The role of Bcl-2 and its pro-survival relatives in tumorigenesis and cancer therapy

Introduction

Nearly 40 years ago Kerr, Wyllie and Currie first proposed that cellular hyperplasia, which often constitutes a forerunner of a malignant tumour, could result from abnormally decreased cell death and not only from abnormally increased cell proliferation.¹ The demonstration of cell death in normal adult tissues confirmed the hypothesis that cells must continuously be lost to balance cell proliferation for maintaining homeostasis in healthy tissues.¹ Programmed cell death (apoptosis) is now widely recognised as an evolutionarily conserved, genetically controlled process for killing damaged, infected, superfluous or potentially dangerous cells that is essential for the normal development and function of multi-cellular organisms (reviewed in²). Defects in the control of apoptosis causing either the survival of unwanted cells or inappropriate killing of vital cells underlie a multitude of disorders, including autoimmunity, degenerative diseases and cancers (reviewed in³). Indeed, defects in apoptosis are now considered to be a hallmark of most, if not all, cancers.⁴ Members of the Bcl-2 protein family are critical regulators of apoptosis and include three sub-groups of proteins that either promote cell survival (e.g. Bcl-2 and Bcl-x_L), initiate cell killing (e.g. Bim, Puma or Bid) or activate the effector pathways of apoptosis (Bax, Bak) (Figure 1). Observations in human tumours and studies with genetically modified (transgenic or knock-out) mice have shown that tumorigenesis can be driven by gain-of-function mutations in cell death antagonists (e.g. Bcl-2 over-expression) or loss-of-function mutations in cell death activators (e.g. loss of Bim). These mutations can serve as either initiating (primary) or secondary (propagating) oncogenic events to promote tumour development and progression to metastatic disease. Unfortunately but not surprisingly, mutations that deregulate apoptotic cell death also render tumour cells refractory to cancer therapeutics. This review provides an overview of the pro-survival subgroup of the Bcl-2 family of proteins and their functions in cell death control, particularly in the context of tumorigenesis and responses of tumour cells to anti-cancer therapy. The functions of the two pro-apoptotic Bcl-2 sub-groups in normal physiology, tumorigenesis and cancer therapy are also discussed.

Figure 1. The mammalian Bcl-2 protein family.

Bcl-2 family members share regions of homology called Bcl-2 homology (BH) domains, and may contain a trans-membrane (TM) domain that mediates insertion into the outer membrane of the mitochondria and to the endoplasmic reticulum. The pro-survival family members, including Bcl-2, Bcl-x_L, Bcl-w, Mcl-1 and A1, share four BH (Bcl-2 Homology) domains plus a transmembrane (TM) region. The pro-apoptotic family members can be sub-divided into two sub-groups: the multi-BH domain proteins, and the BH3-only proteins. Multi-BH domain proteins contain up to four BH domains (and some also have a TM region) and include Bax, Bak, Bok, Bcl-x_S, Bcl-g_L and Bfk. The BH3-only proteins include Bad, Bik, Bid, Hrk, Bim, Puma, Noxa and Bmf, and are so-called because they contain only the BH3-domain. Some also contain a TM region.

Bcl-2: discovery of the first cell death regulator and its role in tumorigenesis

The first insights into the role of apoptosis in tumour development came from cytogenetic analysis of human B cell lymphomas. A strong correlation was observed between the t[14;18] chromosomal translocation and human follicular centre B cell lymphoma.⁵ This rearrangement places a then novel gene, bcl-2 (B cell lymphoma-2), under the control of the immunoglobulin heavy chain (IgH) gene enhancer Eµ. Subsequent functional studies with cytokine-dependent cell lines have shown that enforced expression of Bcl-2 inhibits growth factor-deprivation induced death of these cells but does not enhance their proliferation.⁶ These results demonstrated that the molecular mechanisms that control cell survival and cell proliferation in response to cytokine stimulation must be distinct and demonstrated for the first time that defects in the control of cell death can cause cancer.

The in vivo function of Bcl-2 was first probed by the generation of transgenic mice that were engineered to express bcl-2 under control of the IgH gene enhancer Eµ, thereby mimicking the t[14;18] chromosomal translocation characteristic of human follicular center B lymphoma. Such Eµ-bcl-2 transgenic animals were found to harbor abnormally increased numbers (3- to 5-fold) of B lymphoid cells, a large (30- to 200-fold) excess of antibody forming cells and increased serum Ig levels.^7–9 These immune system abnormalities progressed with relatively high incidence (at least on certain genetic backgrounds, such as mixed C57BL/6×SJL) to fatal systemic lupus erythematosus (SLE)-like autoimmune nephritis,⁹ demonstrating for the first time that apoptosis of lymphocytes imposes a critical barrier against autoimmune disease.

However, despite the marked B lymphoid survival advantage conferred by Bcl-2 overexpression, Eµ-bcl-2 transgenic mice displayed only a low incidence of lymphoma at ~5% within the first year of life.^10–12 This long latency period indicated that the stochastic acquisition of additional oncogenic mutations was required to promote the transition from low-grade polyclonal hyperplasia to monoclonal high-grade malignancy. Indeed, over half of the plasmacytomas and lymphomas from the Eµ-bcl-2 transgenic mice were found to harbour translocations of the c-myc proto-oncogene into the IgH gene locus.^{11, 12}

The transcriptional regulator c-Myc controls a diverse array of target genes that regulate cell cycle progression, cell volume growth, inhibition of terminal differentiation and, under conditions where survival signals are limiting, apoptosis (reviewed in¹³). Importantly, abnormally high levels of Myc have been observed in ~70% of all human cancers (reviewed in¹³). When transgenic mice were engineered to express both myc and bcl-2 transgenes (Eµ-myc/Eµ-bcl-2 doubly transgenic mice), this provided the first formal demonstration that deregulated cell proliferation and impaired cell death were potently synergistic in tumorigenesis. Indeed, Eµ-myc/Eµ-bcl-2 bi-transgenic mice displayed marked acceleration in B cell lymphoma development, and by seven weeks of age, 100% of these animals had succumbed to lymphoma compared to only ~40% of the Eµ-myc single transgenic mice.¹⁰ In the context of human lymphomagenesis, a similar synergy is seen in human bcl-2/IgH follicular lymphoma where progression to a more aggressive state is in some cases associated with the additional acquisition of a c-myc/IgH gene chromosomal translocation.¹⁴

The functions of pro-survival Bcl-2 family proteins

The discovery of Bcl-2 established a new paradigm in cancer biology, namely that defects in apoptosis bestow cells with a selective survival advantage, that when combined with mutations that facilitate unrestrained proliferation (e.g. deregulated c-myc expression), can promote malignant transformation. This prompted intense research in this area and it soon became clear that Bcl-2 is a member of a substantial family of apoptosis regulatory proteins, which contains a sub-group that inhibit cell death and two that promote cell killing. So far, five pro-survival members have been identified in mammals: Bcl-2, Bcl-x_L, Mcl-1, Bcl-w and A1/Bfl-1 (Figure 1). These proteins share in common four Bcl-2 homology domains (BH1–BH4) and very similar 3D structure (reviewed in¹⁵). Overexpression of any one of these proteins is sufficient to protect cells from the effects of a broad range of apoptotic stimuli in culture and even within the whole animal (reviewed in¹⁵). Conversely, loss of Bcl-2-like pro-survival proteins has profound consequences on normal tissue homeostasis and development (reviewed in¹⁵). For example, Bcl-2 was found to be critical for the survival of renal epithelial stem cells during embryogenesis, melanocyte progenitors and mature B and T lymphocytes.^{16, 17} Bcl-x_L on the other hand is critical for survival of erythroid progenitors and neuronal cells during embryogenesis,¹⁸ whereas Bcl-w appears to have a selective role in spermatogenesis.¹⁹ Studies with complete and tissue restricted “knock-out” mice have shown that Mcl-1 is essential for implantation during early development,²⁰ survival of hematopoietic stem cells, committed lymphoid progenitors, mature B and T lymphocytes, activated germinal centre B cells and several other cell types.^{21, 22} This broad array of defects caused by loss of Mcl-1, its somewhat divergent structure compared to its pro-survival relatives and its rapid turnover (~30 min compared to ~24 h for Bcl-2) indicate that Mcl-1 may have a special role in the control of cell survival. The overall function of A1 in cell survival has not yet been determined because there are four a1 genes in mice that although closely located, are interspersed with other genes, making it challenging to generate mice deficient for all A1 proteins. However, loss of one A1 protein, A1a, was shown to accelerate apoptosis of granulocytes and allergen activated mast cells.²³

The role of pro-survival Bcl-2 family proteins in tumorigenesis

With respect to tumorigenesis, deregulated expression of Bcl-2 and related pro-survival proteins has been found to be a feature of many human cancers, and there is substantial evidence that deregulated expression of Bcl-2-like proteins as either a primary or secondary oncogenic event is critical in tumour development, maintenance and therapeutic resistance (reviewed in²⁴).

Bcl-x_L

Human multiple myelomas have been found to express high levels of Bcl-x_L²⁵ (and also Mcl-1 ²⁶; see below), but comparatively low levels of Bcl-2. Bcl-x_L has been proposed to promote the survival of follicular and germinal centre B cells undergoing Ig class switch recombination and somatic hypermutation of the variable regions of their rearranged Ig genes.²⁷ These cells are the likely targets for impaired DNA double-strand break repair that may generate chromosomal translocations, such as those involving the myc oncogene. Deregulated Myc expression is a consistent feature of plasma cell neoplasms in humans (multiple myeloma).²⁸ Accordingly, enforced expression of both Myc and Bcl-x_L under control of the immunoglobulin heavy chain gene enhancer (Eµ) leads to development of plasmacytoma with considerably higher incidence and more rapid onset compared to mice expressing either Myc or Bcl-x_L transgenes alone.²⁸ Bcl-x_L has also been implicated in the development and therapeutic resistance of Bcr/Abl⁺ chronic myelogenous leukaemia.²⁹ A broad range of signalling pathways are activated by the Bcr/Abl oncogenic tyrosine kinase, and the STAT5 transcription factor, an activator of bcl-x transcription, is known to play a major role in cellular transformation.²⁹ Pertinently, somatically acquired copy number increases in bcl-x have been found in a range of human cancers, including certain lung cancers and giant-cell tumours of the bone.³⁰

Mcl-1

Mcl-1 (myeloid cell leukaemia 1) was identified as an early response gene induced during the differentiation of ML-1 human myeloblastic leukaemia cells³¹ and has since been shown to be expressed at relatively high levels in a broad range of both haematological as well as solid malignancies, including multiple myeloma, acute myeloid leukaemia (AML) and cholangiocarcinomas.^32,33 Transgenic mice expressing Mcl-1 under the control of the endogenous mcl-1 promoter display enhanced survival of B and T cells, and develop myeloid malignancy, albeit with low incidence and long latency.³⁴ Expression of mcl-1 under control of the pan-hematopoietic vav promoter elicited transformation of immature hematopoietic stem/progenitor and pre-B/B lymphoid cells.³⁵ Moreover, enforced expression of Mcl-1 was shown to inhibit Myc-induced apoptosis and therefore synergised with an Eµ-myc transgene in lymphomagenesis.³⁵ A study of 151 clinical isolates of non-Hodgkin’s B lymphoma found that high levels of Mcl-1 expression correlated with increasing grade of severity in follicular lymphoma.³⁶ Interestingly, the BH3 mimetic ABT-737 which binds and inhibits Bcl-2, Bcl-x_L and Bcl-w, but not Mcl-1 or A1 (see also below)³⁷ was unable to inhibit proliferation of B cell lymphomas or acute myeloid leukaemias expressing high levels of Mcl-1 as a single agent, but was highly efficient when Mcl-1 was inactivated, for example by RNAi-mediated knock-down.^{38, 39} Recently, somatically acquired copy number increases in mcl-1 have been found in certain lung and breast cancers and, importantly, studies using RNA interference have shown that in cell lines derived from some of these tumours, Mcl-1 is critical for sustained survival and growth, at least in vitro.³⁰ Collectively, these results provide evidence that for many malignant cell types Mcl-1 may be critical for sustained survival and expansion and appears to impose an important obstacle against anti-cancer therapy.

A1/Bfl-1

A1 (the murine homologue of human Bfl-1) is expressed in a broad range of haematopoietic cell populations, including B and T lymphocytes, macrophages, neutrophils, mast cells and dendritic cells.⁴⁰ In many (possibly all) of these cells, A1 expression is rapidly induced by activation of antigen or cytokine receptors (e.g. BCR, TCR, FcεR) but A1 levels also rapidly decline because of its rapid turnover.²³ Molecular profiling of B cell lymphomas has indicated that A1 overexpression may be a signature of certain B lymphoid malignancies⁴¹ and may therefore constitute a target for the design of novel anti-cancer therapeutics. Indeed, down-regulation of A1 expression using short hairpin RNAs (shRNA) was found to increase the sensitivity of B lymphoblastic cells and diffuse large B cell lymphoma lines to anti-CD20 (Rituximab)-mediated killing and conventional chemotherapeutic agents, including doxorubicin, cisplatin and vincristine.⁴² A1 has also been implicated in the development of Bcr/Abl+ CML, as A1 upregulation was reported to be essential to promote cell cycle transition, IL-3 independent growth and in vivo leukaemic transformation.⁴³

Bcl-w

Although high levels of Bcl-w expression have not to date been reported for haematological malignancies, Bcl-w overexpression has been found to promote survival and influence the migratory and invasive potential of gastric cancer cells.⁴⁴ This study also found that Bcl-w, but not Bcl-2 overexpression, correlated with increased levels of matrix metalloproteinase-2, an enzyme which catalyses the destruction of the basement membrane to facilitate metastasis. Moreover, Bcl-w was also reported to be frequently expressed at relatively high levels in colorectal adenocarcinomas (69/75 tumours) with higher Bcl-w levels detected in advanced stage cancers as opposed to localised tumours with better prognosis.⁴⁵

Which endogenous Bcl-2-like pro-survival protein(s) is/are required for the development and sustained growth of tumour cells?

Only a few cancers (e.g. human follicular centre B lymphoma) have mutations that activate pro-survivalBcl-2 family members. It therefore appears likely that endogenously expressed pro-survival Bcl-2 family members are critical to keep cells alive during the process of neoplastic transformation and may also be essential to sustain the survival and growth of malignant cancers. Knowing which pro-survival Bcl-2-like protein(s) is/are critical in which type of tumour will undoubtedly provide vital clues for the development novel cancer therapies. Initial insight was provided from studies with mice harbouring a doxycyline-inducible bcl-2 transgene and a constitutive c-myc transgene. When mice were maintained on doxycycline-supplemented drinking water, their B lymphoid cells overexpressed both Myc and Bcl-2, leading to the rapid emergence of pre-B/B cell lymphoma. Remarkably, shutdown of the inducible bcl-2 transgene (by termination of doxycycline administration) in lymphoma-burdened bi-transgenic mice resulted in tumour regression and significantly prolonged animal survival in many (although not all) cases.⁴⁶ These findings demonstrate that sustained Bcl-2 overexpression is required for the continued growth of lymphomas that were elicited (in part) by Bcl-2 overexpression. But what about tumours that were not elicited by enforced overexpression of Bcl-2 or one of its pro-survival relatives? In this regard, our group recently carried out the first study to address the role of endogenous Bcl-2 in tumorigenesis. Since Bcl-2 is expressed in many B lymphoid cell subsets, including early progenitors⁴⁷ and since Bcl-2 overexpression enhances survival of B lymphocytes at all stages of development,^{7, 9, 48} we hypothesised that endogenous Bcl-2 may be critical for Eµ-myc-induced lymphomagenesis.

Although Bcl-2-deficient mice complete embryonic development, they succumb to polycystic kidney disease between 3–5 weeks of age^{16, 17} prohibiting long-term studies on lymphoma development in bcl-2^−/− mice expressing an Eµ-myc transgene. We overcame this impediment by reconstituting lethally irradiated wild-type mice with an Eµ-myc/bcl-2^−/− or, as controls, an Eµ-myc/bcl-2^+/+ (referred hereafter as Eµ-myc) haematopoietic system. Analysis of the sequential stages of B cell development in pre-leukemic Eµ-myc/bcl-2^−/−; mice revealed that endogenous Bcl-2 was largely dispensable in pro-B, pre-B and immature (sIgM^hisIgD^lo) B cells, but was critical for the survival of mature B cells, as the Eµ-myc/bcl-2^−/− reconstituted mice were almost completely devoid of sIgM^losIgD^hi B lymphocytes in their spleen and lymph nodes.⁴⁹ Surprisingly, despite the abnormal B cell deficit, the absence of bcl-2 did not reduce the incidence or delay the onset of Eµ-myc lymphoma (Figure 2).⁴⁹ These findings indicate that during the genesis of Myc-driven lymphoma, the acquisition of oncogenic lesions that propagate neoplastic transformation must occur at a stage at which Bcl-2 is dispensable for survival. The pro-B and/or pre-B cells (or perhaps earlier progenitors), but not the mature B lymphocytes, are likely candidates because Bcl-2 is not critical for their survival. Pertinently, pro-B and pre-B cells are subject to genomic instability due to IgH and IgL gene rearrangement, which are error prone processes that can cause oncogenic lesions. Since impaired apoptosis is thought to be an essential step in tumour development,⁴ these results also raise the possibility that an pro-survival protein(s) other than Bcl-2 is/are critical to sustain cell survival during the course of Myc-induced lymphomagenesis (Figure 3). Bcl-x_L and Mcl-1 represent attractive candidates because they are expressed at several stages of B lymphopoiesis (Figure 4)^{21, 27} and both have been shown to be critical for the survival of B lymphoid progenitors and/or precursors.^{18, 21} Moreover, as mentioned above, overexpression of Bcl-x_L or Mcl-1 can cause lymphoid malignancies^{35, 50} and synergises with Myc in lymphomagenesis.^{28, 35}

Figure 2. Loss of endogenous Bcl-2 does not prevent or delay Eµ-myc-induced B lymphoma development.

Kaplan-Meier analysis of tumour latency in lethally irradiated wt (Ly5.1⁺) mice reconstituted with foetal liver cells from Ly5.2⁺ Eµ-myc (closed square), Eµ-myc/bcl-2^+/− (closed diamond) or Eµ-myc/bcl-2^−/− (open square) (E14.5) embryos. Mice were sacrificed when deemed moribund by an animal technician who was blinded to the genotype of the mice. This figure was first published in PN Kelly et al, Blood 2007 and is reproduced here with the permission of the journal.

Figure 3. Proposed model for apoptosis induced by deregulated Myc expression with consequent suppression of Myc-induced lymphomagenesis.

Two major apoptotic pathways contribute to the suppression of Myc-induced tumorigenesis, one involving p19Arf-p53-mediated activation of the BH3-only protein Puma, and the other involving apoptosis mediated by BH3-only relative Bim. The mechanisms by which Myc activates Bim expression are presently unclear. As pro-survival Bcl-2 family members are thought to be essential for the initiation of Myc-induced lymphoma development and as Bcl-2 was shown to be dispensable for this, we predict that Bcl-x_L and Mcl-1 stand at the convergence point of these two pathways, possibly by keeping Bim and Puma in check.

Figure 4. Expression of Bcl-2, Bcl-x_L and Mcl-1 during B lymphocyte differentiation.

The levels of expression of Bcl-2, Bcl-x_L and Mcl-1, based on published date (see papers cited in the text) during B lymphocyte differentiation in the mouse are indicated.

The demonstration that removing Bcl-2 decimated the lymphomas arising in Eµ-myc/inducible Eµ-bcl-2 doubly transgenic mice⁴⁶ supports the concept that inactivation of Bcl-2 constitutes a promising new approach to cancer therapy.³⁷ However, our finding that endogenous Bcl-2 is dispensable for Eµ-myc induced lymphoma development⁴⁹ may also indicate that different pro-survival Bcl-2 family proteins may have to be targeted for treatment of different tumour types.

Bax/Bak proteins: essential activators of the effector phase of apoptosis

Pro-survival Bcl-2 family members maintain cell survival (at least in part) by keeping in check the so-called pro-apoptotic multi-BH domain Bcl-2 family members Bax, Bak (and possibly also the poorly studied Bok) (reviewed in¹⁵). These proteins contain three BH domains and share with their pro-survival relatives surprisingly extensive structural similarity. Activation of Bax and Bak involves homo-dimerisation and oligomerisation within the outer mitochondrial membrane, which leads to release of apoptogenic proteins, such as cytochrome c and Smac/DIABLO, from the mitochondrial inter-membrane space (reviewed in⁵¹). This in turns promotes activation of the caspase cascade that culminates in proteolysis of hundreds of intra-cellular proteins and consequent cellular demolition (Figure 5). Bax and Bak appear to have largely overlapping function. Mice lacking Bak appear normal⁵² and those lacking Bax have a minor increase in spleen weight and the males are sterile because Bax-dependent death of early stem cells in the testes are required to initiate spermatogenesis.⁵³ Remarkably, Bax/Bak doubly deficient mice have severe developmental defects, including webbed feet, and cells from these animals are highly resistant to a broad range (possibly all) stimuli that activate the so-called Bcl-2-regulated (also called “mitochondrial”, “intrinsic” or “stress”) apoptotic pathway.⁵² In mouse model systems, such as Eµ-myc transgenic mice, Bax loss can accelerate tumorigenesis,⁵⁴ but loss of Bax or Bak has so far only rarely been observed in human cancer, most likely because they have overlapping function and hence four alleles would need to be lost in nascent neoplastic cells, which would be exceedingly rare. Intriguingly, somatically acquired copy number variations in bok have been found in certain human cancers,³⁰ indicating that it may function as a tumour suppressor.

Figure 5. Mammals have two distinct but ultimately converging pathways to apoptosis.

Signalling via death receptors is indicated on the right. The “death receptor” pathway is activated by the stimulation of extracellular “death receptors” (such as Fas/CD95) by their cognate “death ligands” (FasL/CD95L). The adaptor protein FADD then promotes recruitment and activation of the “initiator caspase”, caspase-8. Caspase-8 in turn proteolytically activates “effector caspases” (caspase-3, -6 and -7). Caspase-8 mediated proteolytic activation of the BH3-only protein Bid connects the “death receptor” pathway to the “Bcl-2 regulated” pathway and serves to amplify the apoptosis cascade. This amplification mechanism is essential for Fas-induced killing in so-called type 2 cells (e.g. hepatocytes) but dispensable in type 1 cells (e.g. thymocytes).

The Bcl-2-regulated pathway is indicated on the left, and is activated in response to a broad range of cell stressors, such as growth factor deprivation or chemotherapeutic drugs. The pro-apoptotic BH3-only proteins (members of the Bcl-2 family; red boxes) serve as molecular sensors to initiate apoptosis in response to these cellular stresses. Their ability to kill cells is dependent on the multi-BH domain pro-apoptotic Bax/Bak-like Bcl-2 family members. BH3-only proteins are thought to activate Bax/Bak either directly or indirectly by unleashing them from the pro-survival Bcl-2 family members. Regardless of mode of activation, activated Bax/Bak cause permeabilisation of the mitochondrial outer membrane (MOMP) with consequent release of apoptogenic factors (e.g. cytochrome c, Smac/DIABLO). Together with the adaptor protein Apaf-1, cytochrome c promotes activation of the “initiator caspase”, caspase-9 which in turn leads to “effector caspase” activation and subsequent cell demolition.

BH3-only proteins: essential initiators of apoptosis that can function as tumour suppressors

Apoptosis is initiated in response to a broad range of stress stimuli, including those frequently encountered during tumour development, such as oncogene activation, DNA damage, hypoxia (oxygen deprivation), loss of appropriate growth signals and anoikis (loss of cell attachment) (Figure 6; reviewed in¹⁵). BH3-only proteins (Bim, Puma, Bid, Bad, Bik, Noxa, Bmf, Hrk), a pro-apoptotic subgroup of the Bcl-2 family that share with each other and the family at large only the ~23 aa BH3 domain are essential for initiation of apoptotis signalling (reviewed in⁵⁵). BH3-only proteins trigger apoptosis by binding with their BH3 region to a groove on the surface of their pro-survival relatives, thereby unleashing Bax and Bak, but at least some of them have also been reported to directly bind and activate Bax/Bak (reviewed in¹⁵). Studies with gene-targeted mice have shown that different apoptotic stimuli require distinct BH3-only for cell killing (reviewed in⁵⁵). For example, Puma is essential for p53-mediated apoptosis induced by DNA damage, anoxia or Myc over-expression,^{56, 57} whereas Bim is critical for growth factor deprivation induced killing in diverse cell types.⁵⁸ BH3-only proteins function as crucial barriers against the development of malignant disease by serving as molecular sentinels to kill abnormal cells harbouring potentially neoplastic lesions (reviewed in⁵⁵). Accordingly, loss of either Bim⁵⁹ or Puma,^{60, 61} two key initiators of cytokine withdrawal induced apoptosis,^56–58 inhibit the apoptosis of Myc-overexpressing B cells and accelerates Eµ-myc-induced lymphomagenesis. Importantly, loss of both alleles of bim has been found in ~20% of human mantle cell B lymphoma⁶² and ~40% of Burkitt lymphoma express only very low levels of Puma, which in (at least) some cases is thought to be due to epigenetic silencing.⁶⁰ Moreover, somatically acquired loss of copy number for puma was found in a range of human cancers.³⁰ Collectively, these results show that pro-apoptotic BH3-only Bcl-2 family members can function as tumour suppressors.

Figure 6. Activation of BH3-only proteins by distinct apoptotic stimuli including those elicited by oncogene activation.

Different apoptotic stimuli, including oncogene activation, DNA damage, hypoxia (oxygen deprivation), loss of appropriate growth signals and anoikis (loss of cell attachment), initiate apoptosis signalling by activating different BH3-only proteins. Some apoptotic stimuli activate more than one BH3-only protein (e.g. cytokine deprivation can activate both Bim and Puma). Also, there are cell type specific differences (e.g. Puma is more important than Bim in cytokine deprivation induced apoptosis in mast cells, whereas Bim is more important than Puma in lymphoid cells).

Two models to explain the functional interactions between the three Bcl-2 family sub-groups

BH3-only proteins bind and occupy the hydrophobic groove on the surface of their pro-survival relatives, such as Bcl-x_L (formed by their BH1 BH2 and BH3 domains), thereby neutralising their survival promoting ability (reviewed in¹⁵). This physical interaction has been shown to be dependent upon a functional BH3 domain within the BH3-only protein. It was originally believed that all pro-survival Bcl-2 family members have identical biochemical action. It has, however, been discovered that they differ markedly with respect to their binding affinities for different BH3-only proteins.⁶³ Bim, Puma and caspase activated Bid (tBid) (so called promiscuous BH3-only proteins) bind with high affinity to all pro-survival Bcl-2 family members, whereas in striking contrast, all other (so called selective) BH3-only proteins bind only a subset. For example, Bad binds to Bcl-2, Bcl-x_L and Bcl-w but not to Mcl-1 or A1, whereas Noxa interacts strongly with Mcl-1 and A1 but not with Bcl-2, Bcl-x_L or Bcl-w.^{63, 64} These observations provide an explanation for why enforced expression of either Bim, Puma or tBid on their own potently kills cells whereas, for example, enforced Bad or Noxa expression alone do not.^{63, 64} Interestingly, co-expression of “selective” BH3-only proteins with complementary binding patterns (e.g. Bad plus Noxa) can kill cells as potently as Bim or Puma.^{63, 64} Two models have been proposed to explain the functional interactions between the pro-survival Bcl-2 family members, pro-apoptotic BH3-only proteins and Bax/Bak. The ‘direct’ model predicts that BH3-only proteins exist as either ‘activators’ or ‘derepressors’. Bim and tBid (and according to one study perhaps also Puma) represent activator proteins and can initiate apoptosis by directly binding to Bax and/or Bak.⁶⁴ Bim and tBid can also be bound and thereby be kept in check by pro-survival Bcl-2-like proteins. Thus, the ‘derepressor’ BH3-only proteins, promote apoptosis by binding to the pro-survival Bcl-2 family members, thereby liberating Bim, tBid and Puma to activate Bax/Bak.⁶⁴Conversely, according to the ‘indirect’ model BH3-only proteins do not bind and directly activate Bax and/or Bak. Instead, it proposes that in healthy cells Bax and Bak are kept in check by the pro-survival Bcl-2 family members and the binding of BH3-only proteins to the pro-survival Bcl-2 family members unleashes Bax/Bak.⁶⁵ Thus, for apoptosis initiation all pro-survival Bcl-2 family members present in a cell must be neutralised by BH3-only proteins. Interestingly, a recent study with knock-in mutant mice in which the BH3 region of Bim was modified to alter its binding specificity to that of Bad, Noxa or Puma indicated that aspects of both the “direct” and “indirect” models may actually operate in developmentally programmed death (at least with respect to Bim).⁶⁶

Targeting pro-survival Bcl-2 proteins for cancer therapy: an emerging strategy for the management of malignant disease

The ability to trigger tumour cell apoptosis is (at least in part) responsible for the therapeutic effects of chemotherapeutic drugs, γ-radiation and even novel designer cancer drugs, such as inhibitors of oncogenic kinases. For example, the killing of Eµ-myc lymphoma cells by DNA damage inducing chemotherapeutic drugs required, as expected, the p53 targets Puma and Noxa, but surprisingly optimal lymphoma cell killing required in addition the BH3-only protein Bim,⁶⁷ which is not activated in a p53 dependent manner. Bim is also critical for the killing of tumour cells by inhibitors of oncogenic kinases, such as the response of CML to the BCR-ABL inhibitor Gleevec,⁶⁸ the response of small cell lung cancer cells bearing EGF-R mutations to Gefitinib or Tarceva⁶⁹ or the response of B-Raf mutant melanoma and colon carcinoma cells to shut-down of this oncogenic pathway.⁷⁰

Since BH3-only proteins are critical for the responses to cancer therapeutics but many cancers have aberrations that preclude optimal induction of these killer proteins (e.g. p53 mutations, Bcl-2 overexpression), small molecule mimetics of BH3-only that can directly target pro-survival Bcl-2 family members are being developed as a novel therapeutic approach. One of the most promising candidates is the small molecule BH3 mimetic ABT-737 and the closely related but orally available ABT-263, which were designed to target the hydrophobic groove of Bcl-x_L, and can also bind Bcl-2 and Bcl-w with very high affinity.³⁷ In preclinical studies, ABT-737 promoted tumour regression in murine xeno-transplanation models of certain human lymphomas or small cell lung carcinomas and in primary-patient derived follicular lymphoma cells.³⁷ Mechanistic studies indicated that, as expected for a BH3 mimetic, ABT-737 requires Bax/Bak for cell killing, activating them indirectly by neutralising the pro-survival proteins Bcl-2, Bcl-x_L and/or Bcl-w.³⁹ Not surprisingly given its binding specificity, on its own ABT-737 is inefficient at killing tumour cells that express substantial levels of Mcl-1, but it can still efficiently kill them when combined with other cancer therapeutics.^{38, 39, 68} There is therefore great anticipation about the efficacy of BH3-mimetics in cancer therapy.