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

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)

(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

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

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

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

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

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

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

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

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

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.

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

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-

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

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

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

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

Research UK.
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