Sabutoclax ( BI 97 C 1 ) and BI 112 D 1 , Putative Inhibitors of MCL-1 , Induce Mitochondrial Fragmentation Either Upstream of or Independent of Apoptosis 1 , 2

Owing to the high levels of antiapoptotic B-cell lymphoma 2 (BCL-2) family members observed in several cancers, there has been a major effort to develop inhibitors of the BCL2-family as chemotherapeutic agents. Of the different members in the BCL-2 family, myeloid cell leukemia sequence 1 (MCL-1) is commonly amplified in human tumors and is associated with their relapse and chemoresistance. As a result, specific inhibitors of MCL-1 are being designed to treat resistant tumors. However, there is increasing evidence for other nonapoptotic roles of the BCL-2 family, ranging from ionic homeostasis and autophagy to the regulation of fission-fusion dynamics in subcellular organelles, including the endoplasmic reticulum and mitochondria. In this study, we characterize the specificity of two novel putative MCL-1 inhibitors, BI97C1 (Sabutoclax) and BI112D1, in inducing apoptosis in a BAX/BAK-dependent manner and in an MCL-1–dependent system. In addition to their being proapoptotic, these inhibitors also cause enhanced mitochondrial fragmentation that accompanies a time-dependent loss of optic atrophy 1 (OPA1), suggesting an impairment of mitochondrial fusion. This mitochondrial fragmentation occurs independently of dynamin-related protein 1 (DRP1)–mediated fission activity and, unlike most apoptotic stimuli, occurs upstream of and/or independent of BAX, BAK, and other BH3-only proteins. Furthermore, thismitochondrial fragmentation occurred rapidly and preceded other hallmarks of apoptosis, including the loss in mitochondrial membrane potential and the release of cytochrome c. Although such mitochondrial fragmentation did not deplete total cellular adenosine triphosphate (ATP) or alter other mitochondrial complexes, there was significant accumulation of reactive oxygen species. Neoplasia (2013) 15, 568–578 Introduction Several diseases including cancer, autoimmunity, and neurodegeneration have been attributed to a defective/ineffective apoptotic program, a major mechanism by which cells in the body undergo self-destruction. Apoptosis can be triggered by the activation of death receptors on the cell surface (extrinsic pathway) or by perturbation of mitochondrial integrity (intrinsic pathway) [1,2]. Apoptosis is primarily regulated by the B-cell lymphoma 2 (BCL-2) family of proteins, which comprise the apoptotic effector molecules, BAX and BAK, which are activated by BH3-only proteins (including BIM, BAD, PUMA, and NOXA) and antagonized by antiapoptotic BCL-2 family proteins (including BCL-2, BCL-XL, BCL-w, myeloid cell leukemia sequence 1 (MCL-1), and BCL2A1) [3,4]. BCL-2 family members act primarily to control the integrity of the outer mitochondrial membrane, thereby regulating cellular susceptibility to apoptosis induced by the intrinsic pathway Abbreviations: DKO, double knockout; ROS, reactive oxygen species Address all correspondence to: Gerald M. Cohen, PhD, MRC Toxicology Unit, Hodgkin Building, PO Box 138, Leicester LE1 9HN, United Kingdom. E-mail: gmc2@le.ac.uk M.P. thanks the National Institutes of Health (NIH; grant CA 149668) for support. S.V., M.B., D.D., and G.M.C. declare no conflict of interest. J.W. and M.P. declare a possible conflict of interest because Sanford-Burnham Medical Research Institute has licensed Sabutoclax and related compounds to Oncothyreon, Inc (Seattle, WA). However, we do not believe that this has in anyway influenced the presentation and interpretation of any of the results in the manuscript. This article refers to supplementary materials, which are designated by Figures W1 and W2 and are available online at www.neoplasia.com. These authors made an equal contribution to the work. Received 16 January 2013; Revised 26 February 2013; Accepted 4 March 2013 Copyright © 2013 Neoplasia Press, Inc. All rights reserved 1522-8002/13/$25.00 DOI 10.1593/neo.13230 www.neoplasia.com Volume 15 Number 5 May 2013 pp. 568–578 568