Báo cáo khoa học: Apoptosis and autophagy: BIM as a mediator of tumour cell death in response to oncogene-targeted therapeutics pptx

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
Introduction
The conserved, ‘cell intrinsic’ or ‘mitochondrial’ apop-
tosis pathway is controlled by the interplay between
three groups of B-cell lymphoma 2 (BCL-2) proteins
[1,2]. The multidomain, pro-apoptotic proteins BCL-2
Keywords
B-cell lymphoma 2 (BCL-2); breakpoint
cluster region ⁄ Abelson murine leukaemia
viral oncogene (BCR ⁄ ABL); BCL-2-
interacting mediator of cell death (BIM);
v-raf murine sarcoma viral oncogene
homologue B1 (BRAF); epidermal growth
factor receptor (EGFR); extracellular signal-
regulated kinase 1 ⁄ 2 (ERK1 ⁄ 2); mitogen-
MAPK or ERK Kinase 1 ⁄ 2 (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:
(Received 16 March 2009, revised 23 June

2009, accepted 9 July 2009)
doi:10.1111/j.1742-4658.2009.07329.x
The BCL-2 homology domain 3 (BH3)-only protein, B-cell lymphoma
2 interacting mediator of cell death (BIM) is a potent pro-apoptotic protein
belonging to the B-cell lymphoma 2 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 1 ⁄ 2
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.
Abbreviations
AML, acute myeloid leukaemia; b-TrCP, b-transducin repeat containing protein; BAD, BCL-x
L
⁄ BCL-2-associated death promoter; BAK, BCL-2
homologous antagonist ⁄ killer; BAX, BCL-2-associated x protein; BCL-2, B-cell lymphoma 2; BCL-x
L,
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, non-
small 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
associated x protein (BAX) and bcl-2 homologous
antagonist ⁄ killer (BAK) can activate caspase-
dependent 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-x
L
) or mye-
loid cell leukaemia 1 (MCL-1). The third group of
BCL-2 proteins, the BCL-2 homology domain 3
(BH3)-only proteins (BOPs), includes BCL-2-interact-
ing mediator of cell death (BIM), p53-upregulated
modulator of apoptosis (PUMA), NOXA (‘damage’),
BCL-2 modifying factor (BMF) and BCL-x
L
⁄ BCL-2-
associated death promoter (BAD); they are activated
(that is, expressed de novo, post-translationally modi-
fied and ⁄ or stabilized) in response to various pro-
apoptotic 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, seques-
tering them and allowing BAX and⁄ or BAK to pro-
mote cell death [3]. One observation that favours this
model is that the relative toxicity of different BOPs
segregates well with the repertoire of prosurvival BCL-
2 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 toxic-
ity [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 occur-
ring dominant negatives or do they point to additional
functions for BIM that are unrelated to the promotion
of cell death? The most extensively studied splice vari-
ants, BIM
S
, BIM
L
and BIM
EL

(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 BIM
L
and BIM
EL
, 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 regu-
lation of BIM by the prosurvival extracellular signal-
regulated kinase 1 ⁄ 2 (ERK1 ⁄ 2) and protein kinase B
(PKB) pathways that act downstream of oncogenic
protein kinases [10,11]. It is increasingly apparent that
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 oncogene-
targeted therapeutics cause loss of ERK1 ⁄ 2 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 impor-
tant 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 ⁄ 2 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 pre-
venting it from activating BIM transcription [16]. In
addition, direct ERK1 ⁄ 2-dependent phosphorylation
targets FOXO3A for proteasome-dependent degrada-
tion [17]. These studies provide relatively simple expla-
nations for the fact that inhibition of either the
ERK1 ⁄ 2 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, BIM
EL
, is the most
abundant isoform and undergoes the most dynamic
changes in expression upon withdrawal of survival
factors [14]. In addition, expression of BIM
EL
often
precedes that of BIM

S
or of BIM
L
[14,18], suggesting
that BIM
EL
is subject to some unique mode of regula-
tion. Indeed, many studies have now shown that
BIM
EL
is phosphorylated at multiple sites in response
to activation of the ERK1⁄ 2 pathway, and this has the
effect of promoting its ubiquitination and proteasome-
dependent degradation [7,19–21]. BIM
EL
is phosphory-
lated on at least three Ser-Pro motifs, including Ser69
(Ser65 in mouse and rat) (Fig. 1). The first effect of
A. S. Gillings et al. BIM as a mediator of tumour cell death
FEBS Journal 276 (2009) 6050–6062 ª 2009 The Authors Journal compilation ª 2009 FEBS 6051
this phosphorylation appears to be to promote the dis-
sociation of BIM
EL
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 BIM
EL
serves as a cell-sur-
vival mechanism. In addition, this may constitute part

of the signal for BIM
EL
degradation because a BIM
EL
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 BIM
EL
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 BIM
EL
degradation result in its serine phosphorylation. Subse-
quently, other studies have failed to demonstrate any
role for CBL by showing that BIM
EL
is not phosphor-
ylated on tyrosine, CBL and BIM fail to interact, and
ERK1 ⁄ 2-driven BIM
EL
turnover proceeds normally in
cells lacking CBL [24,25]. These studies reveal that
CBL is not the E3-ubiquitin ligase responsible for tar-
geting BIM

EL
for degradation in fibroblasts and epi-
thelial 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 BIM
EL
in response to treatment
with paclitaxel [26]. Initially, dynein light chain 1
(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,
BIM
EL
, CIS and Cullin 2 (CUL2), with assembly of
some components being enhanced by paclitaxel. RNA
interference (RNAi)-mediated knockdown of RACK1
or DLC1 resulted in BIM
EL
accumulation [26]. How-
ever, a recent study failed to reproduce the CUL2–
BIM
EL
interaction but rather demonstrated co-immu-
noprecipitation of BIM
EL

with CUL1, leading to the
proposal that BIM
EL
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 pro-
tein S6 kinase (RSK), activated downstream of
ERK1 ⁄ 2, phosphorylates BIM
EL,
providing a binding
site for the F-box proteins beta-transducin repeat con-
taining protein (bTrCP)1 and bTrCP2, which promote
the poly-ubiquitination of BIM
EL
[27]. It is known that
ERK1 ⁄ 2 can phosphorylate BIM
EL
at Ser55, Ser69
and Ser73 within cells, and Ser69 seems to contribute
to BIM
EL
turnover [20,28]. The new study proposes
that ERK1 ⁄ 2-dependent phosphorylation of BIM
EL
at
Ser69 facilitates optimal phosphorylation by RSK at
Ser93, Ser94 and Ser98, and this motif then serves as
the binding site for bTrCP1 ⁄ 2 [27] (Fig. 1). This attrac-
tive model may explain why mutation of a single

ERK1 ⁄ 2 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
UPS 26S
BIM
RAS
RAF
MEK
ERK
RSK
TrCP1/2
BIM
EL
BIM
EL
P
P
P
MCL-1
BIM
EL
P
P
P
P
P
P
Ub
Ub

Ub
Ub
BIM
EL
BIM
EL
P
P
P
P
P
P
P
P
P
P
P
P
Fig. 1. Regulation of BIM-binding properties
and stability by ERK1 ⁄ 2. 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. BIM
EL
, the most abundant BIM
splice variant, is phosphorylated directly by
ERK1 ⁄ 2 on up to three different sites. This
promotes the dissociation of BIM

EL
from
prosurvival proteins [14,22]. ERK1 ⁄ 2-cataly-
sed phosphorylation may also ‘prime’ BIM
EL
for phosphorylation by RSK1 or RSK2,
providing a binding site for the bTrCP E3
ubiquitin ligase [27]; bTrCP promotes the
poly-ubiquitination of BIM
EL
, thereby
targeting it for destruction by the 26S
proteasome. See the text for details.
BIM as a mediator of tumour cell death A. S. Gillings et al.
6052 FEBS Journal 276 (2009) 6050–6062 ª 2009 The Authors Journal compilation ª 2009 FEBS
previously mapped ERK1 ⁄ 2 docking domain [29]. Pre-
sumably this must mean that the binding of ERK1 ⁄ 2,
RSK and bTrCP1 ⁄ 2 is subject to fine temporal coordi-
nation within the cell. Does ERK1 ⁄ 2 dissociate rapidly
after phosphorylating BIM
EL
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 ⁄ 2 path-
ways can repress BIM transcription, whilst activation

of ERK1 ⁄ 2 can selectively target the major BIM splice
variant, BIM
EL
, by reducing its binding to prosurvival
BCL-2 proteins and promoting its proteasome-depen-
dent destruction (Fig. 1). As the ERK1 ⁄ 2 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 sig-
nalling, 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 accu-
mulate dozens of mutations during their lifetime. How-
ever, 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 muta-
tions 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
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 onco-
proteins results in inactivation of the ERK1 ⁄ 2 and
PKB pathways, increased expression of BIM and cell
death. These agents do not target BIM directly; expres-
sion or activation of BIM occurs indirectly, resulting
from the inactivation of signalling pathways that nor-
mally repress BIM. It is increasingly clear that whilst
drug-induced expression of BIM alone may not be suf-
ficient 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 1 ⁄ 2 (MEK1 ⁄ 2)–
ERK1 ⁄ 2 signalling cascade has received much atten-
tion 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 con-
cogene (RAS) [33] and v-raf murine sarcoma viral
oncogene homolog B1 (BRAF) [34] mutations identi-
fied in certain human cancers. Within the ERK1 ⁄ 2
pathway, MEK1 ⁄ 2 are attractive targets for pharmaco-
logical 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 ⁄ 2 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 ⁄ 2 pathway and was the first MEK1 ⁄ 2 inhibi-
tor to demonstrate oral anticancer activity in a preclin-
ical 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
A. S. Gillings et al. BIM as a mediator of tumour cell death
FEBS Journal 276 (2009) 6050–6062 ª 2009 The Authors Journal compilation ª 2009 FEBS 6053
for the MEK1⁄ 2–ERK1 ⁄ 2 pathway and show similar
oral activity, with AZD6244 undergoing clinical evalu-
ation 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 BRAF
600E
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 ⁄ 2
pathway for repression of BIM and growth factor-
independent 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 BRAF
600E
with PD184352

or the BRAF
600E
-selective inhibitor PLX4720 also syn-
ergized with growth factor withdrawal to increase BIM
expression, and cell death was partially dependent
upon BIM [42]. Similar results in both colorectal can-
cer and melanoma cell lines have also been reported
[43], and in all cases BIM
EL
was the predominant iso-
form expressed upon MEK inhibition. Indeed,
ERK1 ⁄ 2-dependent turnover of BIM
EL
via the protea-
some was primarily responsible for the repression of
BIM
EL
in all three studies [41–43] (Fig. 2). BIM, alone
or in combination with other BOPs, has been impli-
cated in melanoma cell death, arising from MEK1 ⁄ 2
inhibition, in several other studies [44–46]
It is interesting to note that in all cases MEK inhibi-
tor-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 sur-
vival pathways, such as the phosphoinositide 3¢-kinase
(PI3K)–PKB pathway, may be required to cooperate
with MEK1 ⁄ 2 inhibition for optimal tumour cell death,

BIM
EL
MCL-1
FOXO3A
PI3K
PDK
PKB
RAS
BIM
Mut
BIM
EL
BRAF
MEK1/2
ERK1/2
BAX
BAX
AZD6244
PD184352
PD0325901
cyt-c
CASP
BAX
BCR-ABL
EGFR
BCL-2
Gefitinib / Erlotinib
Imatinib
Dasatinib
Nilotinib

ABT-737
Mut
Mut
PLX4720
Mut
Fig. 2. 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 expres-
sion of BIM. Inactivation of the ERK1 ⁄ 2 pathway seems to be particularly important for upregulation of the most abundant BIM isoform,
BIM
EL
. Dephosphorylated BIM
EL
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-x
L
found in some tumours. The co-administration of BH3 mimetics, such as ABT-737, inhibits BCL-2 and
BCL-x
L
and synergizes effectively with oncogene-targeted therapeutics that mobilize BIM to promote tumour cell killing and tumour regres-
sion. See the text for details. CASP, caspase-9; cyt-c, cytochrome c.
BIM as a mediator of tumour cell death A. S. Gillings et al.
6054 FEBS Journal 276 (2009) 6050–6062 ª 2009 The Authors Journal compilation ª 2009 FEBS
providing a rationale for the use of combinations of
MEK1 ⁄ 2 inhibitors and PI3K–PKB pathway inhibitors.
Indeed, rapamycin, an inhibitor of mammalian target of

rapamycin (mTOR) downstream of PKB, can synergize
with the MEK1 ⁄ 2 inhibitor, PD0325901, to promote
regression of established melanomas in a mouse model
in which melanoma is driven by Braf
600E
and phospha-
tase 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 ⁄ 2 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 cyclin-
dependent kinases (CDKs), checkpoint protein 1
(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 ⁄ 2 and in the phosphorylation
and loss of BIM
EL
; however, co-administration of the
MEK1 ⁄ 2 inhibitor PD184352 stabilized BIM
EL
and
effectively synergized with UCN-01 to promote tumour
cell death [49,50]. These observations suggest that

whilst MEK inhibition is sufficient to cause BIM
EL
accumulation and subsequent abrogation of the anti-
apoptotic properties of BCL-2 ⁄ BCL-x
L
, other UCN-
01-inducible signals are required to cooperate with
BIM to induce apoptosis.
The results of clinical trials utilizing some MEK1 ⁄ 2
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 treat-
ments. 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 ⁄ 2
pathway plays in controlling BIM expression. Treat-
ment of ERK1 ⁄ 2 pathway-addicted cancer cells with a
BRAF or MEK inhibitor, resulting in an accumulation
of BIM, may serve to sensitize cells to other pro-apop-
totic stimuli or therapeutics.
Non-small cell lung cancers with
epidermal growth factor receptor
mutation
Over-expression of the epidermal growth factor recep-
tor (EGFR) is frequently observed in a variety of
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 corre-
late 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 associ-
ated with secondary mutations in the kinase domain
(the most frequent being the T790M gatekeeper muta-
tion, 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]. Erloti-
nib 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 dis-
tinct, 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 ⁄ 2
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
A. S. Gillings et al. BIM as a mediator of tumour cell death
FEBS Journal 276 (2009) 6050–6062 ª 2009 The Authors Journal compilation ª 2009 FEBS 6055
ERK1 ⁄ 2 signalling contributes substantially to the
increase in BIM expression. In the majority of NSCLC
cell lines treated with erlotinib or gefitinib, BIM
EL
was
the predominant isoform induced [52–54] and was
expressed predominantly as the dephosphorylated, sta-
bilized, active form, correlating with loss of ERK1 ⁄ 2

activity. Finally, inhibitors of PI3K or PKB did not
cause accumulation of BIM, whereas inhibitors of
MEK–ERK1 ⁄ 2 signalling did [54]. However, despite
causing increased BIM expression, inhibition of the
ERK1 ⁄ 2 pathway alone caused little cell death in com-
parison 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 trans-
location, giving rise to the breakpoint cluster region–
Abelson murine leukaemia viral oncogene (BCR–ABL)
fusion oncoprotein [56]. The mutant BCR–ABL tyro-
sine kinase activates several signalling pathways, includ-
ing the ERK1 ⁄ 2 pathway, the PKB pathway and the
Janus kinase ⁄ signal transducer and activator of tran-
scription (JAK-STAT) pathway, to promote prolifera-
tion, survival and transformation [56,57]. The
importance of the BCR–ABL tyrosine kinase in the sur-
vival 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 inhibi-
tor) 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 inhibi-
tion. Downregulation of BIM is of key importance in
cytokine-mediated survival in murine haematopoetic
progenitor cells [61]. Expression of BCR–ABL in inter-
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 apopto-
sis 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–ABL-

expressing 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 pan-
aurora 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 do
increase BIM expression, it is surprising that MK-0457
does not; does this suggest that the additional inhibi-

tory 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; vorino-
stat increases BIM expression and BIM plays a signifi-
cant 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 synergisti-
cally 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 ⁄ 2 ⁄ 5 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
BIM as a mediator of tumour cell death A. S. Gillings et al.
6056 FEBS Journal 276 (2009) 6050–6062 ª 2009 The Authors Journal compilation ª 2009 FEBS
predominantly by the constitutive activity of the
ERK1 ⁄ 2 and perhaps by the PI3K pathway.
Regulation of BIM by other oncogenes/
pathways
FMS-like tyrosine kinase 3 (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]. Activa-
tion 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 inhibi-
tion, only loss of BIM was able to preserve clonogenic
survival in FDC-p1 cells expressing mutant FLT3 pro-
teins. 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 regu-
lating 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-depen-
dent 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 demon-
strated in a mouse model of androgen-independent
prostate cancer [71]. In this instance, the combination
of the MEK1 ⁄ 2 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 rapa-
mycin was more effective than PD0325901 alone, and
death arising from the combination therapy was at
least partially BIM-dependent [71]. Both of these stud-
ies suggest that TOR activity normally represses BIM
and that therapeutic inhibition of TOR will increase
BIM levels and contribute to tumour cell death. How-
ever, 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
mTORC2 (composed of mTOR, mLST8 and rictor)
phosphorylates PKB at Ser473, contributing to its acti-
vation. Rapamycin binds to FKBP12 and, in this
form, inhibits preformed mTORC1 complexes but not
preformed mTORC2; as a result, the effects of rapa-
mycin 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 do not, of course, detract from the strik-
ing synergy seen between MEK1 ⁄ 2 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 impor-
tant 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
- ⁄ -
back-
ground. 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 3
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 kill-
ing, when assayed by overexpression [4]. Despite this,
oncogene-targeted therapeutics alone can often cause
quite significant increases in BIM expression, but rela-
tively modest tumour cell death. Similarly, the
A. S. Gillings et al. BIM as a mediator of tumour cell death
FEBS Journal 276 (2009) 6050–6062 ª 2009 The Authors Journal compilation ª 2009 FEBS 6057
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-x
L
and BCL-2-like protein 2 with
high affinity and is thought to act by liberating BAX
and BAK from these proteins; in addition, BOPs dis-
placed 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 some-
times quite striking synergistic tumour cell killing
(Fig. 2).
Tumours with BRAF mutations
Even in tumour cells with BRAF
600E
that show strong
addiction to ERK1 ⁄ 2 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 pan-
MEK1 ⁄ 2 ⁄ 5 inhibitor, U0126, in a range of tumour-cell
lines harbouring BRAF
600E
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 SkMel-
28 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 BRAF
600E
.
NSCLC with EGFR mutations
In NSCLC cell lines that are sensitive to erlotinib or
gefitinib and exhibit drug-induced expression of BIM,
the induction of apoptosis is often modest. However,
two studies have now shown that erlotinib or gefitinib-
induced 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-x
L
are often expressed at high levels
in CML cells [62,63,78,79], and this prompted an
investigation of the efficacy of combinations of ABT-
737 and BCR-ABL inhibitors. Indeed, ABT-737 coop-
erates 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 combina-
tions of kinase inhibitors and BH3-mimetics may sub-
stantially 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
5 years has provided a good working model for
how BIM is inhibited by survival signalling pathways,
notably the ERK1 ⁄ 2 pathway, and has led to the
BIM as a mediator of tumour cell death A. S. Gillings et al.
6058 FEBS Journal 276 (2009) 6050–6062 ª 2009 The Authors Journal compilation ª 2009 FEBS

recognition that BIM plays a major response in
tumour cell death arising from inhibition of oncogenic
kinases. Indeed, the increased expression of dephos-
phorylated BIM
EL
could even be viewed as a biomar-
ker for drug-induced inactivation of ERK1 ⁄ 2 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-x
L
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 Asso-
ciation for International Cancer Research, Astra-
Zeneca, the Babraham Institute, the Biotechnology
and Biological Sciences Research Council and Cancer

Research UK.
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