Abstract 548: Birinapant enhances gemcitabine's anti-tumor efficacy in triple-negative breast cancer by inducing intrinsic pathway-dependent apoptosis
Authors: Xie Xuemei , Lee Jangsoon , Pearson Troy , Lu Alexander Y. , Tripathy Debu , Devi Gayathri R. , Bartholomeusz Chandra , Ueno Naoto T. 
Published: September 2020

Abstract
Background: Apoptosis resistance is a hallmark of cancer, and apoptosis regulators have been targeted for cancer treatment. A class of such regulators is inhibitor of apoptosis proteins (IAPs), which suppress caspase activity by inducing degradation of active caspases or blocking interaction of caspases with their substrates. Dysregulated IAP expression has been reported in many cancers, including breast cancer, and is involved in chemoresistance. IAPs have therefore become attractive targets for cancer treatment. Birinapant (TL32711), a biindole-based bivalent mimetic of second mitochondria-derived activator of caspase (SMAC, an endogenous antagonist of IAPs), has been shown to effectively activate apoptotic signaling by targeting IAPs and has potential application for treatment of multiple cancers. In addition, birinapant showed synergistic anti-tumor effects with several widely used chemotherapeutic agents in various cancers. Here, we assessed whether birinapant synergizes with commonly used anti-cancer drugs, including entinostat (class I histone deacetylase inhibitor), cisplatin, paclitaxel, voxtalisib (PI3K inhibitor), dasatinib (Src inhibitor), everolimus (mTOR inhibitor), and gemcitabine, in triple-negative breast cancer (TNBC). Among these drugs, gemcitabine strongly synergized with birinapant (combination index [CI] &lt;1). We hypothesize that birinapant enhances anti-tumor efficacy of gemcitabine in TNBC by inducing intrinsic pathway-dependent apoptosis. Methods: The in vitro anti-tumor efficacy of birinapant, gemcitabine, and their combination was determined using CellTiter-Blue viability and soft agar colony formation assays. The CI (CalcuSyn software) was used to quantify the synergy. The effects of drug treatment on cell cycle distribution were analyzed by flow cytometry; the effects on apoptosis were assessed by annexin V-PE and 7-AAD staining, and induction of apoptosis was further confirmed by pan-caspase inhibitor treatment; and the effects on IAP degradation were examined by Western blotting. The in vivo antitumor efficacy was determined using TNBC xenograft mouse models.
Results: Birinapant as a single agent had varying anti-proliferation effects in TNBC cells (IC50 = 0.21 to &gt;20 µM). It had no synergistic effects with entinostat, cisplatin, paclitaxel, voxtalisib, dasatinib, or everolimus but strongly synergized with gemcitabine (CI &lt;1 µM) in tested TNBC cells. Birinapant and gemcitabine synergistically inhibited the growth of TNBC cells by activating the intrinsic apoptosis pathway, accomplished by inducing degradation of cIAP2 and XIAP, which led to apoptotic cell death. Furthermore, birinapant significantly enhanced anti-tumor effectiveness of gemcitabine in TNBC xenograft mouse models (n = 10; P &lt; 0.05) by inducing apoptosis.
Conclusion: Our findings demonstrate the therapeutic potential of birinapant for enhancing the anti-tumor efficacy of gemcitabine in TNBC by targeting the IAP family of proteins.
Citation Format: Xuemei Xie, Jangsoon Lee, Troy Pearson, Alexander Y. Lu, Debu Tripathy, Gayathri R. Devi, Chandra Bartholomeusz, Naoto T. Ueno. Birinapant enhances gemcitabine's anti-tumor efficacy in triple-negative breast cancer by inducing intrinsic pathway-dependent apoptosis [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 548.