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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, this mitochondrial 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.
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 . As most cancer chemotherapeutic agents induce apoptosis by activation of the intrinsic pathway and many cancers exhibit high levels of antiapoptotic BCL-2 family members, there has been a major effort to develop inhibitors of the BCL2-family.
Despite the many claims in the literature of their supposed specificity, the vast majority of these putative BCL-2 family inhibitors are nonspecific, with two key exceptions, ABT-737 and its orally active and metabolically more stable analog, ABT-263 (Navitoclax), the latter of which has recently entered clinical trials for the treatment of various hematological malignancies [5–10]. Both ABT-737 and ABT-263 inhibit BCL-2, BCL-XL, and BCL-w but do not inhibit MCL-1 or BCL2A1 [6,7]. As MCL-1 is commonly amplified in human tumors and is associated with tumor relapse and chemoresistance, particularly to ABT-737 [8,11–15], specific inhibitors of MCL-1 could be a very valuable addition to aid in treating chemoresistant tumors.
In addition to their role in the regulation of apoptosis, there is increasing evidence for nonapoptotic roles of the BCL-2 family [16,17] in particular in the regulation of mitochondrial structure and partial control of mitochondrial fusion and fission [18,19]. In this regard, a recent report has proposed that one isoform of MCL-1 resides in the outer mitochondrial membrane and antagonizes apoptosis, whereas an N-terminal truncated isoform resides in the mitochondrial matrix where it is required for normal mitochondrial fusion, adenosine triphosphate (ATP) production, and membrane potential . However, the precise mechanisms by which MCL-1 regulates mitochondrial structure and/or function are yet to be determined.
In this study, we use an MCL-1-dependent non-small cell lung cancer cell line, H23, to characterize the specificity of putative novel MCL-1 inhibitors . Owing to the limitations of ABT-737 and ABT-263 to inhibit MCL-1, nuclear magnetic resonance (NMR) binding assays and computational docking studies were used to identify apogossypol derivatives with pan-BCL-2 family inhibitory properties [21,22]. One of these molecules, BI-97C1 (Sabutoclax), is an optically pure apogossypol derivative with improved in vitro and in vivo efficacy and inhibits tumorigenesis in various models of prostate cancer [23,24]. In addition, one optically pure apogossypolone derivative, BI112D1 ((-)BI97D6), is also a potent pan-active BCL-2 family inhibitor and exerts antitumor activity in a prostate cancer xenograft model in mice [25,26]. Both BI97C1 and BI112D1 induced apoptosis in a BAX/BAK-dependent manner and in MCL-1-dependent cells. These inhibitors also caused a time-dependent loss of optic atrophy 1 (OPA1) that accompanied enhanced mitochondrial fragmentation as well as an increased mitochondrial accumulation of reactive oxygen species (ROS).
Wild-type (WT) and BAX/BAK double knockout (DKO) mouse embryonic fibroblasts (MEFs) from Dr A. Strasser (Walter and Eliza Hall Institute, Melbourne, Australia) were cultured in Dulbecco's modified Eagle's medium supplemented with 5 mM l-glutamine and 10% fetal calf serum (all from Life Technologies Inc, Paisley, United Kingdom). H23 cells from Prof. C. Pritchard (University of Leicester, Leicester, United Kingdom) were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum and 5 mM l-glutamine.
BI97C1 and BI112D1 were synthesized as described [22,26]. ABT-263 was obtained from Selleck Chemicals Co (Houston, TX). Antibodies against cytochrome c, HSP60, OPA1, mitofusin 1(MFN1), and dynamin-related protein 1 (DRP1) from BD Biosciences (Oxford, United Kingdom), MFN2, ATP5A, UQCRC2, SDHB, and tubulin from Abcam (Cambridge, United Kingdom), and COX4 and NDUFA9 from Santa Cruz Biotechnology, Inc (Dallas, TX) were used. DRP1 K38A was a kind gift from Dr Bampton (MRC Toxicology Unit, Leicester, United Kingdom). MitoTracker Deep Red, MitoSOX Red, and tetramethylrhodamine ethyl ester (TMRE) were from Molecular Probes, Inc (Eugene, OR). All other reagents, unless mentioned otherwise, were from Sigma-Aldrich Co (St Louis, MO).
For transient transfections, cells were transfected using TransIT-LT-1 transfection reagent (Mirus Bio LLC, Madison, WI) and left for 48 hours, according to the manufacturer's instructions. For siRNA knockdowns, cells were reverse transfected with oligoduplexes (Life Technologies or Thermo Scientific, Waltham, MA), using Interferin Reagent (Polyplus Transfection Inc, New York, NY), according to the manufacturer's protocol and processed 72 hours after transfection. Cells were reverse transfected with 10 nM DRP1 (ID No. s19560), OPA1 (ID No. s9850), MFN1 (ID No. s31220), BCL-XL (ID No. s1920), BCL-w (ID No. s1924), MCL-1 (ID No. s8583), BIM (ID No. s195011), or NOXA (ID No. L-005275).
Cytochrome c release experiments were carried out in cells exposed to different drugs for the indicated times and assessed as previously described . Western blots were carried out according to standard protocols . Briefly, 50 µg of total protein lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently, proteins were transferred to nitrocellulose membrane and protein bands were visualized with ECL reagents (GE Healthcare, Bucks, United Kingdom).
For immunofluorescent staining, cells grown on coverslips were fixed with 4% (vol/vol) paraformaldehyde, permeabilized with 0.5% (vol/vol) Triton X-100 in phosphate-buffered saline, and followed by incubations with primary antibodies and analyzed as previously described . For monitoring mitochondrial fragmentation and changes in mitochondrial membrane potential, cells were stained for 30 minutes with 200 nM MitoTracker Deep Red and 500 nM TMRE before image acquisition. For electron microscopy, cells were fixed and processed as previously described . Electron micrographs were recorded using a Megaview 3 digital camera and iTEM software (Olympus Soft Imaging Solutions GmbH, Münster, Germany) in a Jeol 100-CXII electron microscope (Jeol UK Ltd, Welwyn Garden City, United Kingdom).
Loss in mitochondrial membrane potential (ψm) was assessed as described previously by staining cells with TMRE, a lipophilic fluorescent dye that accumulates in the mitochondria in proportion to the membrane potential . Cell death was assessed by phosphatidylserine (PS) externalization and staining with Annexin V-fluorescein isothiocyanate as described previously . For measuring the extent of ROS accumulation in the mitochondria, cells exposed to DMSO or the inhibitors for the indicated times were incubated for 10 minutes at 37°C with 5 µM MitoSOX Red reagent and assessed for increase in fluorescence intensity.
Total cellular ATP in cells exposed to the different inhibitors for the indicated times was measured using CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega, Madison, WI), according to the manufacturer's instructions.
Since antiapoptotic members of the BCL-2 family antagonize BAX/BAK-dependent release of cytochrome c and other apoptotic factors from the mitochondria, we tested the specificity of the putative MCL-1 inhibitors, BI97C1 and BI112D1, in MEFs, derived from WT or BAX and BAK DKO mice. As a positive control, we used ABT-263 (Navitoclax), a BCL-2 family antagonist, which has recently entered clinical trials in patients with B cell malignancies, as it induces cell death in a BAX/BAK-dependent manner [7,29]. In agreement with previous data , ABT-263 induced a concentration-dependent cell death (assessed by PS externalization) in WT but not in BAX/BAK DKO MEFs (Figure 1A, left panel). Similarly, both BI97C1 and BI112D1 induced a concentration-dependent cell death in a BAX/BAK-dependent manner in MEFs, although BI97C1 induced marked cell death at 3 µM (Figure 1A, center and right panels). Thus, both BI97C1 and BI112D1, like ABT-737 and ABT-263, kill cells specifically in a BAX/BAK-dependent manner, unlike most other putative BCL-2 antagonists described in the literature [5,9,10].
Since BI97C1 and BI112D1 possess improved binding affinities for MCL-1 compared with both ABT-263 and the structurally related apogossypol [7,21,22,26], we examined their efficacy in an MCL-1-dependent cell line, H23 . Both BI97C1 and BI112D1 caused a time-dependent induction of apoptosis, assessed by PS externalization, which was preceded by a loss in mitochondrial membrane potential (ψm) and accompanied by an elevation in cytosolic cytochrome c (Figure 1, B–D). Surprisingly, ABT-263 was more potent than BI112D1 in inducing cell death in H23 cells (Figure 1, B–D), suggesting that at high concentrations it could effectively inhibit MCL-1 in these cells. These data are compatible with the putative MCL-1 inhibitors, BI97C1 and BI112D1, inducing mitochondrial outer membrane permeabilization (MOMP) and apoptosis by activation of the intrinsic pathway.
Resistance to BCL-2 antagonists as well as other chemotherapeutic agents is frequently due to elevated levels of MCL-1 [8,11–14]. To our knowledge, there is currently no bona fide inhibitor of MCL-1 described in the literature. Since both BI97C1 and BI112D1, like ABT-263, induced apoptosis in MCL-1-dependent H23 cells, primarily through the mitochondrial pathway, we examined their effects on mitochondrial structure and function. All compounds caused a dramatic time-dependent loss of the tubular mitochondrial network accompanied by extensive mitochondrial fragmentation, which was evident as early as 1, 2, and 8 hours after exposure to BI112D1, BI97C1, and ABT-263, respectively (Figures 2A and and4D).4D). The rapid induction of mitochondrial fragmentation suggested that these inhibitors could be valuable tools to further understand mechanisms of mitochondrial fragmentation and its possible relationship to cell death. The fragmentation of mitochondria by both compounds was confirmed by electron microscopy (Figure 2B). Despite the statistical improbability of a 70-nm-thick section being cut both coincident and parallel with the long axis of an elongate mitochondrion, several mitochondrial profiles with an aspect ratio >5:1 were found in untreated H23 cells. No such profiles were found in treated cells; indeed, few mitochondria were found with an aspect ratio >2:1, but all appeared to be healthy with no signs of swelling or disruption. There were no indications of autophagy/mitophagy in any of the treated cells (Figure 2B). Since H23 cells primarily express MCL-1 and BCL-w, with barely detectable levels of BCL-XL and no BCL-2  (Figure W1A), it is likely that the mitochondrial fragmentation observed following these compounds is due to the inhibition of MCL-1 and/or BCL-w. However, a recent report has also claimed a novel role for an amino-terminally truncated isoform of MCL-1 in mitochondrial fusion in multiple cell types . In support of this observation, knockdown of MCL-1 but not BCL-XL or BCL-w resulted in mitochondrial fragmentation (Figures 2C and W1A). However, knockdown of MCL-1 in H23 cells also caused significant cell death, thus masking the extent of mitochondrial fragmentation in the remaining viable cells. Taken together, our data supported a role for MCL-1 in either promoting mitochondrial fusion or preventing fission.
The length and continuity of the filamentous mitochondrial network is regulated by a conserved family of GTPases that balance mitochondrial fission and fusion events . MFN1/2 and OPA1 are essential for mitochondrial fusion, whereas Fis1 and the DRP1 are essential for mitochondrial fission . Since BI97C1, BI112D1, and ABT-263 all caused extensive mitochondrial fragmentation, resembling enhanced fission, we examined if down-regulation of DRP1 would prevent mitochondrial fragmentation. However, neither knockdown of DRP1 nor expression of its dominant negative mutant (K38A) affected the ability of ABT-263, BI97C1, or BI112D1 to induce mitochondrial fragmentation (Figure 3). These results suggested that these inhibitors induced mitochondrial fragmentation independent of DRP1.
Recently, it was proposed that MCL-1 regulates mitochondrial fragmentation by facilitating mitochondrial fusion . Compatible with this was our finding that the putative MCL-1 inhibitors induced mitochondrial fragmentation independent of DRP1-mediated fission (Figure 3). Knockdown of either OPA1 or MFN1, two proteins involved in mitochondrial fusion , resulted in similar mitochondrial fragmentation (Figure 4, A and B). Further examination of the effects of ABT-263, BI97C1, and BI112D1 on key proteins involved in mitochondrial fusion revealed a time-dependent loss of the high molecular weight isoforms of OPA1 (Figure 4C) that corresponded with the induction of extensive mitochondrial fragmentation (Figure 2A). Taken together, these results suggest that these compounds inactivate the ability of MCL-1 to promote mitochondrial fusion, possibly by enhancing the proteolytic processing of OPA1.
Next, we wished to explore the relationship if any between the ability of these compounds to induce mitochondrial fragmentation and their ability to induce apoptosis. While the rate of induction of mitochondrial fragmentation was BI11D1 > BI97C1 >> ABT-263, the rate of loss of ψm was BI97C1 > ABT-263 >> BI112D1, suggesting that the mitochondrial fragmentation occurred before changes in ψm, which was most evident in cells exposed to BI112D1 (Figures 4D and W2). ABT-263, BI97C1, and BI112D1 also induced mitochondrial fragmentationinbothWTand BAX/BAK DKOMEFs (Figure 5A), although they all required BAX and BAK to induce apoptosis (Figure 1A), thus placing mitochondrial fragmentation either upstream of or independent of BAX and BAK. Previously, it had been shown that knockdown of the BH3-only proteins, NOXA and BIM, partially rescued H23 cells from apoptosis induced by MCL-1 down-regulation . Using a similar knockdown of BIM and NOXA to prevent apoptosis, all three inhibitors induced profound mitochondrial fragmentation (Figures 5B and W1B) further supporting the conclusion that mitochondrial fragmentation occurs upstream of and/or independent of apoptosis.
In addition to its proposed role in regulating mitochondrial fusion, MCL-1 is also proposed to regulate ATP production, mitochondrial membrane potential, and maintenance of oligomeric ATP synthase, thereby preserving mitochondrial homeostasis . We therefore hypothesized that the dramatic changes in mitochondrial ultrastructure induced by the three inhibitors would disrupt mitochondrial function. Although the inhibitors neither affected ATP levels (Figure 6A) nor certain components of electron transport chain complexes (I, II, III, IV, and V; Figure 6B), they all induced an increase in ROS (Figure 6C), which suggested a possible role for ROS in MCL-1-regulated mitochondrial fragmentation and/or apoptosis.
Non-small cell lung cancer is the most predominant form of lung cancer and is the primary cause of cancer deaths worldwide. Although often treated with monoclonal antibodies or tyrosine kinase inhibitors, new targeted therapies are urgently needed. BCL-2 family members and particularly MCL-1 have been reported in primary tissues from patients as well as in several non-small cell lung cancer cell lines, suggesting that these antiapoptotic proteins may be suitable targets for therapy [33–35]. Recently, there has been considerable success in the development of selective inhibitors of some members of the BCL-2 family. Some of these inhibitors, ABT-737 and its bioavailable analog, ABT-263, bind selectively to BCL-2, BCL-XL, and BCL-w but not to MCL-1 or BCL2A1, whereas other inhibitors, including apogossypol and obatoclax, are considered pan-BCL-2 antagonists [5,7,9,36]. To date, the selective inhibitors developed do not target either MCL-1 or BCL2A1, and as these may be frequent causes of chemoresistance, it is essential to develop inhibitors of these antiapoptotic molecules [5,9,12–14,36,37]. Of the several putative broad spectrum BCL-2 family inhibitors that we tested, apogossypol proved rather better in inducing apoptosis in a BAX/BAK-dependent manner . In the present study, we characterize novel derivatives of apogossypol/apogossypolone, BI-97C1 (Sabutoclax) and BI112D1 ((-) BI97D6) [22,23,26]. Both BI97C1 and BI112D1 induced apoptosis in a BAX/BAK-dependent manner and in an MCL-1-dependent system (Figure 1). Although BI112D1 demonstrated better binding affinities to MCL-1, BI97C1 induced rapid release of cytochrome c, loss of mitochondrial membrane potential, and eventually PS externalization (Figure 1, B–D). Furthermore, while BI112D1 was most selective in the induction of apoptosis in a BAX/BAK-dependent manner, similar to ABT-263, the selectivity was more modest with BI97C1 (Figure 1A).
Recently, we identified a rapid and reversible reorganization of endoplasmic reticulum (ER) membranes following exposure of cells to apogossypol, which was attributed partly to the inactivation of several BCL-2 family members, particularly MCL-1 [28,38]. In support of this, both the putative MCL-1 inhibitors, BI97C1 and BI112D1, induced ER membrane reorganization, albeit to modest extents compared with apogossypol (Figure 2B and data not shown). However, these inhibitors were very potent in inducing extensive mitochondrial fragmentation (Figure 2), in agreement to the proposed role of MCL-1 in regulating mitochondrial fission-fusion dynamics . Moreover, BI112D1 exhibited a greater potency and induced a more rapid mitochondrial fragmentation than BI97C1 or ABT-263, in agreement with the relative binding affinities of these inhibitors to MCL-1 (Figures 2 and and4D)4D) [7,21,22,26]. This was further confirmed by RNA interference in H23 cells, which expressed both MCL-1 and BCL-w, with barely detectable levels of BCL-XL and no BCL-2 ( and Figure W1). Although BCL-XL was not detected by Western blot analysis (Figure W1), we carried out the experiment with the appropriate siRNA because of the report stating the presence of very low levels of BCL-XL in these cells . Silencing the expression of MCL-1, but not BCL-XL or BCL-w, resulted in mitochondrial fragmentation (Figure 2C), although the effect was often masked by the extensive apoptosis that resulted within hours of MCL-1 down-regulation in the MCL-1-dependent H23 cells (data not shown).
Mitochondrial fragmentation may be a consequence of either an enhanced fission of the filamentous mitochondria or a loss in the fusion events to form the lengthy and continuous mitochondrial network [30–32]. The balance between these dynamic processes is brought about by a family of GTPases, some of which regulate fission (DRP1), while some favor mitochondrial fusion (OPA1 and MFNs) [30–32]. Mitochondrial fragmentation observed following several stimuli is successfully inhibited by inactivating DRP1, either by using a specific chemical inhibitor, Mdivi1, by silencing endogenous DRP1 expression, or by overexpressing dominant negative mutants of DRP1 [39,40]. However, none of these approaches prevented the mitochondrial fragmentation mediated by BI97C1, BI112D1, or ABT-263 (Figure 3 and data not shown), arguing against the involvement of DRP1 in this phenotype. Nevertheless, all these inhibitors caused a time-dependent loss of the high molecular weight isoforms of OPA1 that precisely corresponded to the kinetics of mitochondrial fragmentation (Figure 4), which raised the possibility that MCL-1 may regulate OPA1 loss possibly by proteolysis, thereby regulating mitochondrial fusion. OPA1 exists in eight isoforms resulting from alternative splicing with some isoforms being evolutionary conserved and involved in fusion of the mitochondrial network [41,42]. Although we could not establish a direct interaction between MCL-1 and OPA1, they co-eluted on gel filtration analysis (data not shown), raising the possibility that MCL-1 may be one component of a complex responsible for OPA1 loss. Recent studies implicate a role for different proteases, including metalloproteases and overlapping with the m-AAA protease 1 (OMA1) in the proteolysis of OPA1 [43–46]. Attempts to prevent OPA1 proteolysis using o-phenanthroline , a metalloprotease inhibitor, were unsuccessful because o-phenanthroline alone resulted in excessive mitochondrial fragmentation (data not shown). Furthermore, BI97C1, BI112D1, and ABT-263 induced mitochondrial fragmentation to a similar extent in both WT and OMA1 null MEFs (data not shown), which demonstrates that in this scenario OMA1 is not responsible for mitochondrial fragmentation.
The precise mechanism whereby MCL-1 regulates mitochondrial fragmentation is intriguing. Recently, the existence of two distinct isoforms of MCL-1 with each isoform performing specific functions with respect to mitochondrial fusion dynamics and apoptosis has been proposed . It is certainly possible for MCL-1 to perform functions other than its well-characterized role, as an antiapoptotic BCL-2 family member. Other BCL-2 family members have been reported to perform functions not directly related to their role in apoptosis [16,17]. It is feasible that different inhibitors target different MCL-1 isoforms depending on binding affinities and/or cellular distribution and thus may differentially affect the properties associated with these isoforms. Thus, some inhibitors may preferentially affect the antiapoptotic role of MCL-1, whereas others may have a greater effect on other functions, such as mitochondrial fusion.
Changes in mitochondrial fusion-fission dynamics have frequently been associated with apoptosis and are thought to occur downstream of BAK and BAX, resulting in the loss of mitochondrial membrane potential and release of cytochrome c and activation of the intrinsic pathway of apoptosis . To our knowledge, this is the first report in which BCL-2 family antagonists induce mitochondrial fragmentation upstream of a loss in membrane potential and other hallmarks of apoptosis (Figures 1 and and2).2). Although the inhibitors shifted the dynamics from a filamentous mitochondrial network to fragmented mitochondria, careful examination revealed that the fragmented mitochondria still retained intact outer and inner membranes (Figure 2B), unlike the rupture of the outer mitochondrial membrane observed in a novel paradigm of apoptosis following exposure of primary chronic lymphocytic leukemia cells to ABT-737 and ABT-263, specific inhibitors of BCL-2 and BCL-XL [27,48]. Furthermore, there was no evidence of mitochondrial swelling or mitophagy, supporting the notion that the mitochondria remained healthy despite a shift in the fission-fusion dynamics, which may explain why the loss in membrane potential and MOMP was not detected until much later. Although one may expect that some functions of fragmented mitochondria may be compromised, no changes in either complex IV, ATP synthase oligomeric state, or ATP levels were observed (Figure 6, A and B, and data not shown), whereas these parameters were affected in MCL-1-deleted cells . A permanent inability to fuse could result in the accumulation of ROS and mutations of mtDNA, which could eventually kill the cells. Although we observed an accumulation of mitochondrial ROS, we were unable to ascertain if this was responsible either for mitochondrial fragmentation or death, as several ROS scavengers (Trolox, N-acetylcysteine, and DTT) failed to reverse these effects (data not shown).
In summary (Figure 7), we have established the selectivity of two novel putative MCL-1 inhibitors, BI97C1 (Sabutoclax) and BI112D1, in the induction of apoptosis in both a BAX/BAK-dependent manner and in an MCL-1-dependent system. Using these inhibitors, support for the role of MCL-1 in the regulation of mitochondrial fusion was provided by the observation of extensive mitochondrial fragmentation accompanying an enhanced loss of OPA1, possibly by proteolyis. This mitochondrial fragmentation occurred independently of DRP1-mediated fission and, unlike most apoptotic stimuli, was observed upstream of and/or independent of BAX, BAK, and other BH3-only proteins and preceded other hallmarks of apoptosis. Further work is required to understand the control, regulation, subcellular localization, and functions of different isoforms of MCL-1. This will be aided by the design and synthesis of isoform-specific inhibitors of MCL-1, although current indications suggest that this may be extremely difficult. Such inhibitors could be particularly valuable in the treatment of chemoresistant tumors, as well as shedding valuable insight into the regulation of mitochondrial fusion.
We thank Judy McWilliam and Tim Smith for technical assistance and the Medical Research Council for core support. We thank C. Pritchard and A. Strasser for the cells.
1M.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.