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1 CTM Review Cancer Translational Medicine Cancer Transl Med 2016;2(1):7 20 doi: / BCL2 Family, Mitochondrial Apoptosis, and Beyond Haiming Dai 1, X. Wei Meng 2, Scott H. Kaufmann 2 1 Center of Medical Physics and Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, Anhui, China; 2 Division of Oncology Research, Mayo Clinic, Rochester, MN, USA Address for correspondence: Dr. Haiming Dai, Center of Medical Physics and Technology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, 350 Shushanhu Road, Hefei , Anhui, China. daih@cmpt.ac.cn; Dr. X. Wei Meng, Division of Oncology Research, Gonda , Mayo Clinic, 200 First St., S.W., Rochester, MN 55905, USA. Meng.xue@mayo.edu; Dr. Scott H. Kaufmann, Division of Oncology Research, Gonda , Mayo Clinic, 200 First St., S.W., Rochester, MN 55905, USA. Kaufmann.scott@mayo.edu Received November 15, 2015; Accepted January 24, 2016 Apoptosis is a morphologically and biochemically distinct form of cell death that plays an essential role in development, immune response, and tissue homeostasis. Diminished apoptosis is also considered a hallmark of cancer whereas many cancer treatments induce apoptosis in susceptible cells. Classically, this induction of apoptosis occurs through two major signaling pathways: the extrinsic pathway and the intrinsic pathway. It has been known for 20 years that B cell lymphoma 2 (BCL2) family proteins control the intrinsic apoptotic pathway by regulating the process of mitochondrial outer membrane permeabilization (MOMP) through protein protein interactions. Recent studies have elucidated how BCL2 antagonist/killer (BAK) and BCL2 associated X protein (BAX) are activated by BCL2 homology 3 (BH3) only proteins and how activated BAK and BAX permeabilize MOM, providing increased understanding of how BCL2 family proteins control MOMP. Moreover, both structural and biochemical studies have revealed dual roles for anti apoptotic BCL2 family proteins in inhibiting BH3 only proteins and restraining activated BAK and BAX. Here, we review recent advances in understanding how BCL2 family proteins control MOMP as well as new nonapoptotic functions for these proteins. Key words: Apoptosis, B cell lymphoma 2 family, BAK/BAX activation, BH3 domain, mitochondrial outer membrane permeabilization INTRODUCTION Cell death plays an essential role in development, immune response, and tissue homeostasis. 1,2 Apoptosis is a morphologically and biochemically distinct form of cell death that can be initiated by various stimuli. Morphologically, it is characterized by cell shrinkage followed by the formation of apoptotic bodies, membrane enclosed cell fragments that are rapidly cleared by phagocytes after they recognize eat me signals on the outer surface of the apoptotic cell s plasma membrane. 3 During cancer development, apoptosis is often deregulated. In particular, anti apoptotic proteins are often overexpressed during cancer formation. 4 6 Conversely, a large number of anti cancer drugs can induce apoptosis in susceptible cells. 7,8 Thus, apoptosis also plays an important role in cancer development and treatment. Apoptosis can proceed through two different pathways [Figure 1]: the extrinsic pathway and the intrinsic pathway. 1,2 The extrinsic pathway, also called the death receptor pathway, is initiated when certain members of the tumor necrosis factor super family bind to their cognate receptors, leading to the formation of an intracellular death inducing signaling complex (DISC), DISC mediated activation of a caspase cascade, and subsequent disassembly of the cell Death receptor initiated apoptosis can either directly initiate the demise of some cells (type I cells, such as lymphocytes) or initiate cell death that requires BID mediated activation of the intrinsic apoptosis pathway (type II cells, such as hepatocytes) due to lower expression levels of death receptors in these cells. 12 The intrinsic apoptotic pathway, also termed the mitochondrial pathway, is controlled by members of the B cell lymphoma 2 (BCL2) protein family, which regulate mitochondrial outer membrane permeabilization (MOMP). Once MOMP occurs, mitochondrial intermembrane space proteins, including cytochrome c and Smac, are released. Cytosolic cytochrome c interacts with apoptotic protease activating factor 1 (Apaf-1) and procaspase 9 to form a complex This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 3.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms. For reprints contact: reprints@medknow.com How to cite this article: Dai H, Meng XW, Kaufmann SH. BCL2 Family, Mitochondrial Apoptosis, and Beyond. Cancer Transl Med 2016;2(1): Cancer Translational Medicine Published by Wolters Kluwer Medknow 7

2 Cancer Transl Med 2016;2(1) Figure 1. Overview of apoptotic pathways: the death receptor and mitochondrial pathways. The death receptor pathway is initiated by members of tumor necrosis factor superfamily, which bind their receptors and cause activation of caspase 8. The mitochondrial pathway is controlled by the BCL2 protein family. Two members of this protein family, BAK and BAX, are directly responsible for breaching the mitochondrial outer membrane. Once this occurs, cytochrome C, Smac, and other mitochondria intermembrane proteins are released and facilitate the activation of downstream caspases, eventually leading to apoptosis. Cyto C: cytochrome c; BCL2: B cell lymphoma-2; BAK: B-cell lymphoma-2 antagonist/killer; BAX: B-cell lymphoma-2-associated X protein called apoptosome, which facilitates caspase 9 activation. 13 Smac can bind and inactivate X linked inhibitor of apoptosis (XIAP), which would otherwise inhibit caspase 9 activation. 3,13,14 Thus, MOMP initiates two processes that facilitate caspase activation. BCL2 FAMILY PROTEINS As indicated in Figure 1, BCL2 family proteins play critical roles in the intrinsic apoptotic pathway. This family of proteins can be divided into three groups based on their structures and intracellular functions [Figure 2 and Table 1]. One group includes BCL2 antogonist/killer (BAK) and BCL2 associated X protein (BAX), which are known as apoptosis effectors. Also called multidomain pro apoptotic BCL2 family proteins, BAX and BAK contain BCL2 homology (BH) domains 1 3 and can directly permeabilize MOM when activated. Whether BCL2-related ovarian killer (BOK) belongs to this same subfamily is not clear. Structurally it is similar to BAK and BAX; 44 however, functionally it does not have the ability to permeabilize the MOM by itself but instead induces apoptosis only in the presence of BAK or BAX. 45 The second group, called anti apoptotic or pro survival BCL2 family members, includes BCL2, BCL X large (BCLX L ), BCL2-like protein 2 (BCLW), BCL2-like protein 10 (BCLB), Figure 2. Overview of the BCL2 protein family. The BCL2 protein family is divided into three groups such as multidomain pro apoptotic BCL2 family proteins, BAK, and BAX; multi domain anti apoptotic BCL2 family proteins, including BCL2, BCLX L, BCLW, BCLB, MCL1 and A1; and BH3-only pro apoptotic BCL2 family proteins, including BIM, PUMA, BID, BAD, NOXA, BIK, BMF, and HRK. The anti apoptotic BCL2 family members generally have four BH domains. BAK and BAX have classically been thought to share BH 1 3 domains, although some studies have suggested that BAK and BAX also contain a BH4 domain as well BCL2: B-cell lymphoma-2; BAK: B-cell lymphoma-2 antagonist/killer; BAX: B-cell lymphoma-2-associated X protein; BCLX L : B-cell lymphoma-x large; MCL1: Myeloid cell leukemia 1; BH: B-cell lymphoma-2 homology myeloid cell leukemia 1 (MCL1), and BCL2-related protein A1 (BFL1) (A1 in mouse). These proteins, which contain four BH domains (BH1 BH4), inhibit apoptosis by binding and sequestering their pro apoptotic counterparts. The final group, termed BH3 only proteins, includes BIM, PUMA, BID, BAD, NOXA, BIK, BMF, and HRK. These polypeptides share only a residue BH3 domain in common with other BCL2 family proteins. This BH3 domain, however, is critical for the interactions of these proteins with other BCL2 family proteins to regulate MOMP. BAK and BAX effectors of mitochondrial permeabilization Structure of BAK and BAX monomers Structural studies have demonstrated that BAK and BAX monomers are globular structures consisting of a central hydrophobic core helix (α5) surrounded by eight alpha helices In the BAK monomer, four of these helices (α1, α3, α4, and α6) are long helices that form a circle around the central helix α5 while the others (α2, α7, and α8) are shorter and link either the longer helices or the main structure to the transmembrane (TM) domain which consists of helix α9. 46 The major structural difference between monomeric BAK and BAX is the orientation of helix α9. In BAX, this helix is buried in a hydrophobic groove formed by helices α3, α4, and α5. 48 In contrast, the hydrophobic groove of BAK is empty; and the α9 helix of BAK extends away from the remainder of the globular protein. Consequently, BAK is normally tethered to cytoplasmic surface of the MOM through this hydrophobic TM domain whereas BAX resides in cytosol. In addition, the hydrophobic groove of BAX must accommodate the α9 helix in the unactivated state. Therefore, the hydrophobic groove of BAX opens wider than that of BAK. 8

3 BCL2 family overview Table 1: BCL2 family protein interactions and their regulation in cancer BCL2 family member Category Main functions Main interacting proteins Up /down regulation in various cancers BAK Apoptosis effector When activated, permeabilizes MOM directly BAX Apoptosis effector When activated, permeabilizes MOM directly BOK Similar to apoptosis effectors structurally but not functionally Induction of apoptosis depends on BAK or BAX BCL2 Anti apoptotic factor Inhibits both apoptosis effectors and BH3 only proteins BCLX L Anti apoptotic factor Inhibits both apoptosis effectors and BH3 only proteins MCL1 Anti apoptotic factor Inhibits both apoptosis effectors and BH3 only proteins BCLW Anti apoptotic factor Inhibits both apoptosis effectors and BH3 only proteins BFL1 Anti apoptotic factor Inhibits both apoptosis effectors and BH3 only proteins BCLB Anti apoptotic factor Inhibits both apoptosis effectors and BH3 only proteins MCL1, BCLX L, BCL2, BIM, PUMA, tbid, and NOXA MCL1, BCLX L, BCL2, BIM, PUMA, tbid MCL1 BAK, BAX, BIM, PUMA, tbid, BAD, BIK, and BMF BAK, BAX, BIM, PUMA, tbid, BAD, BIK, BMF, and HRK BAK, BAX, BOK, BIM, PUMA, tbid, and NOXA BAK, BAX, BIM, PUMA, tbid, BAD, BIK, BMF, and HRK BAK, BAX, BIM, PUMA, tbid, NOXA, and HRK BAX, BIM, PUMA, tbid, BAD, BIK, BMF, and HRK Down regulated in breast, 15 head and neck, 16,17 cervix, 18 and lung cancers 19 Down regulated in head and neck, 16,17 cervix, 18 and lung cancers 19 Up regulated in lung, 20 melanoma, 21 breast cancers, 22 and lymphoid malignancies Up regulated in hormone refractory prostate cancer 26 and breast cancer 27 Up regulated in relapsed acute leukemia, 28 CLL, 29 multiple myelomas, and nonsmall cell lung cancer 33 Up regulated in gastric 34,35 and colorectal cancers, 36 as well as glioblast oma multiforme 37,38 Up regulated in ALL, CLL and DLBL Up regulated in DLBL and breast, prostatic, gastric, colorectal, and small cell lung cancers 43 MCL1: Myeloid cell leukemia 1; MOM: Mitochondrial outer membrane; CLL: Chronic lymphocytic leukemia; DLBL: Diffuse large B cell lymphoma; ALL: Acute lymphoblastic leukemia; BAK: B-cell lymphoma-2 antagonist/killer; BAX: B cell lymphoma 2 associated X protein; BOK: BCL2-related ovarian killer; BCLX L : B cell lymphoma X large; tbid: Truncated BID; BH3: B cell lymphoma 2 homology 3; BCLW: BCL2-like protein 2; BFL1: BCL2-related protein A1; BCLB: BCL2-like protein 10 As a result of these differences in the structure of BAK and BAX monomers, the mechanisms of activation of these two proteins are thought to be different Specifically, BAX activation is reportedly initiated through binding of activating ligands to the N terminal domain containing the α1 and α2 helices. 49,52 This results in a conformational change that releases α9 from the BAX hydrophobic BH3 binding groove and contributes to translocation of BAX from the cytoplasm to mitochondria. 49 Beyond this step, further activation occurs through the binding of activating ligands to the vacated BH3 binding groove. 50,52 Recent structural studies have suggested that at least at some steps, specifically the binding of pro apoptotic BH3 only family members to the BH3 binding groove followed by oligomerization are similar for BAK and BAX 44,53 as described in greater detail below. Models of BAK/BAX activation Three different models have been proposed to explain BAK and BAX activation. One is the so called direct activation model, in which certain BH3 only proteins are proposed to directly bind to BAK and/or BAX to induce their activation. 54,56 According to this model, BH3 only proteins are divided into activators and sensitizers. 55,56 Activators directly bind to BAK and/or BAX, causing a BAK/BAX conformational change that leads to the oligomerization of BAK/BAX and subsequent activation. In contrast, sensitizers bind anti apoptotic BCL2 family proteins, releasing the BH3 only activators that are normally restrained by anti apoptotic BCL2 proteins. An alternative model, the indirect activation model (or displacement model), suggests that BAK and BAX are constitutively activated and are prevented from spontaneously inducing MOMP by their binding to anti apoptotic BCL2 family proteins. According to this model, the role of BH3 only proteins is to neutralize the anti apoptotic family members and release preactivated BAK and BAX. 57,58 More recently, a unified model has suggested that both direct activation and displacement models play roles in BAK/BAX activation, with anti apoptotic BCL2 family members inhibiting MOMP by sequestering both direct activators and activated BAK and BAX. 59 This model has been supported by extensive studies Although BAX activation is reportedly initiated at the N terminus, 49 a second step that involves binding of activators to the canonical BH3 binding groove is also required. 52 Different from BAX activation, BAK activation is initiated by BH3 domains binding exclusively at the canonical BH3 binding groove. 51 Biochemical studies have suggested that binding of activators to the canonical BH3 binding groove of BAK, which induces a conformational change in BAK BH3 domain, 51 might induce BAK octamerization. 66 Structural studies have confirmed that BAK activation is initiated at the canonical BH3 binding groove; 67 this model is also supported by a study using full length BAK. 68 Although BH3 only proteins are the major BAK/BAX activators, additional proteins also appear to be able to serve this function. In particular, p53, Casp8p41, and retinoblastoma protein have been reported to be direct activators Moreover, recent evidence suggests that activated BAK and BAX themselves might also serve as activators

4 Cancer Transl Med 2016;2(1) BAK/BAX mediated permeabilization of the mitochondrial outer membrane When cells encounter conditions favoring apoptosis, BAK, and/or BAX are activated. Earlier studies have reported the exposure of cryptic N terminal epitopes and transient exposure of BAK/BAX BH3 domains during the activation. 75,76 After this BH3 domain exposure, BAK or BAX is thought to form a symmetric dimer through binding of the BH3 domain of one molecule to the canonical BH3 binding groove of its partner. This binding, which is similar to binding of a BH3 domain to anti apoptotic BCL2 family proteins, is thought to be the basis for formation of higher order oligomers Oligomerization has been further found to involve the α6 helix of BAK or BAX. 79,80 Structural studies have not only confirmed that symmetric dimers can form but also provided detailed information about the interaction interfaces in these dimers. 53,67 Moreover, dissociation of the BAK α1 helix from the core domain before dimer formation is required for further activation. 81 However, the structure of higher order BAK/BAX oligomers remains unclear. Another unresolved question is how activated BAK and BAX permeabilize the MOM. Structural studies have demonstrated that BAX 48 and BAK 46 share a similar fold with channel forming domains of bacterial toxins such as diphtheria toxin 82 and colicin E1. 83 These toxins also contain 9-10 α helices and can permeabilize bacterial membranes, raising the possibility that BAX and BAK might use similar mechanisms to permeabilize the MOM. 84 Labeling with the cysteine modifying chemical 4 acetamido 4 ((iodoacetyl) amino) stilbene 2,2 disulfonic acid (IASD) has been used to study how these toxins, 85 as well as BAX and BAK, permeabilize target membranes. 86 These assays defined residues along BAX helices α5, α6, and α9 that could not be labeled when BAX was activated, suggesting a hairpin insertion model for BAX when inserted into the MOM. 87 In addition, purified peptides derived from the pore forming domain, which consists of the α5 and α6 helices of BAK or BAX, can independently form pores and permeabilize liposomes in vitro All of these observations suggest an important role for BAK/BAX helices α5 and α6 in permeabilizing the membrane. Moreover, electron paramagnetic resonance studies have suggested partial interaction of BAK α5 and α6 with liposomes. 90 In particular, the observation that only some of the residues in α5 and α6 of activated BAK and BAX could be labeled with IASD also agrees with a partial interaction model, suggesting an in plane model in which α5 and α6 collapse onto the membrane to permeabilize the MOM. 91 However, this model does not agree with previous structural studies reporting that activated BAK and BAX must have their α5 and α6 helices separated when activated. 53,67 Thus, the mechanisms by which BAK and BAX permeabilize the MOM require further investigation. Nonapoptotic functions of BAK and BAX Mitochondria are in a dynamic equilibrium between a fragmented state and a fused state. 92 BAK and BAX activation leads rapidly to mitochondrial fragmentation, which occurs very close in time to MOMP and is associated with mitochondrial cytochrome c release. 96 However, constitutively fragmented mitochondria are also found in BAK( / )BAX( / ) MEFs. 95 Furthermore, reexpression of BAK in these cells, under conditions where apoptosis does not occur, leads to increase in mitochondrial length, suggesting that BAX and/or BAK are required for mitochondrial fusion/elongation in the absence of apoptosis. 95 On the other hand, the large GTPase of the dynamin superfamily, Drp1, can stimulate truncated BID (tbid) induced BAX oligomerization by interfering with mitochondrial membrane tethering and hemifusion. 97 All of these observations suggest that crosstalk between mitochondrial dynamics and the mitochondrial apoptotic pathway may exist. How BAK and BAX regulate mitochondrial fission and fusion is still not clear. Necrosis is a different kind of cell death, characterized by organelle swelling and early plasma membrane rupture. 98 The presence of BAX and BAK proteins is required for mitochondrial permeability transition pore dependent necrotic cell death in murine cells. 99 However, oligomerization and activation of BAK and BAX are not critical for this kind of necrotic cell death, suggesting that this type of cell death is not downstream of the mitochondrial apoptotic pathway, which depends critically on BAK or BAX oligomerization as described above. 99 BAK and BAX expression and regulation In addition to previous studies suggesting that BAX and BAK might have nonredundant functions, a variety of reports have suggested that their expression might also vary among different tissues BAX appears to be widely expressed in normal tissues and tumors, and its promoter can be directly activainted by the tumor suppressor TP Even though BAK expression is somewhat more varied, this pro apoptotic BCL2 family member is expressed in a substantial fraction of cancers of the breast, 15 kidney, 107 colon, 108 head and neck, 16,17 cervix, 18 and lung, 19 as well as Hodgkin s disease 109 and aggressive non Hodgkin s lymphomas. 110 In view of recent results showing that BAX and BAK can undergo autoactivation in a concentration dependent manner, 73,74 further investigation of factors that affect BAX and BAK expression might be warranted. Anti apoptotic BCL2 family proteins Structures and mechanisms of inhibiting apoptosis The BCL2 gene, encoding the founding member of the BCL2 family of proteins, was originally discovered because of the t(14;18) chromosomal translocation in B cell follicular lymphomas. This translocation results in fusion of the immunoglobulin heavy chain promoter and enhancer on chromosome 14 with the BCL2 gene located on chromosome 18, thus driving excessive BCL2 transcription The anti apoptotic role of BCL2 was highlighted by the discovery that unlike many other previously identified oncogenes, overexpression of BCL2 promoted tumorigenesis by inhibiting cell death 114 rather than by promoting cell proliferation. Since the discovery of BCL2, five other closely related family members, including BCLX L, MCL1, BCLW, BFL1, and BCLB (BCL2L10 in mouse) have been identified. Like BCL2, all of these five proteins are anti apoptotic. 44,115,116 As shown in Figure 2, this group of proteins shares four conserved regions of sequence homology termed BH domains. A C terminal hydrophobic TM domain directs each of these proteins to intracellular membranes, particularly the MOM. The anti apoptotic BCL2 proteins possess a remarkably similar globular structure containing a so called BCL2 core. 116 This core consists of a bundle of eight α helices that form a hydrophobic groove flanked by the BH1 and BH3 domains. In BCLW, the core also includes a short C terminal helix α8 attached to the BH2 domain. The hydrophobic groove made by α3 α5 is termed the BC groove 116 because it binds the BH3 region of binding partners. 117 The BC groove is crucial for the biology of 10

5 BCL2 family overview anti apoptotic BCL2 family proteins, as it provides an interface for the interaction with the BH3 domain of BH3 only proteins and the apoptotic effectors BAK and BAX. Based on the structure of the BC groove, several BH3 mimics that can occupy this groove and thus inactivate these proteins anti apoptotic function have been developed and are currently being tested in the clinic The mechanisms by which anti apoptotic BCL2 family proteins inhibit apoptosis have been extensively characterized. As mentioned previously, anti apoptotic BCL2 proteins bind and sequester pro apoptotic BH3 only proteins or activated BAK/BAX, thereby preventing MOMP. 44,117 The ability of the anti apoptotic BCL2 proteins to bind and sequester different pro apoptotic BCL2 family proteins varies Accordingly, the ability of anti apoptotic BCL2 family proteins to protect from various stimuli depends, at least in part, on the BH3 only proteins that are synthesized or activated by the stimuli. BCL2, BCLX L, and BCLW, which bind to almost all pro apoptotic proteins, are thought to be more potent than BCLB, BFL1, and MCL1 as far as protecting cells from apoptosis induced by a wide range of stimuli. In addition, a recent study revealed that the potency of the anti apoptotic BCL2 family proteins reflects their expression levels. 124 Roles of anti apoptotic BCL2 proteins in normal development The physiological roles of anti apoptotic BCL2 family proteins are best demonstrated by the phenotypes of mice deficient in each of these proteins. Based on these studies, it is generally agreed that anti apoptotic BCL2 family proteins control the survival of all mammalian cells. Mice with BCL2 gene deletion can complete embryonic development but exhibit massive apoptotic cell death in the spleen and thymus. Moreover, these BCL2( / ) mice die of renal failure around 6 weeks of age because of abnormal death of renal epithelial progenitor cells. 125 Knockout of BCL2L1, which encodes BCLX, is embryonic lethal due to massive cell death of immature hematopoietic cells and neurons. 126 MCL1 deficiency has an even more severe physiological outcome, as MCL1( / ) mouse embryos fail to implant in utero, 127 making it impossible to assess the role of MCL1 in development. Nonetheless, conditional MCL1 knockout impairs development and maintenance of both B and T lymphocytes. 128,129 Moreover, specific ablation of MCL1 in cardiomyocytes results in rapid, fatal cardiac failure that can be rescued by genetic deletion of both BAX and BAK. 130,131 In contrast to the profound effects of BCL2, BCL2L1 and MCL1 deletion on development, deletion of BCL2L2, which encodes BCLW, does not affect mouse development except for increased apoptosis in sperm cells during spermatogenesis, which leads to male sterility. 132 Studying the function of A1, a human BFL1 homolog in mice, has been hindered by the difficulty in ablating all three functional A1 genes. Nonetheless, deletion of the A1a gene accelerates the death of granulocytes and mast cells. 133 Specific down regulation of all three functional A1 genes in the mouse hematopoietic system impairs the survival of B lymphocytes and granulocytes without affecting T cell survival. 134 In addition, BFL1 seems important for activation induced mast cell survival in humans. 135 The role of BCLB in mouse knockout cannot be studied because its mouse homolog (BCL2L10) carries an inactivating mutation. 136 The role of anti apoptotic BCL2 family proteins in tumorigenesis Continued programmed cell death is essential for maintaining tissue homeostasis. Because many factors that block apoptosis lead to cancer development, resistance to apoptotic cell death has been regarded as one of the hallmarks of tumors. 4 Evidence accumulated over the past 25 years has shown that elevated expression of anti apoptotic BCL2 proteins [Table 1] is one of the major contributors to oncogenesis. BCL2 High BCL2 levels have been detected in a variety of tumor types, including small cell lung, 4,20 melanoma, 21 breast, 22 prostate, 137 colorectal, 138 and bladder cancers, 139 and especially in human lymphoid malignancies Elevated BCL2 expression has also been reported in acute myeloid leukemia (AML) and correlated with poor response to chemotherapy 140 although this has not been a universal finding. 28 There are many mechanisms that contribute to BCL2 up regulation The t(14;18) chromosomal translocation mentioned above, which places the BCL2 gene next to enhancer elements of the immunoglobulin heavy chain promoter, is found in 90% of follicular lymphomas and about 30% of diffuse large BCLs This translocation is a major mechanism for excessive BCL2 transcription. Other mechanisms include BCL2 gene rearrangement 144 and hypomethylation. 145 In addition, loss of mir 15a and mir 16, which target and repress BCL2 mrna, occurs in more than 50% of chronic lymphocytic leukemia (CLL) as a result of chromosome deletions and mutations. 146 Conversely, overexpression of other BCL2 regulating mirnas, including mir 195, mir 24 2, mir 365 2, and mir 204, reportedly increases the efficiency of chemotherapy in breast, 147 lung, and colon cancers. 147,148 BCL2 levels are also regulated by ubiquitination The inhibitor of NF E2 related factor 2 (INrf2) interacts with the BH2 domain of BCL2 and facilitates Cul3-Rbx1-mediated ubiquitination. Inactivating INrf2 mutations found in some lung cancer cell lines stabilize BCL2 and confer resistance to DNA damage induced cell death. 151 BCLX L BCLX L is an important factor for tumor development. Previous studies have shown that the BCL2 L1 locus, which encodes BCLX L, is amplified in a variety of solid tumors. 33 Interestingly, the BCL2 L1 and MYC loci are often co amplified, suggesting that BCLX L may be essential for the survival of MYC driven tumors. 33 Importantly, BCLX L down regulation induces apoptosis and diminishes tumorigenesis in cell lines with BCL2 L1 amplification, but not in those without this copy number gain. More recent studies in mice have further confirmed an important role for BCLX L in MYC induced tumorigenesis. In particular, loss of a single BCL2L1 allele delays MYC driven lymphomagenesis in mice; even normal levels of BCLX L are sufficient to accelerate this process. 152 High levels of BCLX L have been seen in a variety of human tumors including hormone refractory prostate cancers, 26 mesenchymal breast cancers, 27 and ovarian cancers. 33,153 Several mechanisms that lead to elevated expression of BCLX L have been identified in addition to gene amplification. In particular, the BCR/ABL kinase, which is characteristic of chronic myeloid leukemia, phosphorylates STAT5, which subsequently binds to the promoter of the BCL2 L1 gene and enhances its transcription. 154 Several mirnas that target the 3 UTR of BCLX L mrna and repress its expression have been reported as well. Decreased levels of mir let 7c occur in hepatocellular carcinomas 155 and confer resistance of lung cancer cell lines to cisplatin. 156 Like BCL2, BCLX L is also a target of INrf2-Cul3-Rbx1-mediated ubiquitination and degradation, 151 which requires an additional molecule, phosphoglycerate mutase 5, to bridge the interaction between 11

6 Cancer Transl Med 2016;2(1) INrf2 and BCLX L. Whether inactivation of BCLX L ubiquitination plays a role in tumor development remains to be seen. MCL1 MCL1 up regulation, which is seen in multiple neoplasms, likely, contributes to tumorigenesis as well. Mice expressing an MCL1 transgene develop BCLs at high frequency (85%) over a period of 2 years. 157 MCL1 also is important for the survival of acute lymphoblastic leukemia (ALL), AML 158 and CLL cells. 159 Importantly, elevated MCL1 is seen in acute leukemia at the time of relapse compared to diagnosis 28 and confers resistance to chemotherapy. 159 In addition, increased levels of MCL1 in multiple myeloma seem to correlate with disease progression Moreover, increased MCL1 is associated with poor prognosis in a variety of solid tumors as well. 33,160 MCL1 is regulated at multiple levels. MCL1 gene amplification has been observed in clinical lung and breast cancers as well as lines derived from these tumor types. 33 Loss of mir 29, which targets MCL1 mrna and represses its translation, has been found in many cancers. 161 Decreased levels of mir 125b 162,163 and mir 133b, 164 which target the MCL1 3 UTR and inhibit MCL1 expression, are found in hepatocellular carcinoma and lung cancer, respectively. In addition, increased expression of USP9X, a deubiquitinase that removes Lys48 linked polyubiquitin chains from MCL1, correlates with increased MCL1 protein levels in follicular lymphoma, diffuse large BCLs and multiple myeloma. 162 BCLW Several observations point to a potential role for BCLW in tumorigenesis. BCLW potently accelerates the development of MYC driven leukemia in mice. 165 BCLW overexpression also renders murine lymphoid and myeloid cells resistant to several cytotoxic stimuli, including BH3 mimetics. 166,167 Elevated levels of BCLW are frequently detected in human gastric 34,35 and colorectal cancers 36 as well as glioblastoma multiforme. 37,38 BCLW not only plays a role as a pro survival protein but also promotes tumor invasion 38,168,169 by enhancing the expression of the transcriptional factor specificity protein 1, which subsequently upregulates the expression of matrix metalloproteinase ,171 BCLW levels are regulated by several processes. BCLW overexpression can result from enhanced BCLW promoter activity. 172 Amplification of the BCL2 L2 gene, which encodes BCLW, is an additional contributor for elevated BCLW expression and chemoresistance. 173,174 Moreover, down regulation of several mirnas that target BCLW message has been implicated in increased BCLW expression in a number of tumor types. 163,175,176 BFL1 BFL1 has been studied less extensively than other anti apoptotic BCL2 family proteins. High BFL1 levels have been detected in ALL, CLL, and DLBL and are associated with chemoresistance. A1, a homolog of human BFL1 in mice, accelerates development of MYC driven leukemia, but it is the weakest accelerator among all of the six anti apoptotic BCL2 family members. 165 Although A1 was initially identified as a hematopoietic tissue specific gene that is expressed in several hematopoietic cell lineages, 177 several solid tumors aberrantly express this protein as well. Indeed, increased levels of BFL1 mrna have been detected in a number of human solid tumors and confer resistance to chemotherapy induced apoptosis BFL1 is a direct transcriptional target of nuclear factor kappab (NF kb). 181 Accordingly, activation of PI3K and extracellular signal regulated kinase (ERK), which activate NF kb, enhances BFL1 expression. 178,182 In addition, BFL1 is regulated by the ubiquitin/proteasome pathway. 183,184 Whether these regulatory mechanisms contribute to the elevated BFL1 levels in tumors remains to be elucidated. BCLB BCLB is the least studied of the anti apoptotic BCL2 proteins. Nonetheless, forced BCLB overexpression in mice has been shown to accelerate MYC driven leukemia. 165 BCLB is overexpressed in human breast, prostate, gastric, colorectal, and small cell lung cancers, and high levels of this protein correlate with poor prognosis. 43 In addition, BCLB expression levels reportedly correlate with azacitidine resistance in myelodysplastic syndrome and AML. 185 BCLB levels appear to be regulated by protein turnover. BCLB is polyubiquitinated at steady state, exhibits a half life of only min, and is subject to ubiquitin dependent proteasomal degradation. 186 A K/R mutant that cannot be ubiquitinated protects cells from apoptosis induced by conventional chemotherapeutic drugs, BH3 mimetics, and TRAIL, suggesting that the level of BCLB is crucial for its protective function. Neither the E3 ligase nor the deubiquitinase for BCLB has been identified to date. In summary, studies over the past 25 years have led to the conclusion that all six anti apoptotic BCL2 family proteins can facilitate tumorigenesis and confer resistance to various treatments. The anti apoptotic (or pro survival) capacity varies among the family members, but it is generally agreed that BCL2, BCLX L, and MCL1 have a stronger protective effect against death signaling than BCLW, BFL1, and BCLB. This variation in potency may reflect binding selectivity and affinity for pro apoptotic BH3 only proteins, BAK and BAX, as well as the levels of each anti apoptotic protein. Relative potencies of the anti apoptotic proteins may also depend on cellular context, including phosphorylation and ubiquitylation events that modify BCL2 family proteins, as well as the nature of the cytotoxic signaling. BH3 only proteins Activators and sensitizers The most important role of BH3 only proteins such as BIM, PUMA, BID, and NOXA is to act as integrators of various signals to initiate MOMP. The BH3 only proteins are activated by distinct cytotoxic stimuli in various ways, including enhanced transcription and posttranslational modifications. 187 Microtubule or actin targeting agents disrupt cytoskeleton integrity and release some of the BH3 only proteins (such as BIM and BMF) from their interaction partners, allowing them to translocate to mitochondria and promote MOMP Tyrosine kinase inhibitors upregulate BIM and PUMA through the PI3K AKT pathway to induce MOMP. 191 In short, a variety of cellular stresses are integrated by BH3 only proteins. As mentioned previously, these BH3 only proteins can be divided into direct activators and sensitizers As shown in Figure 3, BIM and tbid were first defined as direct activators because of their ability to directly induce BAK and/or BAX activation. 56 PUMA was also able to directly induce BAK/BAX activation although the efficiency is lower than BIM. 60,192 Moreover, BID/BIM/PUMA triple knockout mice displayed developmental defects that were reportedly similar to BAK/BAX double knockout mice. In addition, BID/BIM/PUMA triple knockout cells exhibited 12

7 BCL2 family overview resistance to a variety of apoptotic stimuli, such as potassium deprivation and etoposide although they were sensitive to other triggers such as ionizing radiation and dexamethasone. 193 It had been unclear whether tbid, BIM, and PUMA are the only direct activators. Several studies also identified NOXA as a direct activator that can bind BAK or BAX and induce the BAK/BAX mediated liposome release in vitro. 51,192 Structural studies, however, showed no direct interaction of NOXA BH3 peptide with BAK or BAX. 53,67,72 The difference between these two sets of studies might arise from the use of BH3 peptide versus full length protein. More recently, study of BID/BIM/PUMA/ NOXA quadruple knockout mouse cells revealed increased resistance to most apoptotic stimuli, including cytokine withdrawal, etoposide, dexamethasone, and irradiation, compared with BID/ BIM/PUMA triple knockout cells, providing further support for the concept that NOXA plays a role in BAK/BAX activation in intact murine cells. 73 Moreover, the observation that NOXA directly interacts with BAK and BAX in living cells also suggests that full length NOXA may serve as a direct activator. 194 BIK, BMF, and HRK have also been reported to directly activate BAK/BAX albeit with lower affinities. 192,195 That leaves the BH3 only protein BAD as the only pure sensitizer that reportedly binds solely to anti apoptotic BCL2 family members, thereby releasing activators. When compared, BIM and tbid show greater ability than other BH3 only proteins to activate BAK/BAX. 51,60 Interestingly, while BIM preferentially activates BAX, tbid preferentially activates BAK. 196 Inhibitors of anti apoptotic BCL2 family proteins Despite varied activity as activators, all of the BH3 only proteins have the ability to act as sensitizers. Specifically, activator BH3 only proteins can not only interact with BAK and BAX but also bind the BH3 binding groove of anti apoptotic BCL2 proteins, thereby causing release of the other activator BH3 only proteins as well as activated BAK or BAX. Surface plasma resonance assays measuring binding of anti apoptotic BCL2 proteins without TM domains to immobilized BH3 peptides from BH3 only proteins have shown that BH3 peptides are not all bound equally by anti apoptotic BCL2 family proteins. 58,121 Based on observed differences in Figure 3. Binding profiles BH3 only proteins to anti apoptotic BCL2 family proteins and BAX and BAK. The BH3 only proteins BIM, PUMA, truncated BID, NOXA, and BAD can either inhibit the anti apoptotic BCL2 family proteins (solid line), or directly activate BAK and BAX (dashed line). The thickness of each line reflects the reported strength of the interaction. BCL2: B-cell lymphoma-2; BH3: B-cell lymphoma-2 homology 3; BAX: B-cell lymphoma-2-associated X; BAK: BCL2 antagonist/killer; BCLXL: B-cell lymphoma-x large; MCL1: Myeloid cell leukemia 1 binding affinities, it was originally thought that BIM and BID could induce more apoptosis because of their ability to bind all anti apoptotic BCL2 family proteins. 57,58 The actual situation, however, is more nuanced than this [Figure 3]. As summarized in Table 1, the highest affinity interactions involve binding of BIM, PUMA, and BID BH3 domains to all of the anti apoptotic BCL2 family proteins; BAD and BMF to BCL2, BCLX L, and BCLW; NOXA to MCL1 and A1; and BIK and HRK to BCLX L, BCLW, and A1. 58,121 Nonetheless, it is important to emphasize that abundance of the anti apoptotic proteins also plays a role. Lower affinity interactions (e.g., with dissociation constants in the submicromolar range) can be detected, e.g., between NOXA and BCLX L or BCL2; these interactions can play a role in modulating drug sensitivity under conditions where large amounts of the less preferred anti apoptotic BCL2 family members are present. Mutations in the BCL2 gene can also affect these binding specificities. In particular, some of the mutant BCL2 proteins found in lymphoma cell lines display enhanced affinity for NOXA, 197 which contributes to bortezomib resistance of cells harboring these mutations. A systematic study of BCL2 mutations in follicular lymphoma also found that some mutant BCL2 proteins have increased affinities for BIM and PUMA, and the patients bearing these BCL2 mutations have increased risk of progression to more aggressive lymphoma and subsequent death. 198 Moreover, CDK1 mediated BCL2 phosphorylation also increases its affinity for BIM and BAK when the cells are treated with microtubule targeted chemotherapeutic agents. 61 Nonapoptotic functions of BH3 only proteins BH3 only proteins have also been reported to have nonapoptotic functions. 199 This can vary with the BH3 only protein and the cellular context. Several BH3 only proteins have been implicated in cell cycle regulation. BAD can regulate the cell cycle through its interactions with BCLX L and BCL2, which reportedly cause cell cycle arrest in S phase. 200 BIM is involved in G1/S cell cycle arrest, which is mediated by ERK phosphorylation when cells are deprived of adhesion mediated signaling and induced to undergo anoikis. 201 BAD has been reported to reside in a glucokinase containing complex where it regulates glucokinase activity, mitochondrial respiration, ATP production, and pancreatic beta cell survival Furthermore, BAD is phosphorylated by the inhibitor of kappa B kinase complex independent of NF kb activation and suppresses tumor necrosis factor α induced apoptosis. 205 BH3 only proteins can also regulate autophagy. BECLIN1 interacts with ATG14 L and VPS34, thereby inducing phagophore formation. 206 BECLIN1 and BECLIN2 also contain BH3 domains that can bind to anti apoptotic BCL2 family proteins Because BECLIN1 binds to anti apoptotic BCL2 family proteins with much less efficiency than other BH3 only proteins, 208,209 BAD and NOXA have been reported to induce autophagy by displacing BECLIN1 from BCL2 208 or MCL1, 210 respectively. However, it has been more recently suggested that the effect of anti apoptotic BCL2 family proteins on autophagy occurs downstream of the mitochondrial apoptotic pathway. 211 The BH3 only protein BID has been reported to be involved in the DNA damage response BID is phosphorylated by ataxia telangiectasia mutated following induction of DNA double strand breaks. However, BID( / ) murine cells undergo cell cycle arrest after DNA damage that is indistinguishable from the cell cycle 13

8 Cancer Transl Med 2016;2(1) response of wild type cells, arguing against a major role for Bid in the DNA damage response. 215 Although this inconsistency might reflect differences in the method of immortalizing the cell lines studied, 216 consistent evidence against a role for Bid in DNA damaged induced apoptosis was obtained from nine distinct cell types. 215 Finally, BIM and PUMA have been reported to play important roles in endoplasmic reticulum (ER) stress. ER stress caused by misfolded proteins induces BIM up regulation and phosphorylation, which are mediated by CHOP C/EBPα and are essential for ER stress induced apoptosis. 217 BIM and PUMA also regulate hyperglycemia induced ER and oxidative stress in type 2 diabetes. 218 Moreover, BIM and PUMA can interact with IRE1α and inhibit its effects on XBP 1 mrna splicing, which can in turn regulates ER stress regulated antibody secretion by primary B cells. 219 FUTURE PERSPECTIVES Since the discovery of BCL2 about 30 years ago, great progress has been made in understanding the structures, normal functions, and pathological roles of the BCL2 family proteins. Recent studies have provided the structures of symmetric dimers of activated BAK and BAX. Importantly, however, there are still a lot of unsolved questions regarding this family of proteins. The structure of higher order BAK and BAX oligomers is still unknown as is the mechanism by which these oligomers cause MOMP. Moreover, BCL2 family proteins have also been implicated in mitochondrial dynamics, cell cycle regulation, the ER stress response, autophagy, and DNA damage responses although some of these nonapoptotic functions need to be further clarified. Finally, while selective antagonists of BCL2, BCLX L, and MCL1 have been identified, only the selective BCL2 antagonist is progressing rapidly in the clinic. Further studies are needed to identify more drug like inhibitors of BCLX L and MCL1. Whether the recent structural information regarding BAX and BAK activation can also be utilized to develop new therapeutic agents, e.g. for degenerative diseases also remains to be seen. Financial support and sponsorship The study was supported by Hundred Talent Program of Chinese Academy of Sciences, and the National Natural Science Foundation of China (No ). Conflicts of interest There are no conflicts of interest. REFERENCES 1. Martinou JC, Youle RJ. 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