inhibitor biologics to produce durable cures in mouse models of glioblastoma in which single agent therapy is ineffective. The complementation of activities between these classes of therapeutics is dependent on cytotoxic T-cell activity and is associated with a reduction in immunosuppressive T-cells. Notably, the synergistic effect is dependent on type I IFN and TNF-α signalling. Furthermore, our results implicate an important role for TNF-α-producing cytotoxic T-cells in mediating the anti-cancer effects of immune checkpoint inhibitors when combined with SMCs. Overall, this combinatorial approach could be highly effective in clinical application as it allows for cooperative and complimentary mechanisms in the immune cell-mediated death of cancer 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.

Abstract B034: Smac mimetics synergistically improve the efficacy of cancer immunotherapies including immune checkpoint blockade in preclinical models

Abstract Aim: To demonstrate the immune potentiating effects of small-molecule inhibitor-of-apoptosis (IAP) antagonists, known as Smac mimetics, with current standard-of-care cancer immunotherapies. Background: Smac mimetic compounds (SMCs) are synthetic small-molecule antagonists of the cellular inhibitor of apoptosis, cIAP1 and cIAP2, proteins. The IAPs act at a critical nexus in the cancer cell, capable of suppressing intrinsic cell death and avoiding immune-mediated killing of the target cancer cell. The cIAPs are essential mediators of TNF cytokine superfamily signaling which is responsible for the activation of classical and alternative NF-κB survival pathways. SMCs cause the rapid loss of cIAP1/2 proteins by ubiquitin-induced and proteasomal-mediated degradation. SMCs sensitize tumor cells to TNFα-mediated killing by blocking the formation of the RIP1 signalosome and by generating RIP1 death-inducing complexes, the ripoptosome and the necrosome. In addition, SMCs by depleting cIAPs in immune cells promote anti-tumor immunity by inducing T-cell co-stimulation, resulting in a multi-pronged cytolytic attack against tumor cells. SMCs are in early stage clinical trials for cancer and have proven safe as single agents or in combination with chemotherapy. Methodology: We tested various combination immunotherapies in vitro and in vivo in multiple different orthotopic models of cancer, including glioblastoma, in immunocompetent mice to ascertain tumor responses. In addition, we elucidated the role of cytokines and immune cells in the efficacy observed. Results: SMCs dramatically enhanced the anti-tumor effects of Toll-like receptor agonists, recombinant type-1 interferon, BCG vaccine or oncolytic rhabdoviruses in models of cancer. Importantly, we observed for the first time remarkable synergy between SMCs and immune checkpoint inhibitor biologics (e.g. anti-PD1, anti-CTLA-4 monoclonal antibodies) in brain tumor models for which neither single agent had any significant activity. We now demonstrate that SMCs induce long-term cancer immunity that is dependent on cytotoxic T-cell activity. Furthermore, SMCs cooperate with anti-PD1 immune checkpoint inhibitors to induce durable cures in aggressive tumor. We demonstrate that both the innate and adaptive immune response are responsible for the anti-tumor responses and the generation of durable cures in these models. Cytokine neutralization and immune-cell depletion experiments point to key roles for TNFα, interferons, macrophages and CD8-positive killer T-cells in the combination immunotherapy effects observed. Conclusions: SMCs can make use of both innate and adaptive immunity to eradicate cancers in mice. Smac mimetic small-molecules represent a novel and universal form of cancer immunotherapy that greatly compliments immune checkpoint blockade. This highly effective combination approach is readily translatable to the clinic. (Acknowledgements: This project was supported by an Impact grant co-funded by CCSRI and Brain Canada, and by operating grants from CIHR). Citation Format: Eric LaCasse, Shawn Beug, Cristin Healey, Caroline Beauregard, Tarun Sanda, Tommy Alain, Robert Korneluk. Smac mimetics synergistically improve the efficacy of cancer immunotherapies including immune checkpoint blockade in preclinical models [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr B034.

Abstract 3548: In vivo CRISPR reveals dual activation of apoptosis and necroptosis as means to eradicate drug resistant leukemia

Abstract Dysregulation of apoptosis constitutes a general mechanism for refractory acute lymphoblastic leukemia (ALL) to escape cell death during chemotherapy. We have assessed the potential of SMAC mimetic compounds (SM) to eradicate drug resistant ALL by blocking the pro-survival cellular inhibitor of apoptosis proteins (cIAPs). On the basis of a large xenograft bank of primary ALL samples, obtained by xenotransplantation in immunodeficient NSG mice, we detected exquisite sensitivity to SM in vitro in a third of the cases tested, including drug-resistant and relapsed samples. Potent in vitro activity was highly predictive of in vivo efficacy in the xenograft model. Treatment of transplanted mice with SM not only delayed leukemia progression, but also completely eliminated leukemia burden in established disease for several weeks following treatment. SM activity required the presence of RIP1, since lentiCRISPR mediated gene knockout of RIP1 in patient-derived ALL cells blocked SM-induced cell death in vivo and in vitro. In contrast, RIP1 loss had no significant impact on the response to standard antileukemic therapies, supporting a view that RIP1-dependent death pathways are not likely to be selected against by front line chemotherapy. Furthermore, using a multicolor lentiCRISPR approach for simultaneous disruption of multiple genes, we found that downstream of RIP1, the response to SM was dependent on simultaneous execution of apoptosis and necroptosis, since genetic targeting of both cell death pathways was required to block SM-induced death. Upstream of RIP1, expression levels of TNF receptor II (TNFR2) in primary ALL correlated with sensitivity to SM, and genetic disruption of TNFR2 conferred resistance to SM treatment in vivo. Thus, SM treatment circumvents drug resistance through parallel activation of RIP1-dependent apoptosis and necroptosis, providing a strong rationale for application in salvage treatment of refractory childhood ALL. Citation Format: Júlia Aguadé-Gorgorió, Scott McComb, Gunnar Cario, Martin Stanulla, Cornelia Eckert, Jean-Pierre Bourquin, Beat C. Bornhauser. In vivo CRISPR reveals dual activation of apoptosis and necroptosis as means to eradicate drug resistant leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3548.

SMAC mimetic in combination with HDACi effectively release brakes on cell death of chronic myeloid leukemia K562 cells accelerating ligand-mediated apoptosis.

Abstract Enthusiasm for immunotherapy has reached an all-time high within the last year due to remarkable clinical outcomes using strategies directed at activating T cells. The mechanisms by which newly activated T cells execute tumor target cells have not been described; it is assumed that CD4 and CD8 T cells may utilize ligation of TNF receptor family members, Fas, TNF, and TRAIL. However, not all tumors are susceptible to T cell killing by death receptor ligands. Chronic myeloid leukemia K562 cells are notably resistant to pro-apoptotic, ligand-induced cell death; whereas, T cell leukemia Jurkat cells undergo rapid caspase 3/7 activation and cell death (within 4–6 hours) in response to pro-apoptotic death ligands Fas and TRAIL. We hypothesized that K562 cells may be sensitized to respond similarly by targeting the regulatory proteins controlling activation of executioner and/or initiator caspases, thus accelerating cell death. SMAC mimetic compounds (SMC) are a novel class of drugs that target pro-survival signaling molecules. Single agent treatment with SMC enhanced TRAIL- and TNF-induced caspase activation, but had only modest effects on cell death after 24 hours. Addition of histone deacetylase inhibitors (HDACi) in combination with SMC, accelerated TNF- and TRAIL- mediated caspase activation and led to rapid cell death in K562 cells. By relieving multiple levels of inhibition in the tumor targets, we achieved rapid and efficient killing of previously resistant tumor cells using recombinant death receptor ligands. Combination therapy with SMC and HDACi may enhance current immunotherapy approaches designed to 1) activate tumor specific T cells expressing death receptor ligands or 2) administer recombinant ligands such as TRAIL.

Cytotoxic effects of SMAC-mimetic compound LCL161 in head and neck cancer cell lines.

Head and neck squamous cell carcinoma (HNSCC) is one of the most common tumor entities worldwide. Unfortunately, recent drug developments in other fields of oncology have yielded no efficacy in the treatment of oral squamous cell carcinoma. As a new starting point, we investigated the impact of Fas ligand (FasL) and the SMAC-mimetic compound LCL161 in mono- and combination treatment in HNSCC cell lines. Five different cell lines of HNSCC were treated with FasL and LCL161 in mono- and combination treatment. Cytotoxicity was measured via a crystal violet assay. The cell lines were characterized for CD95 (FasL receptor) expression via flow cytometry. The degradation of cellular inhibitor of apoptosis protein 1 (cIAP1) was detected via Western blot. Incubation with FasL led to a significant decrease in three out of five cell lines. Combination treatment with LCL161 enhanced cytotoxicity significantly. Two cell lines were FasL resistant, but one of them could be resensitized with LCL161. In all cell lines, Western blot analysis showed degradation of cIAP1 after LCL161 application. However, one cell line showed only minor vulnerability to the FasL and LCL161 combination. This is the first study investigating combination treatment of FasL and LCL161 in head and neck cancer cell lines. Pro-apoptotic effects of the combination were detected in the majority of the cell lines. Interestingly, one of two FasL-resistant cell lines was sensitive to the combination therapy with FasL and LCL161. SMAC-mimetic compounds show promising results in the treatment of other tumor entities in vitro and might be useful drugs to improve HNSCC therapy.