Nuclear Imaging Study of the Pharmacodynamic Effects of Debio 1143, an Antagonist of Multiple Inhibitor of Apoptosis Proteins (IAPs), in a Triple-Negative Breast Cancer Model

Background Debio 1143, a potent orally available SMAC mimetic, targets inhibitors of apoptosis proteins (IAPs) members and is currently in clinical trials. In this study, nuclear imaging evaluated the effects of Debio 1143 on tumor cell death and metabolism in a triple-negative breast cancer (TNBC) cell line (MDA-MB-231)-based animal model. Methods Apoptosis induced by Debio 1143 was assessed by FACS (caspase-3, annexin 5 (A5)), binding of 99mTc-HYNIC-Annexin V, and a cell proliferation assay. 99mTc-HYNIC-Annexin V SPECT and [18F]-FDG PET were also performed in mice xenografted with MDA-MB-231 cells. Results Debio 1143 induced early apoptosis both in vitro and in vivo 6 h after treatment. Debio 1143 inhibited tumor growth, which was associated with a decreased tumor [18F]-FDG uptake when measured during treatment. Conclusions This imaging study combining SPECT and PET showed the early proapoptotic effects of Debio 1143 resulting in a robust antitumor activity in a preclinical TNBC model. These imaging biomarkers represent valuable noninvasive tools for translational and clinical research in TNBC.


Background
e World Health Organization (WHO) reported that 1.7 million women were diagnosed with breast cancer in 2012 with a global number of 6.3 million women diagnosed with breast cancer between 2008 and 2012 [1]. Since the last WHO report in 2008, breast cancer incidence and mortality have increased by more than 20% and 14%, respectively. Breast cancer is also the leading cause of cancer-related death among women (522,000 deaths in 2012) and the most frequently diagnosed cancer in 140 of 184 countries worldwide [1]. e combination of surgery, radiation therapy, chemotherapy, and hormone therapy represents the common therapeutic strategies used nowadays in clinic to treat breast cancer. Clinical and pathologic features (based on conventional histology and immunohistochemistry) allow breast cancer classification as hormone-receptor positive (estrogen receptor (ER) and progesterone receptor (PR)), HER2 (human epidermal growth factor receptor 2) positive, and triple negative (ER, PR, and HER2 negative). is classification process is currently necessary for prognosis evaluation and individualized selection of therapy. Triplenegative breast cancer (TNBC) is a heterogeneous disease associated with a high risk of recurrence and poor prognosis. erapeutic options for TNBC are currently limited to cytotoxic therapy, whereas other types of breast cancer expressing receptors are eligible for targeted therapies such as antihormonal or anti-HER2 therapies. erefore, TNBC is considered as a real challenging disease since no targeted therapies has been approved yet. In this context, numerous new targets are currently under investigations for pharmacological purposes such as Notch signaling, Wnt/β-catenin, and Hedgehog pathways; EGFR, PARP1, mTOR, TGFβ, and angiogenesis inhibitors [2]. e targeting of the inhibitors of apoptosis proteins (IAPs), which are key negative regulators of programmed cell death, represents another promising approach in managing TNBC. Indeed, IAPs have been reported to be upregulated in most cancer types contributing to tumor cell survival and resistance to cancer therapy [3]. Among IAPs, four of them, namely, XIAP, cIAP1, cIAP2, and ML-IAP, negatively regulate apoptosis by downregulating the activity of caspases [4]. In addition to apoptosis, IAPs also influence a multitude of other cellular processes, such as ubiquitin-dependent signaling events that regulate activation of the nuclear factor κB (NFκB), which in turn drive the expression of genes important for inflammation, immunity, cell migration, and cell survival. It has been reported that XIAP protein expression was significantly correlated with a more aggressive tumor phenotype and decreased overall and disease-free survival, suggesting a prognostic value of XIAP for invasive ductal breast cancer with triple-negative phenotype [5]. IAPs are antagonized by the endogenous Second Mitochondriaderived Activator of Caspases (SMAC), also called DIA-BLO (Direct IAP-Binding Protein with Low PI). SMAC is released from mitochondria into the cytosol when mitochondria are damaged by apoptotic stimuli such as UV radiation [4]. Such a mechanism has paved the way for the design of SMAC-mimetic agents to promote apoptosis in cancer cells by antagonizing the activity of IAPs and create conditions in which apoptosis can proceed. A number of SMAC mimetics have been advanced into early clinical development for cancer treatment as single agent or in combination. Interestingly, it has been proposed that TNBC may be more sensitive to SMAC-mimetic drugs than other malignancies, suggesting that SMAC-mimetic could represent a targeted therapy of TNBC which remains to be discovered [4]. Recently, Debio 1143, a new potent orally available monovalent SMAC mimetic targeting multiple IAPs member, has been developed and is currently in clinical trials for cancer treatment [6]. Molecular imaging certainly represents a reliable technique to improve such a drug development since it is recognized to expedite cancer drug discovery, predict responders versus nonresponders to specific treatments, and help determine the overall effectiveness of therapies longitudinally [7]. In oncology, molecular imaging of glucidic metabolism with [ 18 F]-FDG PET has already a crucial impact on several aspects from detection/staging to monitoring/predicting therapeutic effects in both preclinical and clinical settings, so that it remains a gold standard procedure in management of various malignancies. Nevertheless, even if [ 18 F]-FDG uptake reflects the viable tumor cell fraction, it also accumulates in noncancer tissues (e.g., inflammatory lesions, brain, and heart) what can induce pitfalls in images interpretation. e combination of [ 18 F]-FDG imaging with other modalities and/or probes able to image a specific biomarker related to the mechanism of action of the anticancer drugs to be tested is then a reliable way to circumvent these drawbacks. Most of anticancer drugs typically induce cell death through induction of apoptosis which can be noninvasively imaged with molecular imaging probe such as 99m Tc-HYNIC-Annexin V. Such a noninvasive imaging measure of apoptosis would therefore be helpful for demonstrating the efficacy of apoptosis-inducing treatments (e.g., Debio 1143) without requiring tissue sampling. As [ 18 F]-FDG, 99m Tc-HYNIC-Annexin V is a well-known radiotracer and has been extensively assessed in preclinical and clinical settings, making it a safe and reliable probe in spite of a certain lack of specificity since it also labels necrosis [8]. In the current study, we combined SPECT and PET imaging techniques as pharmacodynamic biomarkers to measure the early proapoptotic and antitumor effects of Debio 1143 in a preclinical TNBC model. Using MDA-MB-231 xenografted mice, we successfully demonstrated that Debio 1143 induces apoptosis ( 99m Tc-HYNIC-Annexin V) at early time points and reduced glucidic metabolism ([ 18 F]-FDG PET) over time, which was accompanied by a robust antitumor activity. ese imaging biomarkers represent valuable noninvasive tools for translational research and might be useful for SMAC mimetic clinical development in TNBC.

Materials and Methods
Materials and methods are available in detail in Supplemental Methods.

Cell Culture (MDA-MB-231). Breast adenocarcinoma MDA-MB-231 cells (European Collection of Authenticated
Cell Cultures (ECACC), Salisbury, UK) have been cultured as a monolayer in RPMI 1640 containing 2 mM of L-glutamine (Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (Lonza) at 37°C in a humidified atmosphere (5% CO 2 ).    In vivo evaluation of apoptosis was performed with SPECT-CT imaging ( 99m Tc-HYNIC-Annexin V). When tumors reached a mean volume of 340 mm 3 , 99m Tc-HYNIC-Annexin V SPECT-CT imaging was performed 6 and 24 hours after a single administration of vehicle (p.o., n � 8), Debio 1143 (p.o., 100 mg/kg, n � 8), or paclitaxel (IV, 7.5 mg/kg, n � 8, Taxol ® , 6 mg/mL, Bristol-Myers Squibb SpA, France). Mice were anesthetized through isoflurane inhalation for intravenous injection (tail vein) of 10-20 MBq of 99m Tc-HYNIC-Annexin V one hour prior the imaging study. At the end of the last image acquisition, the animals were sacrificed, and tumors were harvested and used for gamma counting in order to confirm image analyses.

MTS
In vivo evaluation of antitumor activity was performed with At the end of the last imaging, the mice were intraperitoneally injected with an overdose of pentobarbital for euthanasia and tumors harvested for gamma counting (Perkin Elmer, France). See details in Supplemental Methods.
2.6. Statistical Analysis. All results are presented as mean ± SEM. Statistical analysis was determined using one-way ( 99m Tc-HYNIC-Annexin V experiments) or two-way ANOVA ([ 18 F]-FDG PET-CT). Analysis was performed with GraphPad Prism 6.0 (GraphPad Software Inc.), and in all cases, a p value less than 0.05 was considered significant.

e Cytotoxic Activity of Debio 1143 on Human Breast Adenocarcinoma Cells Is Comparable to Paclitaxel.
e incubation of MDA-MB-231 cells with increasing concentration of Debio 1143 and paclitaxel demonstrated a dosedependent cytotoxic activity of both drugs on human breast adenocarcinoma cells. e mean IC50 of D1143 was 137 nM, while the mean IC50 of paclitaxel was 7.44 nM (Figure 1(a)). Our results confirm the findings of previous studies which report an IC50 of 144 nM for Debio 1143 [9].

Debio 1143 Induces Apoptosis of Human Breast
Adenocarcinoma Cells. After 6 hours of incubation of MDA-MB-231 cells with Debio 1143, a significant dose-dependent increase of cells in early apoptosis (Annexin-V+/7-AAD-) was observed compared to vehicle-treated cells (Figure 1(b)).
is increase in early apoptosis was observed starting at 0.3 µM with a maximal effect at 3 µM of Debio 1143. Staurosporine, used as positive control in this experiment, also induced a significant increase in early apoptosis in MDA-MB-231 cells (Figure 1(b)). Interestingly, Debio 1143 also induced a significant increase in late apoptosis/necrosis (Annexin-V+/7-AAD+) of MDA-MB-231 cells starting at 1 µM and increased with dose (Figure 1(b)). ese results were confirmed by a dose-dependent increase in proportion of cells harboring active caspase-3, the major effector of apoptosis, after Debio 1143 treatment (Figure 1(c)). Furthermore, gamma counting of MDA-MB-231 cells after staining with 99m Tc-HYNIC-Annexin V, which specifically stains Annexin-V positive cells, demonstrated that Debio 1143 (3 µM) induced an increase in cells presenting Annexin-V (Figure 1(d)). All together, these results highlight the proapoptotic effects of Debio 1143 on human breast adenocarcinoma cells.

Debio 1143 Induces Tumor-Apoptosis In Vivo in a Human Breast Adenocarcinoma Murine Model.
99m Tc-HYNIC-Annexin V SPECT-CT imaging experiments were carried out when tumors reached a mean volume of 340 mm 3 . Imaging was performed at 6 h after treatment for vehicle-treated mice and at 6 and 24 h after treatment for paclitaxel-and Debio 1143-treated mice. One hour after 99m Tc-HYNIC-Annexin V administration, mice from all group showed an apparent similar whole body distribution of radioactivity localized mainly in kidneys, bladder, and liver concentrating more than 80% of overall radioactive signal as previously described in the literature ( [8]; Figures 2(a) and 2(b)). A weak 99m Tc-HYNIC-Annexin V signal was observed in tumors from vehicle-treated mice, comparable with signal observed in paclitaxel-treated mice. Interestingly, a significant increase in tumor 99m Tc-HYNIC-Annexin V signal was observed at 6h following Debio 1143 treatment (Figures 2(c) and 2(d)). An increase in 99m Tc-HYNIC-Annexin V signal was also observed after 24 h of paclitaxel although not significant (Figure 2(d)). ese results were consistent with ex vivo gamma counting of tumors with an increase of 99m Tc-HYNIC-Annexin V tumor uptake 6 h after Debio 1143 compared to vehicle-treated mice (Figure 2 (e)). All together, these results demonstrate that Debio 1143 specifically induces tumor apoptosis in vivo in a human breast adenocarcinoma murine model.  (Figure 3 (a)). Tumor volume increased regularly and similarly in vehicle-treated mice from D11 (treatment initiation) to D32 (end of experiment; Figure 3(b)). Paclitaxel did not induce any decrease in tumor growth throughout the experiment, while Debio 1143 displayed a significant antitumor activity after 2 weeks of treatment (D25) that was sustained up to D32 (Figure 3(b)

Discussion
In order to improve the management of malignancies, it is now well established that an early and reliable assessment of therapy response is a crucial issue. It allows guidance of the oncologist to the best options for the patients: modulations of the doses, treatment switching, or treatment combinations. In the current study, using two different molecular imaging modalities (SPECT-CT and PET-CT), we assessed the effect of Debio 1143, a new potent oral SMAC mimetic, as a single agent in a preclinical model of TNBC, in immunodeficient mice xenografted with MDA-MB-231 cells. e xenografted models still constitute a major preclinical screen for the development of novel cancer therapeutics, included human-targeted therapies. Despite limitations, these models have identified clinically efficacious agents, suggesting that they are still a "workhorse" of the pharmaceutical industry [10]. TNBC represents 15-20% of breast cancers and remains a challenging disease regarding its aggressive nature, its poor prognosis, and the lack of targeted therapies. As no well-defined molecular targets have been described so far, cytotoxic chemotherapy is currently the only treatment option for TNBC whose major drawback is an unacceptable deterioration in the quality of life. Currently, paclitaxel is commonly used in clinical practice to treat TNBC. However, the clinical efficacy of paclitaxel has been weakened by the development of drug resistance and the emergence of side-effects, including neutropenia and neurotoxicity [11]. Paclitaxel induces apoptosis by targeting microtubules and resulting in cell cycle arrest [12]. Although paclitaxel has been shown to eliminate most tumor cells including TNBC, paclitaxel resistance has been estimated to cause treatment failure in more than 90% of patients [13]. erefore, the development of alternative therapeutic strategies is essential. Inhibitor of apoptosis proteins (IAPs) play key roles in resistance to cell death induced by a variety of anticancer drugs in various indications including in TNBC, and thus are promising drug targets [4]. Debio 1143 (a.k.a. AT-406 or SM-406) is a monovalent, orally available, small molecule antagonist of IAPs in clinical development that has demonstrated potent single-agent antitumor activity in multiple models of human cancer such as lung adenocarcinoma [14,15], head and neck squamous cell carcinoma [16], and TNBC [9,17]. Debio 1143 has also been shown to work synergistically with conventional chemotherapeutic agents (such as taxanes) or radiotherapy RT in nonclinical cancer models [14,16]. SMAC mimetics have been shown to promote apoptosis by inhibiting IAPmediated caspase repression [18]. In vitro SMACmimetics treatment has been shown to increase Annexin-V positive cells and activate caspases-3 and -8 in various cancer cell lines [16,19,20] Tc-HYNIC-Annexin V. is tool could represent a reliable way to monitor early apoptosis induced by anticancer agents in order to evaluate early treatment efficacy and allow improvement of therapeutic strategies.
Interestingly, Debio 1143 presented a higher antitumor activity in vivo in comparison with paclitaxel despite an apparent higher intrinsic cytotoxic activity of paclitaxel in vitro suggesting that targeting IAPs may offer the potential  whereas paclitaxel induced-apoptosis was only detectable (although not significant) 24 h after treatment. Apoptosis is an early event expected to occur after successful chemotherapy and is highly predictive of treatment success. us, apoptosis quantification represents a major way to  assess therapy response. Annexin V (A5) has been widely used in basic and clinical research as an apoptosis marker in conjunction with propidium iodide to distinguish between apoptotic and necrotic cells. erefore, it has been labeled with radionuclides for measuring apoptosis in vitro and in vivo in animal models and patients [8,21,22]. 99m Tc-HYNIC-Annexin V, used in the current study, is the most widely applied probe in preclinical and clinical settings for A5 imaging [23]. Kemerink et al. demonstrated that highest uptake of 99m Tc-HYNIC-Annexin V in humans was observed in the kidneys followed by the liver and spleen [24]. ese results are in accordance with our findings in mice where the highest uptake was found in the kidney at 6h and 24h.
Moreover, 99m Tc-HYNIC-Annexin V showed a fast blood clearance with more than 90% of the tracer cleared with a half-life of 24 min [24], allowing imaging at 6 h after injection.
erefore, 99m Tc-HYNIC-Annexin V has been used successfully to assess therapy response in patients after radiation therapy or chemotherapy [25][26][27]. 99m Tc-HYNIC-Annexin V uptake has also been demonstrated to predict prognostic value and efficacy of anticancer therapies. In our study, Debio 1143 induced a significantly higher tumor (MDA-MB-231) uptake of 99m Tc-HYNIC-Annexin V compared to vehicle and paclitaxel. In parallel, Debio 1143 showed an improved efficacy in preventing tumor growth compared to vehicle and paclitaxel after 1 and 2 weeks of treatment and remained 1 week after treatment arrest confirming the predictive value of 99m Tc-HYNIC-Annexin V tumor uptake on therapy efficacy. Unexpectedly, paclitaxel did not induce a strong in vivo apoptosis in our study and, in parallel, did not prevent tumor growth. Despite some controversy, MDA-MB-231 has been demonstrated to be rather insensitive to paclitaxel compared to other TNBC cells [28][29][30]. Interestingly, Panayotopoulou et al. identified, by high throughput screening, that SMAC mimetics were able to eliminate MDA-MB-231 short-term paclitaxel resistance suggesting a benefit of such drugs for TNBC patients [31]. Similar results have also been found in other cancer types including ovarian cancer [32], non-small cell lung cancer [14], and breast cancer [33], in which SMAC mimetics were able to potentiate the effect of standard chemotherapy, including paclitaxel [14,34,35]. However, this study did not evaluate the efficacy of the combination of SMAC mimetics and paclitaxel with [ 18 F]-FDG PET imaging. Moreover, Panayotopoulou et al. identified that longterm paclitaxel was associated with desensitization to SMAC mimetics. erefore, combination therapy of SMAC mimetics and short-term paclitaxel could be an effective therapeutic strategy for TNBC.
Most interestingly, the effect of the SMAC mimetic birinapant on caspase-3 activation has recently been investigated by in vivo imaging [36]. In this study, Yang et al.

Conclusions
[ 18 F]-FDG PET is nowadays the main tool for detection, staging, and monitoring of tumor clinically. However, [ 18 F]-FDG uptake accumulates in noncancer tissues and can be influenced by physiologic uptake of FDG (for example, infection and inflammation) [37].
Moreover, some adenocarcinoma are characterized by low-grade or absence of FDG uptake [38,39]. In our study, we demonstrate that both 99m Tc-HYNIC-Annexin V and [ 18 F]-FDG PET data can be associated to predict therapy efficacy and outcome. erefore, the combination of [ 18 F]-FDG PET and 99m Tc-HYNIC-Annexin V appears as a reliable and noninvasive way to monitor early therapy efficacy and subsequent tumor activity in TNBC patients. Epidermal growth factor receptor ER: Estrogen receptor FDG: Fluorodeoxyglucose HER2: Human epidermal growth factor receptor 2 IAP: Inhibitor of apoptosis proteins IV: Intravenous NIH: National Institute PET: Positron emission tomography PR: Progesterone receptor SMAC: Second Mitochondria-derived Activator of Caspases SPECT: Single-photon emission tomography SUV: Standardized uptake values TGF-β: Transforming growth factor-β TNBC: Triple-negative breast cancer WHO: World Health Organization.
Data Availability e datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical Approval
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Contrast Media & Molecular Imaging
All animal studies were sanctioned by the accredited ethical committees (Oncomet n°91 and C2ea Grand Campus n°105) and were carried out in accordance with the legislation on the use of laboratory animals (directive 2010/63/EU). Experiments were conducted following the European Union's animal care directive (86/609/EEC).

Conflicts of Interest
is work was performed as a part of a collaborative research project with Debiopharm International SA. Anne Vaslin, Hélène Maby-El Hajjami, Claudio Zanna, and Grégoire Vuagniaux are employees of Debiopharm International SA. e other authors declare that they have no conflicts of interest.