MINIREVIEW Apoptosis and autophagy: BIM as a mediator of tumour cell death in response to oncogene-targeted therapeutics Annette S Gillings, Kathryn Balmanno, Ceri M Wiggins, Mark Johnson and Simon J Cook Laboratory of Molecular Signalling, The Babraham Institute, Babraham Research Campus, Cambridge, UK Keywords B-cell lymphoma (BCL-2); breakpoint cluster region ⁄ Abelson murine leukaemia viral oncogene (BCR ⁄ ABL); BCL-2interacting mediator of cell death (BIM); v-raf murine sarcoma viral oncogene homologue B1 (BRAF); epidermal growth factor receptor (EGFR); extracellular signalregulated kinase ⁄ (ERK1 ⁄ 2); mitogenMAPK or ERK Kinase ⁄ (MEK1 ⁄ 2); protein kinase B (PKB); ribosomal protein S6 kinase (RSK) Correspondence Simon J Cook, Laboratory of Molecular Signalling, The Babraham Institute, Babraham Research Campus, Cambridge CB22 3AT, UK Fax: 44-1223-496023 Tel: 44-1223-496453 E-mail: simon.cook@bbsrc.ac.uk (Received 16 March 2009, revised 23 June 2009, accepted July 2009) The BCL-2 homology domain (BH3)-only protein, B-cell lymphoma interacting mediator of cell death (BIM) is a potent pro-apoptotic protein belonging to the B-cell lymphoma protein family In recent years, advances in basic biology have provided a clearer picture of how BIM kills cells and how BIM expression and activity are repressed by growth factor signalling pathways, especially the extracellular signal-regulated kinase ⁄ and protein kinase B pathways In tumour cells these oncogene-regulated pathways are used to counter the effects of BIM, thereby promoting tumour cell survival In parallel, a new generation of targeted therapeutics has been developed, which show remarkable specificity and efficacy in tumour cells that are addicted to particular oncogenes It is now apparent that the expression and activation of BIM is a common response to these new therapeutics Indeed, BIM has emerged from this marriage of basic and applied biology as an important mediator of tumour cell death in response to such drugs The induction of BIM alone may not be sufficient for significant tumour cell death, as BIM is more likely to act in concert with other BH3-only proteins, or other death pathways, when new targeted therapeutics are used in combination with traditional chemotherapy agents Here we discuss recent advances in understanding BIM regulation and review the role of BIM as a mediator of tumour cell death in response to novel oncogene-targeted therapeutics doi:10.1111/j.1742-4658.2009.07329.x Introduction The conserved, ‘cell intrinsic’ or ‘mitochondrial’ apoptosis pathway is controlled by the interplay between three groups of B-cell lymphoma (BCL-2) proteins [1,2] The multidomain, pro-apoptotic proteins BCL-2 Abbreviations AML, acute myeloid leukaemia; b-TrCP, b-transducin repeat containing protein; BAD, BCL-xL ⁄ BCL-2-associated death promoter; BAK, BCL-2 homologous antagonist ⁄ killer; BAX, BCL-2-associated x protein; BCL-2, B-cell lymphoma 2; BCL-xL, B-cell lymphoma-extra large; BCR ⁄ ABL, breakpoint cluster region ⁄ Abelson murine leukaemia viral oncogene; BH3, BCL-2 homology domain 3; BIM, BCL-2-interacting mediator of cell death; BOP, BH3-only protein; BRAF, v-raf murine sarcoma viral oncogene homologue B1; CBL, Casitas B-lineage lymphoma oncogene; CML, chronic myelogenous leukaemia; CUL2, Cullin 2; DLC1, dynein light chain 1; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; FLT3, FMS-like tyrosine kinase 3; FOXO3A, Forkhead box 3A; KIT, oncogene of HZ4 feline sarcoma virus; MCL, myeloid cell leukaemia 1; MEK, MAPK or ERK Kinase; mTOR, mammalian target of rapamycin; NSCLC, nonsmall cell lung cancer; PDGFR, platelet-derived growth factor receptor; PI3K, phosphoinositide 3¢-kinase; PKB, protein kinase B (also known as Akt); PUMA, p53-upregulated modulator of apoptosis; RACK1, receptor for activated C-kinase-1; RAS, rat sarcoma virus concogene; RNAi, RNA interference; RSK, ribosomal protein S6 kinase 6050 FEBS Journal 276 (2009) 6050–6062 ª 2009 The Authors Journal compilation ª 2009 FEBS A S Gillings et al associated x protein (BAX) and bcl-2 homologous antagonist ⁄ killer (BAK) can activate caspasedependent cell death by promoting the release of cytochrome c from the mitochondria; however, in viable cells, BAX and BAK are restrained by their interaction with the prosurvival proteins such as BCL-2, B-cell lymphoma-extra large (BCL-xL) or myeloid cell leukaemia (MCL-1) The third group of BCL-2 proteins, the BCL-2 homology domain (BH3)-only proteins (BOPs), includes BCL-2-interacting mediator of cell death (BIM), p53-upregulated modulator of apoptosis (PUMA), NOXA (‘damage’), BCL-2 modifying factor (BMF) and BCL-xL ⁄ BCL-2associated death promoter (BAD); they are activated (that is, expressed de novo, post-translationally modified and ⁄ or stabilized) in response to various proapoptotic stimuli (including loss of survival signals) and promote cell death in a manner dependent on the presence of BAX and BAK The precise mechanism by which this is achieved remains controversial, but a wealth of data now favours a model in which the BOPs bind to the prosurvival BCL-2 proteins, sequestering them and allowing BAX and ⁄ or BAK to promote cell death [3] One observation that favours this model is that the relative toxicity of different BOPs segregates well with the repertoire of prosurvival BCL2 proteins to which they can bind [4] For example, BIM and PUMA can bind to all BCL-2 proteins with high affinity and are potent killers, whereas NOXA only binds to MCL-1 and BCL2-related protein A1 and is less toxic (except presumably in cells in which MCL-1 and A1 are the predominant BCL-2 proteins?) BIM has a number of properties that set it apart from most other BOPs In addition to its strong toxicity [4], alternative splicing [5,6] gives rise to a variety of BIM isoforms with different intrinsic toxicities and modes of regulation [7] Some BIM splice variants exhibit no apparent toxicity; are these naturally occurring dominant negatives or they point to additional functions for BIM that are unrelated to the promotion of cell death? The most extensively studied splice variants, BIMS, BIML and BIMEL (BIM-short, BIM-long and BIM-extra long, respectively), are all cytotoxic and subject to different modes of regulation by various prodeath and prosurvival signalling pathways [7] Some, such as BIML and BIMEL, are phosphorylated by c-Jun N-terminal kinase in response to various stresses, and this promotes apoptosis [8,9] In addition, particular attention has focussed recently on the regulation of BIM by the prosurvival extracellular signalregulated kinase ⁄ (ERK1 ⁄ 2) and protein kinase B (PKB) pathways that act downstream of oncogenic protein kinases [10,11] It is increasingly apparent that BIM as a mediator of tumour cell death these pathways are utilized by oncogenes to inhibit or neutralize BIM, thereby facilitating tumour cell survival Arising directly from this is the growing appreciation that the new generation of oncogenetargeted therapeutics cause loss of ERK1 ⁄ and ⁄ or PKB signalling and, as a consequence, promote increased expression of BIM and BIM-dependent cell death in tumour cells Here we review recent advances in understanding BIM regulation and analyze the results of studies which suggest that BIM is an important mediator of tumour cell death in response to novel oncogene-targeted therapeutics Regulation of BIM by cell survival signalling pathways Transcriptional regulation of BIM Transcription of the BIM gene is normally repressed by serum, growth factors and cytokines, and increases upon the withdrawal of such survival factors; indeed, expression of BIM is required for optimal cell death following cytokine withdrawal [12–14] Transcription of BIM is promoted by the Forkhead box 3A (FOXO3A) transcription factor [15], which itself is inhibited by both the ERK1 ⁄ and PKB pathways PKB phosphorylates FOXO3A directly at three serine residues and this allows binding to 14-3-3 proteins, thereby sequestering FOXO3A in the cytosol and preventing it from activating BIM transcription [16] In addition, direct ERK1 ⁄ 2-dependent phosphorylation targets FOXO3A for proteasome-dependent degradation [17] These studies provide relatively simple explanations for the fact that inhibition of either the ERK1 ⁄ or PKB pathways is sufficient to increase BIM mRNA in many cell types Post-translational regulation of BIM by phosphorylation The extra long splice variant, BIMEL, is the most abundant isoform and undergoes the most dynamic changes in expression upon withdrawal of survival factors [14] In addition, expression of BIMEL often precedes that of BIMS or of BIML [14,18], suggesting that BIMEL is subject to some unique mode of regulation Indeed, many studies have now shown that BIMEL is phosphorylated at multiple sites in response to activation of the ERK1 ⁄ pathway, and this has the effect of promoting its ubiquitination and proteasomedependent degradation [7,19–21] BIMEL is phosphorylated on at least three Ser-Pro motifs, including Ser69 (Ser65 in mouse and rat) (Fig 1) The first effect of FEBS Journal 276 (2009) 6050–6062 ª 2009 The Authors Journal compilation ª 2009 FEBS 6051 BIM as a mediator of tumour cell death A S Gillings et al RAS RAF Ub Ub Ub P PUb P P P BIM P MEK BIM EL UPS 26S ERK RSK PP P BIM EL BIMEL PP P P PBIM P EL TrCP1/2 PP P P PBIM P EL MCL-1 this phosphorylation appears to be to promote the dissociation of BIMEL from prosurvival BCL-2 proteins [14] (Fig 1); because BIM promotes cell death by binding to prosurvival BCL-2 proteins, this alteration in the binding properties of BIMEL serves as a cell-survival mechanism In addition, this may constitute part of the signal for BIMEL degradation because a BIMEL mutant that fails to bind to BCL-2 proteins is degraded more rapidly in cells [14,22] The nature of the E3 ubiquitin ligase responsible for poly-ubiquitination of BIMEL remains a matter of some debate The really interesting new gene (RING) finger protein, Casitas B-lineage lymphoma oncogene (CBL), was originally proposed as the relevant E3 [23]; however, this suggestion was rather controversial because substrates of CBL are almost invariably phosphotyrosine-containing proteins and to date the only pathways suggested to play a role in BIMEL degradation result in its serine phosphorylation Subsequently, other studies have failed to demonstrate any role for CBL by showing that BIMEL is not phosphorylated on tyrosine, CBL and BIM fail to interact, and ERK1 ⁄ 2-driven BIMEL turnover proceeds normally in cells lacking CBL [24,25] These studies reveal that CBL is not the E3-ubiquitin ligase responsible for targeting BIMEL for degradation in fibroblasts and epithelial cells, and that any role it may play in other cell types is likely to be an indirect one In a separate study, receptor for activated C-kinase-1 (RACK1) and cytokine-inducible SH2 protein (CIS) were reported to be members of an ElonginB ⁄ C-Cullin-SOCS-Box (ECS)–regulator of cullins (Roc) complex responsible for the degradation of BIMEL in response to treatment 6052 Fig Regulation of BIM-binding properties and stability by ERK1 ⁄ The pro-apoptotic BH3-only protein BIM is expressed de novo following cytokine withdrawal and binds to prosurvival proteins, such as MCL-1, thereby releasing BAX or BAK to promote cell death BIMEL, the most abundant BIM splice variant, is phosphorylated directly by ERK1 ⁄ on up to three different sites This promotes the dissociation of BIMEL from prosurvival proteins [14,22] ERK1 ⁄ 2-catalysed phosphorylation may also ‘prime’ BIMEL for phosphorylation by RSK1 or RSK2, providing a binding site for the bTrCP E3 ubiquitin ligase [27]; bTrCP promotes the poly-ubiquitination of BIMEL, thereby targeting it for destruction by the 26S proteasome See the text for details with paclitaxel [26] Initially, dynein light chain (DLC1, a known BIM-binding protein) was found to bind to RACK1 in a yeast two-hybrid screen Further overexpression studies suggested a large E3 ligase complex involving RACK1 in complex with DLC1, BIMEL, CIS and Cullin (CUL2), with assembly of some components being enhanced by paclitaxel RNA interference (RNAi)-mediated knockdown of RACK1 or DLC1 resulted in BIMEL accumulation [26] However, a recent study failed to reproduce the CUL2– BIMEL interaction but rather demonstrated co-immunoprecipitation of BIMEL with CUL1, leading to the proposal that BIMEL degradation occurs via a classic Skp-Cullin-F-box (SCF) E3 ligase [27] The search for the relevant E3 ligase now appears to have been resolved with the report that ribosomal protein S6 kinase (RSK), activated downstream of ERK1 ⁄ 2, phosphorylates BIMEL, providing a binding site for the F-box proteins beta-transducin repeat containing protein (bTrCP)1 and bTrCP2, which promote the poly-ubiquitination of BIMEL [27] It is known that ERK1 ⁄ can phosphorylate BIMEL at Ser55, Ser69 and Ser73 within cells, and Ser69 seems to contribute to BIMEL turnover [20,28] The new study proposes that ERK1 ⁄ 2-dependent phosphorylation of BIMEL at Ser69 facilitates optimal phosphorylation by RSK at Ser93, Ser94 and Ser98, and this motif then serves as the binding site for bTrCP1 ⁄ [27] (Fig 1) This attractive model may explain why mutation of a single ERK1 ⁄ phosphorylation site, Ser69, causes loss of at least two further phosphorylation sites in cells [28] However, it also reveals that one of the important RSK phosphorylation sites, Ser98, lies within the FEBS Journal 276 (2009) 6050–6062 ª 2009 The Authors Journal compilation ª 2009 FEBS A S Gillings et al previously mapped ERK1 ⁄ docking domain [29] Presumably this must mean that the binding of ERK1 ⁄ 2, RSK and bTrCP1 ⁄ is subject to fine temporal coordination within the cell Does ERK1 ⁄ dissociate rapidly after phosphorylating BIMEL to allow binding of RSK, which in turn dissociates to allow binding of bTrCP1 ⁄ 2? Are these events coordinated by a scaffold protein that brings the components together at the outer surface of the mitochondria? No doubt these details will emerge in the future Taken together, a wealth of literature now clearly indicates that activation of the PKB or ERK1 ⁄ pathways can repress BIM transcription, whilst activation of ERK1 ⁄ can selectively target the major BIM splice variant, BIMEL, by reducing its binding to prosurvival BCL-2 proteins and promoting its proteasome-dependent destruction (Fig 1) As the ERK1 ⁄ and PKB pathways are two of the major cell-survival signalling pathways [10,21], it follows that these pathways play a major role in growth factor-dependent cell-survival signalling, including the repression or inhibition of BIM Arising from this is a growing understanding of (a) the role that these pathways play in repressing BIM and in promoting aberrant cell survival in tumours that harbour mutations in oncogenes which control these pathways and (b) the role of BIM in tumour cell death arising from targeted inhibition of such oncogenes or pathways Oncogene addiction and the tumour cell response to novel targeted therapies Research into the molecular basis of cancer in the last 25 years has identified hundreds of genes that can cause malignant transformation when overexpressed or mutated, and it is known that tumours typically accumulate dozens of mutations during their lifetime However, some of these mutations (so-called ‘drivers’) are more important than others (‘passengers’) [30] Driver mutations promote the initiation, development and maintenance of the tumour, whereas passenger mutations confer no selective advantage and probably arise through genomic instability Tumour cells exhibit a series of hallmarks that set them apart from normal cells [31] and driver mutations are thought to promote the acquisition and underpin the maintenance of these tumour-specific traits It seems that tumours evolve to be dependent upon certain key driver mutations and on the signalling pathways they control, to maintain their malignant phenotype – a concept known as ‘oncogene addiction’ [32] This evolved dependency upon particular oncogenes often reflects a loss of BIM as a mediator of tumour cell death signal pathway redundancy, providing a therapeutic window for tumour-selective intervention The new, targeted therapeutics take advantage of this window by targeting the specific driver oncoproteins, or their downstream effector pathways, to which tumours are addicted Because tumour cells typically evolve to be dependent upon their driver oncoproteins for survival signals, tumour cell death is a common and clinically desirable response to these new, targeted therapeutics Recent studies have shown that in certain tumour types pharmacological inhibition of these driver oncoproteins results in inactivation of the ERK1 ⁄ and PKB pathways, increased expression of BIM and cell death These agents not target BIM directly; expression or activation of BIM occurs indirectly, resulting from the inactivation of signalling pathways that normally repress BIM It is increasingly clear that whilst drug-induced expression of BIM alone may not be sufficient to kill these tumour cells, death is at least partly BIM-dependent, with the degree of BIM involvement reflecting the role of other BOPs or other pathways in different cell types Here we review the most pertinent recent examples Tumours with BRAF mutations The v-raf-1 murine leukemia viral oncogene (RAF)–MAPK or ERK Kinase ⁄ (MEK1 ⁄ 2)– ERK1 ⁄ signalling cascade has received much attention in terms of drug discovery because of its role in promoting cell proliferation and survival [10] and as a result of the high frequency of rat sarcoma virus concogene (RAS) [33] and v-raf murine sarcoma viral oncogene homolog B1 (BRAF) [34] mutations identified in certain human cancers Within the ERK1 ⁄ pathway, MEK1 ⁄ are attractive targets for pharmacological intervention because of (a) their strict selectivity for their downstream targets, ERK1 and ERK2, and (b) the presence of a unique hydrophobic inhibitor binding pocket adjacent to the Mg ⁄ ATP-binding site that exhibits little homology to other kinases and explains the high degree of specificity that has been observed with the MEK1 ⁄ inhibitors reported to date [35] The first-generation pan-MEK inhibitors PD98059 and U0126 can also inhibit MEK5 [10] and exhibit poor potency and pharmacokinetic properties PD184352 (CI-1040) is selective for the MEK1 ⁄ 2-ERK1 ⁄ pathway and was the first MEK1 ⁄ inhibitor to demonstrate oral anticancer activity in a preclinical model [36]; however, despite encouraging results in phase I clinical trials [37] it showed inadequate clinical activity to justify development [38] PD0325901 [39] and AZD6244 (ARRY-142886) [40] are both selective FEBS Journal 276 (2009) 6050–6062 ª 2009 The Authors Journal compilation ª 2009 FEBS 6053 BIM as a mediator of tumour cell death A S Gillings et al for the MEK1 ⁄ 2–ERK1 ⁄ pathway and show similar oral activity, with AZD6244 undergoing clinical evaluation at the time of writing AZD6244 can cause a G1 cell cycle arrest and in some cases apoptosis; mouse xenograft studies have revealed both tumour stasis, associated with reduced tumour proliferation, and tumour regression, accompanied by apoptosis [40] An understanding of how and under what circumstances MEK inhibition can promote apoptosis may permit a more targeted clinical use of AZD6244 and related molecules Several studies have recently implicated BIM as a tumour cell executioner in response to inhibitors of the BRAF–MEK–ERK signalling pathway (Fig 2) Colorectal cancer cell lines harbouring a BRAF600E mutation are relatively resistant to death arising from serum starvation and fail to upregulate BIM; however, this is readily overcome by treatment with AZD6244, indicating that these cells are addicted to the ERK1 ⁄ pathway for repression of BIM and growth factorindependent survival RNAi-mediated knockdown revealed a major role for BIM in AZD6244-induced cell death [41] In a separate study, treatment of melanoma cells harbouring BRAF600E with PD184352 or the BRAF600E-selective inhibitor PLX4720 also synergized with growth factor withdrawal to increase BIM expression, and cell death was partially dependent upon BIM [42] Similar results in both colorectal cancer and melanoma cell lines have also been reported [43], and in all cases BIMEL was the predominant isoform expressed upon MEK inhibition Indeed, ERK1 ⁄ 2-dependent turnover of BIMEL via the proteasome was primarily responsible for the repression of BIMEL in all three studies [41–43] (Fig 2) BIM, alone or in combination with other BOPs, has been implicated in melanoma cell death, arising from MEK1 ⁄ inhibition, in several other studies [44–46] It is interesting to note that in all cases MEK inhibitor-induced BIM expression alone was not sufficient to induce a dramatic increase in cell death; pronounced increases in apoptosis were only observed when MEK inhibition was combined with serum deprivation [41,42] This suggests that loss of other serum-dependent survival pathways, such as the phosphoinositide 3¢-kinase (PI3K)–PKB pathway, may be required to cooperate with MEK1 ⁄ inhibition for optimal tumour cell death, EGFR Mut Mut RAS BCR-ABL Mut Gefitinib / Erlotinib Mut PI3K PDK MEK1/2 PKB Imatinib Dasatinib Nilotinib BRAF PLX4720 ERK1/2 ABT-737 AZD6244 PD184352 PD0325901 BCL-2 BIM EL BIMEL FOXO3A MCL-1 BIM BAX BAX BAX CASP cyt-c Fig BIM-dependent tumour cell death – a common response to oncogene-targeted therapeutics Tumours with mutated oncogenic kinases, such as BCR–ABL (in CML), EGFR (in NSCLC) or BRAF (in melanoma and colorectal cancer), typically evolve to be addicted to these oncoproteins for cell survival Selective oncogene-targeted therapeutics, such as imatinib (BCR–ABL), erlotinib (EGFR) or PLX4720 (BRAF), inhibit these kinases or, in the case of MEK inhibitors (such as AZD6244), inhibit one of the key signalling pathways they control; in all cases inhibition of the oncogenic kinase results in the loss of downstream survival signalling pathways and a consequent increase in the expression of BIM Inactivation of the ERK1 ⁄ pathway seems to be particularly important for upregulation of the most abundant BIM isoform, BIMEL Dephosphorylated BIMEL then binds to prosurvival BCL-2 proteins, such as MCL-1, to release BAX and ⁄ or BAK to promote cell death Tumour cell death arising from such drug treatments requires BIM to varying degrees; in many cases BIM acts in concert with other BOPs, such as BAD Despite this, cell death in such circumstances can be quite modest as a result of buffering by high levels of prosurvival proteins, such as BCL-2 and BCL-xL found in some tumours The co-administration of BH3 mimetics, such as ABT-737, inhibits BCL-2 and BCL-xL and synergizes effectively with oncogene-targeted therapeutics that mobilize BIM to promote tumour cell killing and tumour regression See the text for details CASP, caspase-9; cyt-c, cytochrome c 6054 FEBS Journal 276 (2009) 6050–6062 ª 2009 The Authors Journal compilation ª 2009 FEBS A S Gillings et al providing a rationale for the use of combinations of MEK1 ⁄ inhibitors and PI3K–PKB pathway inhibitors Indeed, rapamycin, an inhibitor of mammalian target of rapamycin (mTOR) downstream of PKB, can synergize with the MEK1 ⁄ inhibitor, PD0325901, to promote regression of established melanomas in a mouse model in which melanoma is driven by Braf600E and phosphatase and tensin homologue deleted on chromosome 10 (Pten) loss In this case a cell line established from this mouse model exhibited increased BIM expression upon treatment with PD0325901 [47] Furthermore, the MEK1 ⁄ inhibitor, AZD6244, can cooperate with PI3K inhibitors to inhibit the growth of otherwise refractory colorectal cancer cell lines [48] Synergistic interactions between MEK inhibitors and other kinase inhibitors have also been reported [49,50] UCN-01 is a reversible and ATP-competitive inhibitor, which targets several protein kinases such as cyclindependent kinases (CDKs), checkpoint protein (CHK1), 3¢-phosphoinositide-dependent kinase-1 (PDK1) and protein kinase Cs (PKCs) Treatment of multiple myeloma cells with UCN-01 alone resulted in the activation of ERK1 ⁄ and in the phosphorylation and loss of BIMEL; however, co-administration of the MEK1 ⁄ inhibitor PD184352 stabilized BIMEL and effectively synergized with UCN-01 to promote tumour cell death [49,50] These observations suggest that whilst MEK inhibition is sufficient to cause BIMEL accumulation and subsequent abrogation of the antiapoptotic properties of BCL-2 ⁄ BCL-xL, other UCN01-inducible signals are required to cooperate with BIM to induce apoptosis The results of clinical trials utilizing some MEK1 ⁄ inhibitors as a monotherapy [38] highlight the need for a greater understanding of how these compounds act to initiate cell death, which could guide the selection of suitable therapeutic partners for combination treatments BRAF or MEK inhibition has emerged as a pivotal mediator of synergistic effects in combination with other therapeutics Early indications suggest that this can be attributed in part to the role the ERK1 ⁄ pathway plays in controlling BIM expression Treatment of ERK1 ⁄ pathway-addicted cancer cells with a BRAF or MEK inhibitor, resulting in an accumulation of BIM, may serve to sensitize cells to other pro-apoptotic stimuli or therapeutics Non-small cell lung cancers with epidermal growth factor receptor mutation Over-expression of the epidermal growth factor receptor (EGFR) is frequently observed in a variety of BIM as a mediator of tumour cell death epithelial malignancies, including non-small cell lung cancer (NSCLC) A subgroup of NSCLC patients exhibit somatic activating mutations in the EGFR tyrosine kinase domain; these primary mutations correlate well with clinical responses to the EGFR-specific, ATP-competitive tyrosine kinase inhibitors gefitinib and erlotinib, indicating that human NSCLC cells are addicted to these mutant EGFR oncoproteins [51] Several studies have now shown that human NSCLC cell lines harbouring these primary EGFR mutants undergo apoptosis upon treatment with gefitinib or erlotinib [52–55] Acquired resistance to gefitinib and erlotinib is a very real issue clinically, and most EGFR-mutant tumours that respond well in the first instance eventually become resistant, allowing disease progression Acquired resistance is frequently associated with secondary mutations in the kinase domain (the most frequent being the T790M gatekeeper mutation, which impairs drug binding) but may also arise as a result of the amplification of other oncogenes Gefitinib- or erlotinib-induced NSCLC cell death proceeds via the cell-intrinsic mitochondrial pathway, and increased expression of BIM is invariably an early event following treatment with these drugs in NSCLC harbouring primary EGFR mutations [52–54] Erlotinib treatment also blocks the formation of tumours in transgenic mice that conditionally express the L858R EGFR mutation and inhibits the growth of NSCLC cells as xenografts; in both cases this is associated with increased BIM expression [52] In NSCLC cell lines harbouring primary EGFR mutations, knockdown of BIM by RNAi significantly, but not completely, reversed the cell death induced by gefitinib or erlotinib The partial protection afforded by knockdown of BIM may reflect a role for other BOPs, such as BAD or PUMA, or may simply reflect incomplete knockdown of BIM Furthermore, NSCLC cell lines expressing the secondary T790M mutant EGFR were resistant to gefitinib and erlotinib and failed to upregulate BIM; in such cases, BIM induction and cell death were re-imposed by administration of the structurally distinct, covalent EGFR inhibitor, CL-387785 [53] These studies demonstrate that NSCLCs are addicted to the activity of their primary EGFR mutant for repression of BIM and cell survival, and demonstrate that BIM, whilst not acting alone, is an important key effector of gefitinib- or erlotinib-induced cell death (Fig 2) Virtually all NSCLCs harbouring primary EGFR mutations exhibit strong activation of the ERK1 ⁄ and PKB pathways, and treatment with gefitinib or erlotinib causes inactivation of both pathways Thus, expression of BIM and cell death could reflect loss of either pathway, or both In fact drug-induced loss of FEBS Journal 276 (2009) 6050–6062 ª 2009 The Authors Journal compilation ª 2009 FEBS 6055 BIM as a mediator of tumour cell death A S Gillings et al ERK1 ⁄ signalling contributes substantially to the increase in BIM expression In the majority of NSCLC cell lines treated with erlotinib or gefitinib, BIMEL was the predominant isoform induced [52–54] and was expressed predominantly as the dephosphorylated, stabilized, active form, correlating with loss of ERK1 ⁄ activity Finally, inhibitors of PI3K or PKB did not cause accumulation of BIM, whereas inhibitors of MEK–ERK1 ⁄ signalling did [54] However, despite causing increased BIM expression, inhibition of the ERK1 ⁄ pathway alone caused little cell death in comparison to that seen with gefitinib, suggesting that loss of other signalling pathways (and activation of other BOPs?) must also contribute to gefitinib-induced cell killing, as discussed elsewhere [10] BCR-ABL inhibitors and chronic myeloid leukaemia Chronic myeloid leukaemia (CML) is characterized by the presence of the t(9;22)(q34;q11) reciprocal translocation, giving rise to the breakpoint cluster region– Abelson murine leukaemia viral oncogene (BCR–ABL) fusion oncoprotein [56] The mutant BCR–ABL tyrosine kinase activates several signalling pathways, including the ERK1 ⁄ pathway, the PKB pathway and the Janus kinase ⁄ signal transducer and activator of transcription (JAK-STAT) pathway, to promote proliferation, survival and transformation [56,57] The importance of the BCR–ABL tyrosine kinase in the survival of CML cells led to the development of the tyrosine kinase inhibitor imatinib (STI571, Gleevec), which is a potent inhibitor of BCR–ABL, platelet-derived growth factor receptor (PDGFR) and oncogene of HZ4 feline sarcoma virus (KIT) and has produced impressive results in clinical trials in CML [57,58] Resistance to imatinib has proved to be a problem clinically, and 40% of patients who relapse on imatinib therapy have point mutations in the BCR–ABL kinase domain, including the T315I gatekeeper mutation that impairs imatinib binding [58,59] Accordingly, new therapies are being tested and these include second-generation tyrosine kinase inhibitors, such as dasatinib (inhibits BCR–ABL, KIT, PDGFR and SRC family kinases), nilotinib (a more potent inhibitor of BCR–ABL, KIT and PDGFR), INNO406 (a dual BCR-ABL and Lyn inhibitor) and PPY-A and PHA-739358 (which can inhibit the T315I mutant of BCR–ABL) [57,60] Several observations suggest that BIM is important in promoting cell death following BCR–ABL inhibition Downregulation of BIM is of key importance in cytokine-mediated survival in murine haematopoetic progenitor cells [61] Expression of BCR–ABL in inter6056 leukin-3-dependent Baf-3 cells represses BIM and allows interleukin-3–independent survival; treatment of these cells with imatinib reverses the effects of BCR– ABL, resulting in increased expression of BIM and cell death [62,63] RNAi has shown that BIM expression is required, at least in part, for imatinib-induced apoptosis in BCR–ABL-transformed murine progenitor cells [64] and in BCR–ABL+ K562 CML cells in response to both imatinib and nilotinib [65] INNO-406, a more potent inhibitor of BCR–ABL, also induces apoptosis in BCR–ABL+ K562 cells, and whilst BCR–ABLexpressing myeloid progenitor cells from BIM- ⁄ - mice are partially protected against INNO-406-induced apoptosis, substantially greater protection is seen in BIM- ⁄ - BAD- ⁄ - double knockout cells or upon BCL-2 overexpression [66] These results reveal that a variety of first-generation and second-generation BCR–ABL inhibitors increase BIM expression and elicit BIM-dependent cell death in CML cells, with BIM acting in concert with other BOPs, such as BAD (Fig 2) A notable exception to this is the third-generation dual BCR–ABL and panaurora kinase inhibitor MK-0457, which can inhibit both wild-type and imatinib-resistant BCR–ABL mutants (including T315I) Despite inhibiting BCR– ABL, MK-0457 predominantly induces polyploidy, rather than apoptosis, in BCR–ABL+ CML cells, probably reflecting its activity against the aurora kinases; the lack of cell death induced by MK-0457 correlates with its inability to increase BIM expression [67] However, because other BCR–ABL inhibitors increase BIM expression, it is surprising that MK-0457 does not; does this suggest that the additional inhibitory activity against aurora kinases antagonizes BIM expression, or does it suggest other targets for MK-0457? Regardless, MK-0457 can synergize with the histone deacetylase inhibitor vorinostat to promote apoptosis, and this synergy does involve BIM; vorinostat increases BIM expression and BIM plays a significant role in the induction of apoptosis observed with the combination of the two drugs [67] MEK inhibitors alone tend to cause only modest cell death in CML cells, but PD184352 can act synergistically with imatinib or dasatinib in BCR–ABL+ cells, leading to a substantial increase in apoptosis [68,69] Treatment of BCR–ABL-expressing Baf-3 cells with either the pan-MEK1 ⁄ ⁄ inhibitor, PD98059, or the pan-PI3K inhibitor, LY294002, could induce BIM expression [62], although other studies showed that PD98059, but not LY294002, could increase BIM expression in BCR–ABL-expressing Baf-3 cells and primary CML cells [63] In summary, the repression of BIM downstream of BCR-ABL appears to be mediated FEBS Journal 276 (2009) 6050–6062 ª 2009 The Authors Journal compilation ª 2009 FEBS A S Gillings et al predominantly by the constitutive activity of the ERK1 ⁄ and perhaps by the PI3K pathway Regulation of BIM by other oncogenes/ pathways FMS-like tyrosine kinase (FLT3), a receptor tyrosine kinase related to PDGFR and KIT, is frequently mutated in acute myeloid leukemia (AML) and this correlates with poor prognosis; mutant FLT3 proteins typically exhibit ligand-independent dimerization and activation Treatment of primary AML cells with either of two FLT3 inhibitors (AG1295 or PKC412) caused a substantial cell-death response [70] Activation of the PI3K–PKB pathway downstream of FLT3 was the major pathway responsible for repressing FOXO3A and BIM expression, and whilst both BIM and PUMA were upregulated following FLT3 inhibition, only loss of BIM was able to preserve clonogenic survival in FDC-p1 cells expressing mutant FLT3 proteins Thus, AML cells expressing mutant FLT3 are addicted to FLT3-dependent signalling via the PI3K– PKB pathway for repression of BIM and cell survival Whilst there is a well-defined role for the PI3K– PKB pathway in repressing FOXO3A (see above), studies have also suggested a role for mTOR in regulating BIM expression The earliest study to suggest this demonstrated that treatment of haematopoietic progenitor cells with the mTOR inhibitor, rapamycin, increased BIM expression and overcame RAS-dependent survival signals to promote cell death, arguing that mTOR was an important survival signal that acted, in part, by repressing BIM [61] Most recently, prominent effects of rapamycin on BIM were demonstrated in a mouse model of androgen-independent prostate cancer [71] In this instance, the combination of the MEK1 ⁄ inhibitor, PD0325901, and rapamycin was remarkably effective at inhibiting the growth of prostate cancer cell lines and the growth of prostate cancer in vivo in Pten-deficient mice Interestingly, although rapamycin alone failed to increase BIM expression, the combination of PD0325901 and rapamycin was more effective than PD0325901 alone, and death arising from the combination therapy was at least partially BIM-dependent [71] Both of these studies suggest that TOR activity normally represses BIM and that therapeutic inhibition of TOR will increase BIM levels and contribute to tumour cell death However, a recent study suggests that repression of BIM might not be a direct effect of TOR mTOR exists in mammalian cells as two distinct complexes: mTORC1 (composed of mTOR, mLST8 and raptor) regulates cell growth via the effectors S6K1 and 4E-BP1, whilst BIM as a mediator of tumour cell death mTORC2 (composed of mTOR, mLST8 and rictor) phosphorylates PKB at Ser473, contributing to its activation Rapamycin binds to FKBP12 and, in this form, inhibits preformed mTORC1 complexes but not preformed mTORC2; as a result, the effects of rapamycin are frequently attributed to inhibition of mTORC1 alone However, FKBP12–rapamycin can bind to free mTOR, and Sabatini and co-workers have recently shown that prolonged treatment of cells with rapamycin can actually cause disassembly of mTORC2, loss of PKB Ser473 phosphorylation and apoptosis [72] Thus, it is quite possible that the ability of rapamycin to contribute to BIM expression during prolonged treatments with drug [61,71] may actually reflect loss of PKB phosphorylation and activation of FOXO3A, rather than loss of a direct effect of mTOR Such details not, of course, detract from the striking synergy seen between MEK1 ⁄ inhibitors and rapamycin [47,71], but are important in understanding the mechanisms by which these drugs cooperate to kill tumour cells In addition to promoting cell proliferation and transformation, the c-Myc proto-oncogene is renowned for its ability to promote cell death [73] and there is now good evidence to indicate that (a) BIM is important in Myc-induced cell death and (b) that this may be an arbiter of tumour progression B-lymphoid cells from El-Myc transgenic mice exhibited increased expression of BIM and an increased propensity to undergo apoptosis, which was lost on a BIM- ⁄ - background Loss of even a single BIM allele accelerated Myc-induced tumour progression, giving rise to acute B-cell leukaemia These results demonstrate that Myc can promote expression of BIM and show that BIM is a tumour suppressor in this system [74] Myc-induced BIM expression may be therapeutically relevant in other tumour models For example, in human glioma cell lines, several distinct glycogen synthase kinase inhibitors cause activation of c-Myc, expression of Myc target genes (including BIM) and glioma death, although the role of BIM, as opposed to other Myc target genes, was not defined [75] BH3 mimetics: giving BIM a helping hand By virtue of its ability to engage with and inhibit all of the prosurvival BCL-2 proteins, BIM is one of the most potent and effective BOPs, in terms of cell killing, when assayed by overexpression [4] Despite this, oncogene-targeted therapeutics alone can often cause quite significant increases in BIM expression, but relatively modest tumour cell death Similarly, the FEBS Journal 276 (2009) 6050–6062 ª 2009 The Authors Journal compilation ª 2009 FEBS 6057 BIM as a mediator of tumour cell death A S Gillings et al response observed in the clinic may often be cytostatic (i.e associated with tumour stasis, rather than with cytotoxicity and tumour regression) This may be because the level of BIM upregulation achieved is not sufficient for apoptosis and ⁄ or because tumours often exhibit elevated expression of certain prosurvival BCL-2 proteins, providing an effective buffer to the drug-induced expression of BIM or other BOPs In this context, recent studies suggest that the use of new oncogene-targeted therapeutics, in combination with BH3 mimetics, may prove particularly effective BH3 mimetics are small, cell-permeant molecules that mimic BOPs by binding and inhibiting prosurvival BCL-2 proteins [11,76,77] The prototype, ABT-737, binds to BCL-2, BCL-xL and BCL-2-like protein with high affinity and is thought to act by liberating BAX and BAK from these proteins; in addition, BOPs displaced by treatment with ABT-737 may bind and inhibit MCL-1, providing further activation of BAX ⁄ BAK ABT-737 can kill certain tumour cells as a single agent or when administered with conventional cytotoxic chemotherapeutics; more importantly, it can cooperate with oncogene-targeted therapeutics to provide sometimes quite striking synergistic tumour cell killing (Fig 2) Tumours with BRAF mutations Even in tumour cells with BRAF600E that show strong addiction to ERK1 ⁄ signalling for proliferation, MEK inhibition alone can often induce quite striking increases in BIM expression but only modest tumour cell death [41–43] Cragg et al [43] noted that high levels of the anti-apoptotic BCL-2 protein correlated with low levels of cell death in response to the first-generation panMEK1 ⁄ ⁄ inhibitor, U0126, in a range of tumour-cell lines harbouring BRAF600E and found that ABT–737 synergized with U0126 to promote extensive apoptosis in BRAF mutant SkMel-28 melanoma and Colo205 colon cancer cells; this was associated with the ABT-737-dependent redistribution of BIM from BCL-2 to MCL-1 Striking synergy was also observed when ABT-737 and the second-generation MEK1 ⁄ 2-specific compound, PD0325901, were combined to treat SkMel28 and Colo205 xenografts in nude mice, resulting in partial tumour regression [43], providing compelling support for the use of MEK inhibitors in combination with BH3 mimetics in tumours with BRAF600E NSCLC with EGFR mutations In NSCLC cell lines that are sensitive to erlotinib or gefitinib and exhibit drug-induced expression of BIM, 6058 the induction of apoptosis is often modest However, two studies have now shown that erlotinib or gefitinibinduced cell death can be greatly enhanced by co-administration of ABT-737 In the case of erlotinib, synergy with ABT-737 was observed in PC-9 and H2355 cells [52], whilst in the case of gefitinib, synergy with ABT-737 was most pronounced in H358, H1975 and H1650 cells, gefitinib alone being more efficacious in H3255 cells [54] CML with BCR-ABL Prosurvival BCL-2 family proteins such as MCL-1, BCL-2 and BCL-xL are often expressed at high levels in CML cells [62,63,78,79], and this prompted an investigation of the efficacy of combinations of ABT737 and BCR-ABL inhibitors Indeed, ABT-737 cooperates effectively with imatinib [64] and INNO-406 [66] to promote death of CML cells This cooperation was also seen in Baf-3 cell lines expressing two different mutant BCR-ABL proteins (E255K and H396P), but not in those expressing the T315I gatekeeper mutant These authors also demonstrated that ABT-737 could enhance apoptosis in response to 17-AAG [66], which inhibits the activity of the HSP90 chaperone required for correct BCR–ABL folding Together these examples indicate the powerful synergy that is observed when therapies targeting oncogenic kinases are combined with BH3 mimetics, giving rise to substantially greater tumour cell killing in vitro and tumour regression in vivo [77] (Fig 2) Obviously this is a desirable outcome in its own right, but it may have other advantages For example, the predominantly cytostatic effects of oncogenic kinase inhibitors alone mean that tumour cells stay alive and receive prolonged exposure to the drug; this may explain the frequent emergence of acquired resistance in CML and NSCLC In contrast, the more substantial and precipitate cell-death response seen with combinations of kinase inhibitors and BH3-mimetics may substantially shorten the window of opportunity for acquisition and ⁄ or selection of secondary mutations, making acquired resistance less likely to arise Answers to such speculation may be informed by tissue culture and animal models, but ultimately will come from clinical studies Conclusions A combination of basic and applied biology in the last years has provided a good working model for how BIM is inhibited by survival signalling pathways, notably the ERK1 ⁄ pathway, and has led to the FEBS Journal 276 (2009) 6050–6062 ª 2009 The Authors Journal compilation ª 2009 FEBS A S Gillings et al recognition that BIM plays a major response in tumour cell death arising from inhibition of oncogenic kinases Indeed, the increased expression of dephosphorylated BIMEL could even be viewed as a biomarker for drug-induced inactivation of ERK1 ⁄ and engagement of the BCL-2 axis Whether this increase in BIM gives rise to substantial tumour cell death will depend on the activation of other survival pathways and expression of other BOPs or prosurvival BCL-2 proteins In instances of high BCL-2 or BCL-xL expression, drug-induced, BIM-dependent cell death will be greatly enhanced by combination with BH3 mimetics, giving tumour regression and potentially less opportunity for resistance to arise Moving forward, if we are to take advantage of this clinically, tumours will be sampled for oncogene mutations, biochemical signatures of signal pathway activation (to address pathway redundancy [10]) and expression of BCL-2 family proteins to match the treatment combination to the tumour fingerprint of the patient Acknowledgements We apologise to colleagues in the field whose work we have had to omit because of space constraints Work in the Cook laboratory is funded by the Association for International Cancer Research, AstraZeneca, the Babraham Institute, the Biotechnology and Biological Sciences Research Council and Cancer Research UK References Adams J & Cory S (2007) The Bcl-2 apoptotic switch in cancer development and therapy Oncogene 26, 1324– 1337 Youle RJ & Strassser A (2009) The BCL-2 protein family: opposing activities that mediate cell death Nat Rev Mol Cell Biol 9, 47–59 Willis SN, Fletcher JI, Kaufmann T, van Delft MF, Chen L, Czabotar PE, Lerino H, Lee EF, Fairlie WD, Bouillet P et al (2007) Apoptosis initiated when BH3 ligands engage multiple Bcl-2 homologs, not Bax or Bak Science 315, 856–859 Chen L, Willis SN, Wei A, 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