Acting on the BCL2 dependence of cancer cells






Olivier GENESTE, PhD
Pôle d’innovation Oncologie
Institut de Recherche Servier
FRANCE

Acting on the BCL2 dependence of cancer cells

by O. Geneste, France

Apoptosis is a form of programmed cell death that is essential for development and tissue homeostasis, but is almost systematically impaired in tumor cells, allowing their survival despite subjection to many apoptotic stimuli. Deregulation of the B-cell chronic lymphocytic leukemia/lymphoma 2 (BCL2)-family proteins, which represent a key point of control in apoptosis, clearly plays a major role in the aberrant survival of tumor cells. This protein family functions by engaging a network of interactions. Notably, the prosurvival BCL2 members prevent apoptosis by binding to and, in effect, sequestering the proapoptotic members of this family. Therefore, such interactions with the prosurvival BCL2 members in tumor cells are attractive targets for new cancer treatments. Despite the challenging nature of this class of target, structurally guided drug discovery efforts have seen the emergence of compounds that have very promising results in early clinical trials. This review focuses on these strategies to directly target members of the prosurvival BCL2 family, with particular emphasis placed on the yet “undrugged” member myeloid-cell leukemia sequence 1 (MCL-1).

Medicographia. 2014;36:319-325 (see French abstract on page 325)



Apoptosis represents one form of programmed cell death and is conserved throughout evolution. The extrinsic1 (death receptors) and intrinsic2 (mitochondrial) apoptotic pathways trigger a cascade of caspase activation, which is responsible for the execution of apoptotic cell death.3 The intrinsic pathway is characterized by mitochondrial outer membrane permeabilization (MOMP), which is regulated by the B-cell chronic lymphocytic leukemia (CLL)/lymphoma 2 family (BCL2 family) of proteins (Figure 1, page 320). Escape from apoptosis is a hallmark of cancer cells that contributes to tumor progression and to drug resistance. It is therefore believed that understanding how apoptosis is bypassed will enable the identification of highly valuable therapeutic targets for the treatment of cancer. Alterations in the activity of BCL2 family members are frequent in cancer cells. These alterations allow increased survival of cancer cells under various stressful conditions and cell death signals such as antitumor therapy, a tumor microenvironment, and oncogene signaling. Thus, acting on the BCL2 dependency of cancer cells appears to be a promising therapeutic approach to selectively kill cancer cells.

The BCL2 family network

The BCL2 family is divided into 3 functionally distinct groups (Figure 1): (i) The prosurvival proteins (BCL2, BCL2-like protein 1 [BCL-XL], myeloid-cell leukemia sequence 1 [MCL-1], BCL2-like protein 2 [BCL-W], and BCL2-related protein A1 [A1]).


Figure 1
Figure 1. Schematic representation of the BCL2 family of proteins.

Proteins of the BCL2 family can be divided into 3 major subgroups: the prosurvival BCL2-like
proteins, the proapoptotic multidomain BAX-like proteins, and the proapoptotic BH3-only proteins.
The prosurvival members are playing an essential role by sequestering the proapoptotic
members. The proapoptotic BAX-like proteins are necessary for the activation of the mitochondrial
apoptotic cell death pathway and the BH3-only proteins are crucial for the initiation of this
death pathway in response to various stressful conditions. Some of these BH3-only proteins
contain a transmembrane domain.
Abbreviations: BAD, BCL2-associated death promoter; BAK, BCL2 antagonist/killer; BAX,
BCL2-associated X protein; BCL2, B-cell chronic lymphocytic leukemia/lymphoma 2; A1, BCL2-
related protein A1; BCL-XL, BCL2-like protein 1; BCL-W, BCL2-like protein 2; BH, BCL2
homology (domain); BID, BH3-interacting-domain death agonist; BIK, BCL-2-interacting killer;
BIM, BCL2-interacting mediator of cell death; BMF, BCL2-modifying factor; BOK, BCL2-related
ovarian killer; HRK, harakiri; MCL-1, myeloid-cell leukemia sequence 1; NOXA, phorbol-12-
myristate-13-acetate–induced protein 1; PUMA, p53-upregulated modulator of apoptosis;
TM, transmembrane domain.



These proteins share 4 BCL2-homology domains (BH1-4) and preferentially localize at the mitochondrial outer membrane due to the presence of a hydrophobic domain at their carboxyl-terminal ends. (ii) The multidomain proapoptotic proteins BCL2- associated X protein (BAX), BCL2 antagonist/killer (BAK), and BCL2-related ovarian killer (BOK). These proteins have only 3 BH domains (BH1-3) even though their aminoterminal ends contain a helix resembling a BH4 domain. When synthesized, BAX and BAK are essentially inactive and upon activation, they acquire the ability to translocate to and insert into mitochondrial membranes, oligomerize, and trigger MOMP (Figure 2). The absence of both BAX and BAK renders cells resistant to MOMP, highlighting an essential role of these proteins in apoptosis. BOK has a more restricted expression pattern than BAX and BAK and its precise function is much less understood. For instance, it remains unclear whether BOK can functionally substitute for BAX or BAK.4 (iii) The proapoptotic BH3-only proteins. These proteins function upstream of BAX and BAK and are activated by various stress stimuli by transcriptional, translational, and posttranslational mechanisms. The proapoptotic activity of BH3-only proteins requires their interaction with the prosurvival proteins. BH3-only proteins show clear binding affinity differences for the various prosurvival proteins. BCL2- interacting mediator of cell death (BIM), p53-upregulated modulator of apoptosis (PUMA), and a truncated form of BH3-interacting-domain death agonist (tBID; formed by caspase-8–mediated cleavage) binds with high affinity to all prosurvival proteins, whereas BCL2-associated death promoter (BAD) binds to BCL2, BCL-XL, and BCL-W, and phorbol-12-myristate-13-acetate–induced protein1(NOXA) binds to MCL-1 and A1 specifically.5 Moreover, a subset of the BH3-only proteins (BIM, PUMA, and tBID), called activator BH3-only proteins, are able to bind and activate the multi– BH-domain proapoptotic BAX or BAK proteins.6-8 Precise structural changes occurring during BAX activation have recently been resolved.8





The prosurvival BCL2 members exert their function by making high-affinity interactions with proapoptotic members. These interactions rely on the binding of the BH3 domain (about 25 amino-acid–long α-helix structure) of proapoptotic members to a hydrophobic groove at the surface of the prosurvival proteins that is formed by their BH1, BH2, and BH3 domains.9 The activation of proapoptotic family members, such as BAX and BAK, leads to exposure of their BH3 domain, which increases their interaction with prosurvival BCL2 proteins.

In many cancers, the balance between the proapoptotic and antiapoptotic (ie, prosurvival) BCL2 members is tipped toward survival by genetic or epigenetic changes, signaling pathway alterations, and posttranslational modifications. For example, increased expression of BCL2, BCL-XL, and MCL-1 has been reported both in hematological malignancies and solid tumors.10-12 Numerous mechanistic studies support the notion that prosurvival BCL2 members maintain survival of cancer cells by sequestering either the activator BH3-only proteins (such as BIM) or BAX and BAK themselves. Therefore, the discovery and development of small-molecule inhibitors targeting the BH3-binding groove of prosurvival BCL2 members, thereby releasing proapoptotic BH3-containing proteins, appears to be a highly valuable approach to flip an apoptotic switch in cancer cells.


Figure 2
Figure 2. The mitochondrial apoptotic pathway is triggered in different
ways by different BH3-only proteins.

BH3-only proteins bind to prosurvival BCL2 family members with different specificities.
While BIM, PUMA, and tBID bind with strong affinity to all prosurvival
BCL2 members, BAD binds specifically to BCL2, BCL-XL, and BCL-W, and
NOXA binds specifically to MCL-1 and A1. In addition, some BH3-only proteins
(BIM, tBID, and PUMA) can directly activate the BAX-like proteins.
Abbreviations: BAD, BCL2-associated death promoter; BAX, BCL2-associated
X protein; BAK, BCL2 antagonist/killer; BCL2, B-cell chronic lymphocytic leukemia/
lymphoma 2; A1, BCL2-related protein A1; BCL-XL, BCL2-like protein 1; BCL-W,
BCL2-like protein 2; BH, BCL2 homology (domain); BIM, BCL2-interacting mediator
of cell death; MCL-1, myeloid-cell leukemia sequence 1; PUMA, p53-
upregulated modulator of apoptosis; tBID, truncated BH3-interacting-domain
death agonist; MOMP, mitochondrial outer membrane permeabilization; NOXA,
phorbol-12-myristate-13-acetate–induced protein 1.


Targeting prosurvival BCL2 family members

Binding of the BH3 domains to the prosurvival BCL2 family members involves 4 conserved hydrophobic residues on one face of the BH3 α-helix. These residues form electrostatic interactions with 4 hydrophobic pockets (P1-P4) in the binding grooves of the prosurvival proteins. A number of either crystal or solution structures of prosurvival BCL2 family members bound to various BH3 peptides have been solved (Protein Data Bank entries 1PQ1, 1PQ0, 2NL9, 1WSX, and 1BXL).13-16 The promiscuous binding of the BH3-only proteins BIM, PUMA, and BID to all prosurvival BCL2 proteins can be explained by the fact that the BH3-binding grooves of these proteins share many features due to sequence similarities in their BH1, BH2, and BH3 domains and by mutual conformational adjustments of both partners upon binding. However, the selectivity of some of these interactions relies on subtle differences in a few key residues within both the BH3 domain of the proapoptotic proteins and the BH3-binding grooves of the prosurvival proteins. Structural information about BH3- mediated interactions has provided the necessary insights to rationally design inhibitors of the BCL2 prosurvival proteins. How structural data concerning BH3-domain binding modes and selectivity have been used to discover prosurvival–BCL2- protein inhibitors, so called BH3 mimetics, is well described in reference 17.

What we have learned from currently available prosurvival–BCL2-protein inhibitors
Targeting the BCL2 family members directly with small molecules is a highly challenging task for several reasons. First of all, these intracellular protein-protein interactions are of very high affinity (subnanomolar range for some of them), and the BH3 binding site on the prosurvival BCL2 proteins is rather shallow, flexible, and does not form an easily tractable pocket. It is therefore a major challenge to discover small-molecule inhibitors with suitable drug-like properties for clinical development.

A number of small-molecule inhibitors of prosurvival BCL2 proteins have been characterized (Table I), but most of them bind their target with too poor affinity to be able to functionally inhibit BCL2 proteins in cells and actually trigger cell death independently of the mitochondrial apoptotic pathway, which strongly suggests that most of their biological activity is not due to their inhibition of prosurvival BCL2 proteins. From published work, 4 compounds with sufficient affinity for their target appear to convincingly trigger the mitochondrial apoptotic pathway through inhibition of prosurvival BCL2 proteins. These compounds are ABT-737,18 which binds to BCL2, BCLXL, and BCL-W; ABT-26319 (called navitoclax), an orally active clinical derivative of ABT-737; ABT-199,20 which selectively binds BCL2; and WEHI-539, which selectively binds BCL-XL.21


Table I
Table I. Inhibitors of prosurvival BCL2 proteins.

Abbreviations: BCL2, B-cell chronic lymphocytic leukemia/lymphoma 2; A1,
BCL2-related protein A1; BCL-W, BCL2-like protein 2; BCL-XL, BCL2-like protein
1; MCL-1, myeloid-cell leukemia sequence 1.



ABT-737 exhibits single-agent activity, especially on blood cancer models such as B-cell lymphomas, CLLs, acute myelocytic leukemias (AMLs), acute lymphoblastic leukemias, a subgroup of multiple myelomas, and some small cell lung carcinomas.18 In lymphoid malignancies, the crucial target of ABT- 737 has been shown to be the BCL2-BIM complexes.22,23 The single-agent activity of ABT-737 may be the consequence of constitutive death signals that are induced by some oncogenes. For example, deregulated MYC oncogene can trigger proapoptotic signals and favors MOMP.24 Thus, cancer cells with such MYC alterations require a compensatory survival signal that can be provided by overexpression of the prosurvival BCL2 family members. In agreement with this notion are the facts that some highly aggressive lymphomas harbor both a MYC (t8;14) and a BCL2 (t14;18) translocation,25 and two thirds of cancers with MCL1 or BCL2L1 (coding for BCL-XL) amplifications also have amplifications in the chromosomal region carrying MYC.12

There is rationale for combining BH3 mimetics with cytotoxic drugs (eg, genotoxic drugs, which can increase PUMA and NOXA expression; histone deacetylase inhibitors, which can increase BIM expression), but there is also a risk that such a combination would increase the toxicity of the chemotherapy for the healthy tissues. More promising combinations would be to use BH3-mimetic drugs with targeted therapies such as inhibitors of oncogenic kinases, which have been shown to induce BIM expression (or other BH3-only proteins). Such combinations should be specifically synergistic in cancer cells versus healthy cells, as only the cancer cells have deregulated kinase activity due to oncogenic mutations.2627 This suggests that inhibitors specifically targeting BCL-XL, such as WEHI-539 (or derivative) could be of particular interest in the context of RAS-mutant tumors. More generally, it is believed that in many solid tumors, BCL-XL plays a more important prosurvival role than BCL2.

In the clinic, early trials of ABT-263 have focused on lymphoid malignancies and small cell lung cancer. A clinical trial focusing on CLL patients showed promising results for ABT-263 used as a single agent, where a partial response was observed in 31% (9/29) of patients.28 Results obtained with ABT-263 in small cell lung cancer patients were disappointing, with only 1 partial response out of 39 patients.29 The dose-limiting toxicity of ABT-263 is a transient acute thrombocytopenia resulting from the inhibition of BCL-XL, which controls platelet lifespan.30 This finding led to the discovery and development of ABT-199, which is specific for BCL2.20 A recent phase 1 trial of ABT-199 showed highly promising results in CLL patients (55 evaluable patients) with 84% overall response rate, 65% with a partial response, and 18% with a complete response. The response in CLL patients was independent of the 17pdeletion (missing short arm of chromosome 17) high-risk marker. Preliminary results of the ABT-199 phase 1 trial in non- Hodgkin lymphoma (especially mantle-cell lymphoma) patients were also very encouraging.31 As expected from its binding selectivity for BCL2, ABT-199 did not show platelet toxicity; instead, tumor lysis syndrome (reflecting rapid apoptosis of tumor cells) was a dose-limiting toxicity.

Targeting the yet “undrugged” prosurvival protein MCL-1
MCL-1 is overexpressed in multiple types of cancer and participates in tumor development and resistance to anticancer therapies. Moreover, a recent study reported that the MCL-1 gene is one of the most frequently amplified genes in a large number of tumor types in human and that MCL-1 is required for survival of breast cancer and non-small cell lung carcinoma (NSCLC) cell lines.12 Therefore, MCL-1 is recognized as an important potential therapeutic target in cancer.32 So far, no compound that directly targets MCL-1 convincingly has entered clinical development (ie, MCL-1 is as yet “undrugged”), and MCL-1 has clearly been shown to be a resistance mechanism to the compound ABT-737, which targets BCL2/BCLXL/ BCL-W,33 and to other anticancer drugs.34 Studies using either small interfering RNA (siRNA) or indirect approaches to down regulate protein level have shown that MCL-1 is a major survival factor in tumor cells.

ML-1 has specific features compared with other prosurvival BCL2 members regarding the regulation of its expression. Indeed, MCL-1 expression is remarkably tightly controlled by multiple mechanisms. These include transcriptional, posttranscriptional, and posttranslational levels of regulation (Figure 3). At the transcriptional level, MCL-1 is regulated by various transcription factors, including signal transducer and activator of transcription (STAT) 5, E2F transcription factor 1 (E2F1), STAT3, PU.1, hypoxia-inducible factor-1 (HIF-1), and ternary complex factor (TCF)–serum response factor (SRF), which are themselves downstream targets of key oncogenic pathways.35 At the posttranscriptional level, MCL-1 messenger RNA (mRNA) is subjected to regulation by microRNAs (miRNAs), most notably by miR-29b. Loss of miR-29b has been shown to be a mechanism involved in MCL-1 overexpression, especially in AML, and restoration of miR-29b in AML cells induces apoptosis and reduces tumorigenicity.11 On the other hand, the phosphatidylinositol 3-kinase (PI3K)/Akt (also known as protein kinase B)/ mammalian target of rapamycin (mTOR) complex 1 (mTORC1) pathway promotes tumor-cell survival in a mouse model by stimulating the translation of Mcl-1.36 At the posttranslational level, MCL-1, in contrast to other prosurvival BCL2 family members, is a very short-lived protein with a halflife of 20 minutes to a few hours depending on cell type.37 MCL-1 protein stability is controlled by the ubiquitin/proteasome pathway and 3 distinct E3 ubiquitin ligases (MCL-1 ubiquitin ligase [MULE], β-transducin repeat-containing protein [β-TrCP], and F-box and WD repeat domain containing 7 [FBW7]) have been shown to contribute to MCL-1 ubiquitination, leading to its degradation.38-40 Conversely, MCL-1 protein can be stabilized by the X-linked ubiquitin-specific peptidase 9 (USP9X), which catalyzes the removal of ubiquitin chains from MCL-1.41 FBW7 and USP9X are regulators of MCL-1 protein level of particular interest in the context of cancer. FBW7 lossof- function mutations are common in multiple cancer types and promote tumor-cell survival by increasing MCL-1 protein levels.40 On the other hand, USP9X overexpression in human lymphoma samples correlates with high MCL-1 protein levels and could participate in increased tumor-cell survival.41 In addition to being regulated by ubiquitination, MCL-1 protein is also subjected to phosphorylation. Phosphorylation of MCL-1 on threonine residue (Thr) 163 by extracellular signal–regulated kinase (ERK) increases its half-life,42 whereas phosphorylation of serine residue (Ser) 159 by glycogen synthase kinase 3 (GSK3) promotes its ubiquitination and degradation.43 Proteasomal degradation of MCL-1 has also been shown to be enhanced by phosphorylation of Thr92 by the cyclin-dependent kinase 1 (CDK1)/cyclin B1 complex during mitosis.44


Figure 3
Figure 3. MCL-1 expression and stability are controlled by multiple mechanisms.

MCL1 gene transcription is regulated by multiple transcription factors (such as STAT5, HIF-1, and
TCF-SRF) that are downstream targets of key oncogenic pathways. MCL1 messenger RNA is
regulated by the microRNA miR-29b. The PI3K-AKT-mTORC1 pathway stimulates MCL1 translation.
Stability of the MCL-1 protein is controlled by the ubiquitin-proteasome pathway and 3 E3
ubiquitin ligases (MULE, β-TrCP, and FBXW7) have been shown to contribute to MCL-1 ubiquitination
and subsequent degradation. On the contrary, USP9X stabilizes MCL-1 by catalyzing its
deubiquitination. Phosphorylation of MCL-1 on threonine residue (Thr) 163 by ERK increases the
half-life of MCL-1, whereas the phosphorylation of serine residue (Ser) 159 by GSK3 promotes
MCL-1 ubiquitination and degradation. Proteasomal degradation of MCL-1 has also been shown
to be increased by the phosphorylation of Thr92 by the cyclin B1–cyclin-dependent kinase 1 (CDK1)
complex during mitosis.
Abbreviations: β-TrCP, β-transducin repeat-containing protein; AKT, AKT or protein kinase B;
CDK, cyclin-dependent kinase; E2F1, E2F transcription factor 1; ERK, extracellular signal-regulated
kinase; FBW7, F-box and WD repeat domain containing 7; GSK3, glycogen synthase kinase 3;
HIF-1, hypoxia-inducible factor-1; MCL-1, myeloid-cell leukemia sequence 1; miR-29b, microRNA
29b; MULE, MCL-1 ubiquitin ligase; mTORC1, mammalian target of rapamycin (mTOR) complex 1;
PI3K, phosphatidylinositol 3-kinase; PU.1, Ezb transformation-specific sequence (ETS)-family transcription
factor PU.1; STAT, signal transducer and activator of transcription; TCF-SRF, ternary complex
factor (TCF)–serum response factor (SRF); USP9X, ubiquitin-specific peptidase 9, X-linked.



Altogether, the multiple mechanisms controlling MCL-1 expression and stability could lead to various potential approaches to down regulate MCL-1 for anticancer therapy. A number of compounds globally inhibiting either transcription (eg, CDK9 inhibitors) or translation have been shown to exert their cytotoxic effects by down regulating MCL-1.45 Indeed, because MCL-1 is a very short-lived protein, tumor cells that are dependent on MCL-1 for survival die rapidly in response to global transcription or translation inhibitors. In theory, this finding points to several possible opportunities to indirectly target MCL-1, but these approaches present several issues. The lack of specificity for compounds such as global transcription blockers is almost certain to cause significant toxicity and an acceptable therapeutic window for such treatment might be difficult to achieve. Another important limitation for compounds that down regulate MCL-1 by globally inhibiting transcription (or translation) regards their use in combination with other anticancer drugs. For instance, transcription or translation blockers have, in fact, been shown to counteract the effects of anticancer drugs such as the proteasome inhibitor bortezomib or the histone deacetylase inhibitor vorinostat, which need to induce expression of proapoptotic proteins such as NOXA, BIM, or BCL2-modifying factor (BMF) to exert their cytotoxicity.45 Rather than blocking its synthesis, accelerating degradation of the MCL-1 protein could also be an interesting approach to target MCL-1. In that respect, USP9X, which has been shown to stabilize MCL-1 by catalyzing its deubiquitination is an attractive target.41 Targeting USP9X could be a more specific approach than the ones discussed above, but one open question is whether accelerating MCL-1 degradation will lead to sufficient MCL-1 down regulation to induce apoptosis. So, even if, due to its complex regulation, MCL-1 can be targeted by multiple mechanisms, the best strategy to target MCL-1 is most probably the direct inhibition by BH3-mimetic drugs, which is more specific and more likely to induce a quicker apoptotic response in tumor cells. From work published so far, compounds with only moderate affinity for MCL-1 (<100nM) have been described.46

Conditional knockout experiments in mice have shown that Mcl-1 is an essential survival protein in hematopoietic stem cells47 and cardiomyocytes.48,49 Therefore, the question of therapeutic window is of particular importance when targeting MCL-1. This point was nicely addressed in a recent work using models of AML in mice where the Mcl-1 gene can be selectively targeted in an inducible fashion.50 Importantly, this study showed that leukemic cells were significantly more sensitive to Mcl-1 loss than normal hematopoietic stem cells and progenitor cells. The difference in sensitivity to Mcl-1 depletion between AML cells and normal hematopoietic cells was measurable in terms of percentage of remaining viable cells, but perhaps more clearly in terms of kinetics of cell death. These data suggest that there should be a therapeutic margin with anti–MCL-1 drugs for AML treatment. In addition, it is important to realize that the irreversible loss of MCL-1, such as that resulting from gene-knockout experiments, is not equivalent to its pharmacological inhibition in a timely fashion. While long-term inhibition of MCL-1 may not be tolerated, there should be a therapeutic window due to a higher level of MCL-1 dependency in tumor cells versus normal cells resulting in a more rapid death of tumor cells. So, in the clinic, a therapeutic window could be achievable using an intermittent schedule combined with strategies to increase the level of MCL-1 dependency in tumor cells. Such a strategy could be combinations with treatment that prime tumor cells to MCL-1–dependent cell death, especially in terms of kinetics of cell death. One complicating factor for the BH3-mimetic approach is the high MCL-1 turnover, meaning that a stock of intact MCL-1 protein will be rapidly renewed once the inhibitory drug has disappeared from the tumor cells. So, the therapeutic window is likely to result from a “fine tuning” between drug pharmacokinetics, schedule of administration, kinetics of tumor cell death, and MCL-1 turnover, which could turn out to be challenging for drug discovery and clinical development. Nevertheless, the BH3-mimetic approach, because of its specificity and rapid mechanism of action, probably represents the best opportunity to target MCL-1 for therapeutic benefit.

Conclusions

Acting on the BCL2 dependency of tumor cells is a highly innovative and attractive mode of intervention for cancer treatment. No drugs directly targeting prosurvival BCL2 family members have yet been approved, but the very promising results obtained in early clinical trials, especially for the BCL2- specific inhibitor ABT-199, strongly indicate that such BH3- mimetic compounds will soon be part of anticancer therapy. However, for such compounds to become efficient therapeutic agents, it will be necessary to better understand the BCL2- like dependency of tumor cells at the molecular and cellular levels so that relevant diagnostic biomarkers could be identified. The discovery of BH3-mimetic compounds specifically targeting each prosurvival BCL2 family member will clearly be helpful to characterize this phenotype of BCL2-like dependency and therefore to design better treatments.■

Acknowledgments: I wish to thank all members of my group and colleagues from The Oncology Discovery Research Unit at Institut de Recherches Servier for their work and intellectual input. I also wish to thank Nathalie Noboa for her excellent assistance and for her help in typing this manuscript.






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Keywords: apoptosis; BCL2 family; cancer; protein-protein interactions