ME65CH20-Wang ARI 11 October 2013 11:20

V I E W
E Review in Advance first posted online
R

on N ovember 4, 2013. (Changes may

S
still occur before final publication
online and in print.)
C E
I N

A
D V A

Targeting Apoptosis Pathways

for New Cancer Therapeutics
Longchuan Bai and Shaomeng Wang
University of Michigan Comprehensive Cancer Center and Departments of Internal
Medicine, Pharmacology, and Medicinal Chemistry, University of Michigan, Ann Arbor,
Annu. Rev. Med. 2014.65. Downloaded from www.annualreviews.org

Michigan 48109; email: shaomeng@umich.edu

by Grand Valley State University on 11/13/13. For personal use only.

Annu. Rev. Med. 2014. 65:20.1–20.17 Keywords

The Annual Review of Medicine is online at
med.annualreviews.org BCL-2, BCL-XL, XIAP, cIAPs, MDM2, p53
This article’s doi: Abstract
10.1146/annurev-med-010713-141310
The past decade has witnessed tremendous advances in the discovery
Copyright ⃝ c 2014 by Annual Reviews.
All rights reserved and development of novel small-molecule inhibitors targeting apoptosis
pathways for cancer treatment, with some compounds now in clinical
development. Early promising clinical data have been reported with
these new classes of anticancer drugs. This review highlights the recent
advancements in the development of small-molecule inhibitors target-
ing three major classes of antiapoptotic proteins: antiapoptotic B cell
lymphoma 2 (BCL-2) proteins, inhibitor of apoptosis proteins (IAPs),
and murine double-minute 2 (MDM2). Special emphasis is given to
those that have been advanced into clinical trials. The challenges and
future directions in the further preclinical and clinical development of
these new anticancer drugs are also discussed.

20.1

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 ME65CH20-Wang ARI 11 October 2013 11:20

EXPLOITING APOPTOSIS cytosol. B cell lymphoma 2 (BCL-2) family

PATHWAYS FOR CANCER proteins govern the MOMP (1). The release
TREATMENT of cytochrome c from the mitochondria initi-
ates the formation of a multiprotein complex
Cancer is a multifaceted disease comprised
(apoptosome) consisting of cytochrome c, ap-
of complex genetic and epigenetic aberrations
optotic protease activating factor 1 (APAF1),
that disrupt the normal balance of cellular life
and procaspase-9, resulting in the activation
and death. Apoptosis (programmed cell death)
of caspase-9 and downstream caspase cascade.
represents one of the most important forms
The release of SMAC from mitochondria fur-
of cell death in multicellular organisms and
ther stimulates caspase activation by neutral-
is typically dysregulated in human cancers.
izing IAPs, which regulate apoptosis through
Dysfunction of apoptosis pathways can con-
inhibition of caspases. The extrinsic pathway
fer cancer-treatment resistance, as most con-
is activated following the binding of death li-
ventional chemotherapies as well as radiother-
gands of the tumor necrosis factor (TNF) fam-
apy rely on their ability to provoke apoptotic
ily [e.g., TNFα, TRAIL (TNF-related apopto-
cell death in cancer cells. Therefore, apopto-
Annu. Rev. Med. 2014.65. Downloaded from www.annualreviews.org
by Grand Valley State University on 11/13/13. For personal use only.

sis inducing ligand), and Fas ligand] to their cell

sis pathways can be therapeutically exploited
surface receptors of the TNF receptor (TNFR)
for cancer treatment. Apoptosis is tightly regu-
superfamily (e.g., TNFR1 and 2, death recep-
lated by the balance between pro- and antiapop-
tor 4 and 5, and Fas). Upon ligand-receptor in-
totic proteins and becomes dysregulated when
teraction, a multiprotein complex is formed at
the balance between pro- and antiapoptotic
the plasma membrane, which provides a signal-
proteins is altered. In human cancers, overex-
ing platform for activation of initiator caspase-8
pression of antiapoptotic proteins is frequently
and subsequent executioner caspases. Caspase-
observed and has been linked to tumor progres-
8 also cleaves other noncaspase substrates, such
sion, treatment resistance, and poor progno-
as Bid. Truncated Bid (tBid) then translocates
sis. Therefore, targeting antiapoptotic proteins
to mitochondrial membrane to stimulate cy-
with the goal to restore the apoptotic balance
tochrome c release and activation of the intrin-
has been considered as a promising cancer ther-
sic pathway. IAPs thus play an important role in
apeutic strategy in the past decade. Drugs tar-
regulating both intrinsic and extrinsic apoptosis
geting antiapoptotic proteins may directly elicit
pathways.
apoptosis or lower the threshold for cell death
A wide array of apoptosis-related genes is
induction when combined with other cancer
transcriptionally regulated by tumor suppres-
therapeutics.
sor p53 protein. It has been well established
Two main apoptotic pathways have been
that p53 plays a central role in the regulation
extensively investigated: the intrinsic (mito-
of apoptosis and cell cycle transition, among
chondrial) and the extrinsic (death receptor)
many other cellular processes. p53 is mutated
pathways (Figure 1). The intrinsic pathway is
and inactivated in ∼50% of all human can-
activated by a myriad of stress signals, such
cers (http://www-p53.iarc.fr), implying the
as DNA damage caused by chemo- and ra-
importance of disabling p53 function during
diotherapies. Upon cellular stresses, a signal
tumorigenesis. The abundance and activity of
is relayed to the mitochondria, leading to
p53 are tightly controlled under normal physi-
the mitochondrial outer membranes perme-
ologic conditions. Changes in the regulation of
abilization (MOMP) and the release of ap-
p53 can result in the disturbance of p53 func-
optotic proteins, such as cytochrome c and
tion. By directly binding to the N terminus
second mitochondrial-derived activator of cas-
of p53 and promoting p53 ubiquitination and
pases (SMAC) [also known as DIABLO, di-
degradation, murine double-minute 2 (MDM2)
rect inhibitor of apotosis protein (IAP)-binding
[HDM2 (human homolog of MDM2) in hu-
protein with low pI] from mitochondria into
mans] is a crucial negative regulator of p53 (2).

20.2 Bai · Wang

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 ME65CH20-Wang ARI 11 October 2013 11:20

Death receptors (extrinsic pathway)

SMAC mimetics

cIAP1/2
GDC-0152
AT-406
RIP1 Cas-8 Cas-3/7 Apoptosis
TL32711
LCL161 cIAP1/2
HGS1029 degradation

XIAP
BCL-2 BH3 mimetics
BCL-XL ABT-263
NF-kB ABT-199
Annu. Rev. Med. 2014.65. Downloaded from www.annualreviews.org

GX15-070
by Grand Valley State University on 11/13/13. For personal use only.

MCL-1 Cas-9
Cytokines AT-101
SMAC mimetics
GDC-0152
AT-406
NOXA TL32711
MDM2 p53 PUMA BAX LCL161
HGS1029
BIM BAK
Cytochrome c
BID SMAC
MDM2 antagonists BAD
RO5045337
RO5503781
MI-77301/SAR405838
CGM097

Cellular stresses (intrinsic pathway)

Figure 1
Schematic representation of the extrinsic and intrinsic apoptotic pathways. The extrinsic (death receptor)
apoptotic pathway is initiated at the cellular membrane by the binding of cell death ligands to their
respective receptors, which triggers the formation of a death-inducing signaling complex to initiate caspase
activation cascade. cIAP1/2 can ubiquitinate RIP1 to prevent it from forming a caspases-8-activating
complex. cIAP1/2 also regulate canonical and noncanonical NF-κB pathways. The intrinsic (mitochondrial)
apoptotic pathway can be activated by various stimuli such as chemotherapeutics, which leads to the
compromise of mitochondrial membrane integrity and release of cytochrome c and SMAC into the cytosol.
Cytochrome c stimulates the formation of the apoptosome complex to activate initiator caspase-9 and
subsequent activation of effector caspases. XIAP directly binds to caspases and inhibits their activity. SMAC
blocks XIAP-mediated caspase inhibition. cIAP1/2 can bind to SMAC and sequester it from XIAP. SMAC
interacts with IAPs via its N-terminal AVPI binding motif. SMAC mimetics mimic this motif to antagonize
the inhibitory function of IAPs. BAX and BAK are the “effectors” of mitochondrial permeabilization.
Antiapoptotic members of the BCL-2 proteins counteract the activity of BAX and BAK, whereas the
proapoptotic BH3-only BCL-2 family proteins stimulate apoptosis either by directly interacting with BAX
and BAK or by binding to the antiapoptotic BCL-2 family proteins to release BAX and BAK. BH3 mimetics
competitively disrupt the inhibitory complexes between pro- and antiapoptotic BCL-2 family proteins. p53
promotes apoptosis by transcriptionally regulating apoptosis-related genes or directly activating BAX and
BAK. The stability and transcriptional activity of p53 is tightly regulated by MDM2 in a negative-feedback
loop. MDM2 antagonists competitively displace p53 from the MDM2 complex. Abbreviations: BCL-2,
B cell lymphoma 2; IAP, inhibitor of apotosis protein; MDM2, murine double-minute 2; SMAC, second
mitochondrial-derived activator of caspase.

www.annualreviews.org • Apoptosis-Based Cancer Therapies 20.3

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 ME65CH20-Wang ARI 11 October 2013 11:20
Tremendous efforts have been made by both
academia and the pharmaceutical industry to
design and develop small-molecule inhibitors
to restore apoptosis function in cancer cells.
This review focuses on recent advances in the
development and application of small-molecule
inhibitors targeting three major classes of anti-
apoptotic proteins: BCL-2/BCL-XL, IAP pro-
teins, and MDM2. Special emphasis is given to
those compounds that have been advanced into
clinical development.
TARGETING ANTIAPOPTOTIC
BCL-2 FAMILY PROTEINS FOR
CANCER THERAPY
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MOMP, a defining event in the intrinsic apo-
ptosis pathway, is controlled by BCL-2 family
proapoptotic effector proteins BAX and BAK,
which are activated by proapoptotic BH3-only
BCL-2 family proteins (i.e., BIM, BID, BAD,
PUMA, BMF, BIK, HRK, and NOXA) and an-
tagonized by antiapoptotic BCL-2 family pro-
teins (i.e., BCL-2, BCL-XL, BCL-w, BCL-B,
MCL-1, and A1/BFL-1) (1). Upon activation,
BAX and BAK form large oligomeric pores in
the mitochondrial outer membrane, resulting
in the release of proapoptotic factors and even-
tually cell death. Two different but not mutu-
ally exclusive models have been proposed to de-
scribe the regulation of BAX and BAK activity
by other BCL-2 family proteins. The indirect
model suggests that the sequestration of BAX
and BAK by the antiapoptotic BCL-2 family
proteins inhibits MOMP. Binding of the anti-
apoptotic proteins by some but not all BH3-
only BCL-2 proteins causes the activation of
BAK or BAX. The direct activation model sug-
gests that BIM, tBID, and PUMA, which are se-
questered by antiapoptotic BCL-2 family mem-
bers such as BCL-2, BCL-XL, and MCL-1, can
directly activate BAK and BAX (3, 4).
Members of the antiapoptotic BCL-2 pro-
teins are commonly overexpressed in a wide
range of cancers, including both hematologi-
cal malignancies and solid tumors, where they
contribute to tumorigenesis and the develop-
ment of resistance to chemo- and radiother-
20.4 Bai · Wang
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apies (reviewed in Reference 1). Therefore,
targeting antiapoptotic BCL-2 family proteins
is viewed as an attractive cancer therapeutic
strategy.
Small Molecular Inhibitors of
Antiapoptotic BCL-2 Family Proteins
Several approaches have been utilized to target
the antiapoptotic BCL-2 family proteins, and
one promising approach is to design nonpep-
tide, small molecules to mimic proapoptotic
BH3-only members of the BCL-2 family (BH3
mimetics). The antiapoptotic members of the
BCL-2 family contain a surface hydrophobic
groove that can bind and neutralize the BH3
death domains of proapoptotic members (5).
BH3 mimetics competitively bind into the
hydrophobic groove of these antiapoptotic
BCL-2 family proteins to liberate the proapop-
totic BCL-2 family proteins. A number of
natural product derivatives and rationally
designed compounds have been developed
as BH3 mimetics. These compounds include
ABT-737 and its oral analog ABT-263 (nav-
itoclax), ABT-199, GX15-070 (obatoclax),
R-(−)-gossypol (AT-101), WEHI-539, and
our BM series compounds, among others
(6–12). Although many of the reported com-
pounds exert cytotoxicity through additional
mechanisms, ABT-737/263/199, WEHI-539,
and our newly identified BM series compounds
exhibit antitumor activities by specifically
targeting one or more BCL-2 proteins (10, 13).
ABT-737 and ABT-263 (navitoclax). ABT-
737 was designed using a fragment-based strat-
egy and binds to BCL-XL, BCL-2, and BCL-w
with high affinity (Ki < 1 nM) but with much
lower affinity to BFL-1 and MCL-1 (7). ABT-
263 is an orally available derivative of ABT-737,
and compared with ABT-737, it has a very sim-
ilar binding profile to these antiapoptotic BCL-
2 proteins (7).
Preclinical studies have shown that ABT-
737 and ABT-263 have potent cytotoxicity
against a variety of hematologic malignancies.
However, most solid tumor cell lines that have
 ME65CH20-Wang ARI 11 October 2013 11:20
been studied are insensitive to the drugs, with
the exception of a subset of small-cell lung can-
cer cell lines (7, 14). Resistance to ABT-737 and
ABT-263 is frequently associated with over-
expression of the antiapoptotic BCL-2 family
member MCL-1 (15, 16), which is not targeted
by these compounds. Interestingly, despite its
similar high binding affinities for the antiapop-
totic BCL-2, BCL-XL, and BCL-w proteins in
biochemical assays, ABT-737 discriminates be-
tween the complexes formed between these an-
tiapoptotic proteins and proapoptotic BCL-2
family proteins in cells (17, 18). For instance,
ABT-737 targets BCL-2 with preference over
BCL-XL and BCL-w in leukemia cells (17, 18).
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In vitro, ABT-737 synergizes with conven-
tional chemotherapies, radiotherapy, as well as
tyrosine kinase inhibitors to overcome drug
resistance. Similarly, ABT-263 also signifi-
cantly enhances the activity of chemothera-
peutic agents and targeted therapies (19–22).
For example, ABT-263 enhances the efficacy of
both docetaxel and erlotinib in xenograft mod-
els of ovarian and lung cancers, concomitant
with the downregulation of MCL-1 and/or up-
regulation of BIM (20).
Several clinical trials of ABT-263 have been
conducted in patients with chronic lymphocytic
leukemia (CLL) (23), lymphoma (24), and lung
cancer (25). Partial response was observed in
one-third of patientswith relapsed or refrac-
tory (R/R) CLL (23). However, ABT-263 ex-
hibits limited single agent activity in small-cell
lung cancer (25). BCL-XL is critical for platelet
survival (26). Because ABT-263 potently tar-
gets BCL-XL, thrombocytopenia was observed
across all clinical trials for ABT-263 and is the
dose-limiting toxicity (27). Nevertheless, the
platelet decrease was transient and reversible,
with no clinically significant bleeding (23–25,
28). Several phase II combination trials are
ongoing in lymphoid cancers and solid tumors
(http://clinicaltrials.gov).
ABT-199 (GDC-0199, AbbVie). Given the
dose-limiting toxicity of ABT-263 in patients,
structure-guided reverse engineering of ABT-
263 led to the discovery of ABT-199, the
Changes may still occur before final publication online and in print
first-in-class BCL-2-selective inhibitor with
subnanomolar affinity for BCL-2 (6). ABT-199
potently disrupts BCL-2 function and induces
apoptosis in cancer cells with high BCL-2
expression, such as leukemia and lymphoma
(6). Compared with ABT-263, ABT-199 is
substantially less toxic against platelets (6, 29).
In phase I trials, ABT-199 has demonstrated
substantially greater activity than that seen with
ABT-263 in patients with either R/RCLL or
R/R non-Hodgkin lymphoma and also with no
significant effects on platelets counts (6). Tu-
mor lysis syndrome is dose limiting for ABT-
199 in patients with CLL, and an 85% over-
all response rate was achieved in CLL (30, 31).
Ongoing clinical study of ABT-199 includes a
combination trial with bendamustine and rit-
uximab in patients with R/Rnon-Hodgkin lym-
phoma (http://clinicaltrials.gov).
Obatoclax mesylate (GX15-070) (Gemin
X/Teva). Obatoclax is a pan-BCL-2 inhibitor
that binds to BCL-2, BCL-XL, BCL-w, BCL-
B, BFL-1, and MCL-1 in vitro, albeit with
much lower affinities than those of ABT-263 to
BCL-2, BCL-XL, and BCL-w (32, 33). In light
of the fact that MCL-1 can confer resistance
to ABT-263, the ability of obatoclax to target
MCL-1 represents a potential advantage over
ABT-263. Obatoclax can efficiently diminish
the interactions between MCL-1 and proapop-
totic proteins to activate the intrinsic apopto-
sis pathway (33). However, obatoclax also in-
duces apoptosis in the absence of BAX/BAK
and arrests cancer cells at the G2 phase, sug-
gesting that it exerts cytotoxicity through mul-
tiple mechanisms (13, 34).
Phase I trials of obatoclax in leukemia and
lymphoma have identified transient neurotox-
icity as the most common adverse effect (35).
In these studies, a limited number of ob-
jective responses were observed, along with
hematological improvement in a larger propor-
tion of treated patients (35). Several phase II
combination trials are ongoing in hematolog-
ical malignancies and advanced solid tumors
(http://clinicaltrials.gov).
www.annualreviews.org • Apoptosis-Based Cancer Therapies 20.5
 ME65CH20-Wang ARI 11 October 2013 11:20
AT-101 [R-(−)-gossypol] (Ascenta). AT-
101, a polyphenolic compound derived from
cotton seeds and roots, is an oral pan-BCL-2
inhibitor. AT-101 exhibits affinity for BCL-2,
BCL-XL, and MCL-1 at submicromolar con-
centrations. It induces apoptosis by acting as
a BH3 mimetic as well as a p53-independent
activator of NOXA and PUMA. The cytotoxi-
city of AT-101 may also result from additional
mechanisms, such as DNA cleavage and/or the
generation of reactive oxygen species (36).
AT-101 was well-tolerated in patients and
demonstrated single-agent cytoreductive activ-
ity in several malignancies, including prostate
cancer (37). However, early phase II trials
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in prostate and lung cancers, either used as
monotherapy or in combination with conven-
tional chemotherapies, failed to deliver signif-
icant clinical activity (37–40). Several phase II
combination trials are ongoing in squamous cell
carcinoma of the head and neck and advanced
laryngeal cancer (http://clinicaltrials.gov).
Challenges and Future Directions
Preclinical and clinical studies have provided
valuable information regarding the tolerability
and “on-target” toxicity of BH3 mimetics in
patients. These studies have also demonstrated
promising clinical activity in some patients, as
seen with ABT-263 in CLL and more recently
with ABT-199. However, additional transla-
tional research is required to realize the full po-
tential of this exciting class of targeted drugs.
On-target toxicities. The dependence of
platelets on BCL-XL for survival presents a sig-
nificant challenge to the clinical application of
ABT-263 and other BCL-XL-targeting BH3
mimetics (27). Preclinical studies have demon-
strated that the on-target toxicities of ABT-263
on platelets are greatest at high doses and thus
suggest avoiding a loading dose of ABT-263 to
minimize platelet nadir. Indeed, a regimen with
a low priming dose before dose escalation has
proven safe and effective in early-stage clinical
trials of ABT-263 monotherapy (23–25). The
clinical data on ABT-199 demonstrated that se-
lective targeting of BCL-2 can achieve substan-
20.6 Bai · Wang
Changes may still occur before final publication online and in print
tial antitumor activity in BCL-2-driven tumors,
while avoiding on-target toxicity by ABT-263
(6, 29).
MCL-1-mediated drug resistance. MCL-1
is recognized as one of the major resistance
factors to ABT-737/263. Therefore, strate-
gies are being developed to tackle MCL-
1-mediated resistance. One approach is to
develop high-affinity pan-BCL-2 inhibitors.
Other approaches include combinational treat-
ment with MCL-1-selective inhibitors or with
conventional chemotherapeutics that reduce
MCL-1 levels or antagonize MCL-1 by upreg-
ulating BIM and/or NOXA (19–21, 22).
Biomarkers for therapeutic response. Pre-
clinical studies suggest that the ratio between
BCL-2 and MCL-1 or between antiapoptotic
and proapoptotic BCL-2 family proteins can
predict cancer cell response to ABT-737. For
example, BH3 profiling has demonstrated that
the interactions between and the relative ra-
tios of pro- and antiapoptotic BCL-2 family
members, but not the BCL-2 protein levels,
determine cellular sensitivity to ABT-737 (42,
43). In primary CLL cells, BCL-2 expression
alone does not dictate sensitivity to ABT-737.
Instead, the amount of BCL-2-BIM complex
predicts the sensitivity to ABT-737 (44). Other
preclinical studies have shown that the ratio of
(MCL-1 + BFL-1):BCL-2 or BCL-2:MCL1
predicts the response to ABT-737 in CLL and
MM cell lines (45, 46). In patients with CLL,
low MCL1 expression and high BIM:MCL-1
or BIM:BCL-2 ratios in leukemic cells corre-
lated with response to ABT-263 (23). These
data suggest the importance of profiling both
anti- and proapoptotic BCL-2 family mem-
bers to identify biomarkers for BH3 mimetics
sensitivity.
TARGETING IAPS FOR
CANCER THERAPY
Release of apoptotic proteins, such as cy-
tochrome c and SMAC, from the mitochondria
into the cytosol is a deciding event in the
intrinsic apoptosis pathway. SMAC promotes
 ME65CH20-Wang ARI 11 October 2013 11:20
caspase activation and apoptosis by binding to
cIAP1/2 and XIAP proteins via its N-terminal
Ala-Val-Pro-Ile (AVPI) motif to block their
antiapoptotic activities (47).
Despite their names, IAPs vary in their
ability to block apoptosis. XIAP directly binds
to caspase-3, -7, and -9 through its BIR2 or
BIR3 domains and inhibits their activities.
cIAPs, which are not potent inhibitors of
caspases, bind to SMAC with high affini-
ties and can prevent SMAC from blocking
XIAP-mediated inhibition of caspases (48).
Notably, cIAPs regulate the extrinsic apoptosis
pathway through their E3 ubiquitin ligase
activity. Particularly, cIAPs are responsible
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for RIP1 ubiquitination, preventing it from
forming a complex with caspases-8 and Fas-
associated protein with death domain (FADD),
and subsequent caspase activation (49, 50).
cIAPs are also key regulators of canonical
and noncanonical NF-κB pathways. The E3
ligase activity of cIAPs promotes the activation
of canonical NF-κB signaling by stimulating
K63 ubiquitin on RIP1 (49, 50). Nevertheless,
cIAP1 constitutively suppresses the abundance
of NF-κB-inducing kinase in the noncanonical
NF-κB pathway (51, 52). NF-kB induces the
expression of diverse target genes involved
in inflammation, proliferation, survival, cell
death, angiogenesis, migration, and invasion.
Elevated expression of IAPs has been ob-
served in hematological malignancies and solid
tumors, and it correlates with disease progres-
sion, metastasis, treatment resistance, and poor
prognosis. For example, XIAP overexpression
correlates with chemoresistance and poor prog-
nosis in pancreatic cancer and advanced head
and neck cancer. The chromosome 11q21–q23
region, which harbors cIAP1 and cIAP2 loci, is
amplified in a variety of solid tumors (reviewed
in References 53, 54). Thus, IAPs are attractive
targets for novel cancer therapeutics.
Small-Molecule SMAC Mimetics as
Antagonists of IAPs
The discovery that the IAP-binding motif
of SMAC contains only four amino acids
Changes may still occur before final publication online and in print
(Ala-Val-Pro-Ile) inspired the design of small
molecules mimicking this motif as SMAC
mimetics and antagonists of IAPs. There are
two types of SMAC mimetics: monovalent,
which imitates one AVPI motif, and bivalent,
which contains two APVI-like structures con-
nected by a linker. Resembling the endogenous
active SMAC, bivalent SMAC mimetics have
higher affinity to XIAP because they bind
simultaneously to the BIR2 and BIR3 domains.
As single agents, SMAC mimetics exert cy-
totoxicity in a small subset of cancer cell lines
(55). SMAC mimetics bind to cIAPs, result-
ing in their ubiquitination and subsequent pro-
teasomal degradation followed by stabilization
of NF-κB-inducing kinase and activation of
the noncanonical NF-κB pathway (56). Acti-
vated NF-κB signaling stimulates the transcrip-
tion and production of TNFα, which activate
TNFR1 signaling in an autocrine or paracrine
manner (56). In the absence of cIAPs, un-
ubiquitinated RIP1, together with FADD and
caspase-8, forms a death-signaling activation
platform to activate caspase-8 and provoke ap-
optosis (56). Targeting both cIAPs and XIAP
appears to be critical for efficient cell death in-
duction by SMAC mimetics (57, 58).
Given the limited cytotoxicity of SMAC
mimetics in most cancer cell lines, the antitu-
mor effects of SMAC mimetics have been ex-
tensively examined in combination with other
cancer therapeutics, such as chemo- and ra-
diotherapy, death receptor agonists, small-
molecule inhibitors of kinases, BCL-2, and
proteasome. Synergistic responses have also
been reported in numerous studies using in
vitro and mouse xenograft models (reviewed in
Reference 53). In most cases, the reported syn-
ergy is independent of TNFα but dependent
on the antagonism of XIAP.
GDC-0152 and GDC-0917 (CUDC-427)
(Genentech). GDC-0152 was the first SMAC
mimetic to enter clinical trials. It binds to the
BIR3 domain of XIAP, cIAP1, and cIAP2 as
well as the BIR domain of ML-IAP with Ki
values <50 nM (59). In humans, GDC-0152
exhibited linear pharmacokinetics over a wide
www.annualreviews.org • Apoptosis-Based Cancer Therapies 20.7
 ME65CH20-Wang ARI 11 October 2013 11:20

range of doses with intravenous administration served in patients with diverse tumor types, in-
(59). GDC-0917 is an oral monovalent SMAC cluding breast cancer (66).
mimetic that has shown a favorable safety, PK,
and PD profile in patients with advanced ma- AEG40826/HGS1029 (Aegera/Human Ge-
lignancies (60). nome Sciences). HGS1029 is a bivalent
SMAC mimetic, showing a good tolerability
profile with grade 2 transient lymphopaenia
Birinapant (TL32711) (TetraLogic Phar-
and neutrophilia reported in some patients.
maceuticals). Birinapant is a bivalent SMAC
HGS1029 displays proportional pharmacoki-
mimetic (61). It induces IAP- and caspase-
netics across wide dose ranges. It has also
dependent but TNFa-independent apoptosis.
demonstrated predicted biomarker modu-
It also enhances TRAIL potency in inflam-
lation, such as cIAP1 downregulation and
matory breast cancer models (61). A phase
MCP-1 upregulation (68).
I clinical trial of birinapant has showed that
intravenous birinapant is well tolerated with
SM-406/AT-406/Debio 1143 (University
no dose-limiting toxicities (62). Birinapant also
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demonstrated strong correlation among drug
exposure, predicted biomarker modulation
[e.g., cIAP1 degradation and upregulation of
interleukin-8 (IL-8) and monocyte chemoat-
tractant protein 1(MCP-1)], and apoptosis
induction in tumors (62). Furthermore,
birinapant can be combined with multiple
chemotherapies with excellent tolerability at
standard dosing, and Bell’s palsy was consid-
ered as a dose-limiting toxicity (63). A phase II
study demonstrated clinical benefit of the
combination of birinapant with irinotecan
over irinotecan alone in R/R colorectal cancer
patients, with a 57% overall clinical benefit and
the greatest benefit in KRAS mutant colorectal
cancers (64). An ascending-dose strategy of
birinapant also shows benefits in mitigating
Bell’s palsy risk (64).
LCL161 (Novartis). LCL161, an orally avail-
able IAP antagonist, displays antiproliferative
activities in a subset of tumors from diverse
lineages as a single agent and acts synergisti-
cally with paclitaxel in preclinical models (65,
66). LCL161 demonstrates target antagonism
by causing cIAP1 protein degradation and up-
regulation of circulating chemokines. It is also
well tolerated, with neutropenia, fatigue, and
neuropathy as the principal dose-limiting tox-
icities (66, 67). LCL161 was tested in combi-
nation with paclitaxel in patients with advanced
solid tumors, and objective responses were ob-
of Michigan/Ascenta/DebioPharm). AT-
406 is a monovalent, orally bioavailable SMAC
mimetic that binds to XIAP, cIAP1, and cIAP2
proteins, with a Ki of 66.4, 1.9, and 5.1 nM, re-
spectively (69). Preclinical studies have shown
that AT-406 exerts mechanism-based antitu-
mor activity in selected cancer cell lines and
xenograft tumor models. AT-406 has entered
clinical trials for studies of its safety, pharma-
cological properties, and pharmacodynamics
biomarkers.
Challenges and Future Directions
In the past few years, considerable progress has
been made in the preclinical proof-of-efficacy
studies of SMAC mimetics. Extensive preclini-
cal studies have also identified several outstand-
ing challenges related to developing SMAC
mimetics as a novel therapy for cancer. These
challenges include the knowledge gaps in our
understanding of the biological consequences
of IAP antagonism in normal human tissues, as
well as rational design of SMAC mimetic-based
combination strategies, among others.
TNFα-mediated toxicity. All SMAC mimet-
ics, perhaps with the exception of TL32711
(61), induce TNFα-dependent apoptosis fol-
lowing cIAPs degradation and NF-κB activa-
tion in cancer cells. TNFα is a pleiotropic
cytokine that provokes a variety of cellular
responses, depending on the cellular context.

20.8 Bai · Wang

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 ME65CH20-Wang ARI 11 October 2013 11:20
Pertinent concerns include the potential ad-
verse effects of SMAC mimetic–stimulated NF-
κB activation and cytokine production on nor-
mal tissues. GDC-0152, for example, causes an
acute induction of TNFα in the plasma of dogs
and rats (70). Accordingly, GDC-0152 demon-
strates a toxicity profile consistent with TNFα-
mediated toxicity. The onset and resolution of
these acute toxicities generally track with the
GDC-0152-induced cytokine time course (70).
Despite these potential concerns in preclini-
cal studies, severe cytokine release syndrome,
caused by an acute increase in plasma TNFα
and other inflammatory cytokines, has not been
reported in patients (66–68).
Annu. Rev. Med. 2014.65. Downloaded from www.annualreviews.org
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Nonapoptotic functions of IAPs. Accumu-
lating evidence has shown that IAP proteins
also exert nonapoptotic functions, which mostly
stem from IAPs’ characteristics as E3 ubiquitin
ligases and regulators of NF-kB signaling. For
example, IAPs play a role in cell motility, mi-
gration, invasion, and metastasis. Therefore, by
targeting IAPs, SMAC mimetics can have an
effect on cell motility, migration, invasion, and
metastasis.
Combination with chemotherapeutic
drugs. Chemotherapies, along with radio-
therapy and surgery, are still the mainstays of
cancer treatments today. Genotoxic agents,
such as etoposide, can activate cell death
pathways via proteasome-dependent depletion
of cIAPs and XIAP to promote the assembly of
a cytoplasmic cell death–activating platform,
ripoptosome (71, 72). The core components of
ripoptosome are RIP1, FADD, and caspase-8;
these components are similar to those in
complex IIB originating from an interaction
between the death receptor and death ligand.
However, the formation of ripoptosome is
independent of any death receptors. Notably,
SMAC mimetics stimulate the formation
of ripoptosome in etoposide-treated SMAC
mimetic- -resistant cancer cells (72). In
glioblastoma cells, SMAC mimetics cooperate
with temozolomide (TMZ) to stimulate the for-
mation of a RIP1/caspase-8/FADD complex,
Changes may still occur before final publication online and in print
resulting in the sensitization of TMZ-induced
apoptosis (73). Because chemotherapeutics
are commonly associated with undesirable
side effects, their combination with SMAC
mimetics may lower the therapeutic doses for
chemotherapies without compromising the
efficacy and accordingly may ameliorate the
side effects associated with chemotherapies.
Therefore, combining SMAC mimetics with
conventional chemotherapies may hold con-
siderable clinical promise. Promising evidence
from clinical activity in phase I trials provides
support for this notion: Combining birinapant
with multiple chemotherapeutics at standard
dosing schedules has yielded excellent tolera-
bility in patients with advanced solid cancers
(63).
TARGETING MDM2 FOR
CANCER THERAPY
The p53 tumor suppressor protein plays a cen-
tral role in the regulation of apoptosis, the
cell cycle, and senescence, among many other
cellular processes, through both transcription-
dependent and -independent mechanisms (re-
viewed in 74). p53 promotes apoptosis by
transcriptionally regulating genes directly in-
volved in apoptosis, such as death receptors
(e.g., Fas and DR5), proapoptotic BCL-2 fam-
ily members (e.g., BAX, NOXA, and PUMA),
and APAF1, among many others. Moreover,
cytosolic p53 can directly activate proapop-
totic BAX, leading to mitochondrial mem-
brane permeabilization and cytochrome c re-
lease (75). p53 present at the mitochondria
physically associates with proapoptotic BAK,
disrupting BAK sequestration by MCL-1 and
resulting in BAK activation (76). In addition,
mitochondrial-localized p53 can bind to BCL-
2 or BCL-XL, promoting the release of se-
questered proapoptotic proteins (77).
p53 is normally expressed at low levels so
that it would not cause abnormal cell death
or cell cycle arrest. Multiple mechanisms ex-
ist to control the transcriptional activity, stabil-
ity, and subcellular localization of p53 protein.
MDM2 is a crucial negative regulator of p53.
www.annualreviews.org • Apoptosis-Based Cancer Therapies 20.9
 ME65CH20-Wang ARI 11 October 2013 11:20
MDM2 contains a p53 binding domain at the N
terminus and a RING (really interesting gene)
domain at the C terminus functioning as an E3
ligase. MDM2 inhibits p53 by at least two dif-
ferent mechanisms: (a) It binds to the transac-
tivation domain of p53 and blocks its transcrip-
tional activity (78, 79), and (b) it functions as
an E3 ubiquitin ligase and promotes the ubiq-
uitination and proteasome-mediated degrada-
tion of p53 [1]. p53 also activates the tran-
scription of MDM2. Increased expression of
MDM2 leads to the degradation and inactiva-
tion of p53 in a feedback loop. Gene amplifica-
tion and/or overexpression of MDM2 has been
reported in a wide range of human malignancies
Annu. Rev. Med. 2014.65. Downloaded from www.annualreviews.org
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and is mutually exclusive to p53 mutation in a
significant subset of tumors, underscoring the
role of MDM2 in tumorigenesis (81). There-
fore, antagonizing MDM2 to release p53 from
its negative control is an appealing strategy for
the treatment of cancers with wildtype (WT)
p53.
Small-Molecule Inhibitors of
MDM2-p53 Interaction
Targeting MDM2
Genetic and biochemical studies have mapped
p53-MDM2 binding sites to the N termi-
nus of MDM2 and the transactivation domain
of p53 (78). The crystal structure of a p53-
derived peptide bound to the p53 binding do-
main of MDM2 revealed that three amino acid
residues from the p53 peptide (Phe19, Trp23,
and Leu26) project residues deep into the hy-
drophobic cavity of the p53 pocket in MDM2,
making them critical to the binding of these
two proteins (82). Small molecule inhibitors of
MDM2 have been developed by mimicking the
p53 binding residues, and several such com-
pounds are now in clinical development for can-
cer treatment.
Nutlins (Roche). Nutlins were the first class
of potent, specific, and orally bioavailable
MDM2 inhibitors (83). Nutlins mimic p53 and
bind to the p53-binding pocket in MDM2,
leading to p53 stabilization and activation
20.10 Bai · Wang
Changes may still occur before final publication online and in print
and subsequent cell cycle arrest, apoptosis,
and senescence in cancer cells with WT p53
(84). Nutlin-3 has been used extensively as a
tool compound to investigate the therapeutic
potential and mechanisms of action of MDM2
inhibitors in vitro and in vivo. Nutlin-3
selectively inhibits cell growth in tumor cell
lines with WT p53 over those with mutated or
deleted p53 status. Although Nutlin-3 activates
p53 in all tumor cell lines with WT p53 and in-
duces p53-dependent cell cycle arrest, it induces
apoptosis in only a subset of cancer cell lines.
RG7112 (RO5045337), a derivative of
Nutlin-3 with improved binding affinity to
MDM2 and pharmacokinetics, was advanced
into clinical trials (85, 86). RG7112 was well
tolerated in patients with liposarcoma, some
of whom experienced drug-related hemato-
logical toxicity such as thrombocytopenia,
neutropenia, and neutropenic fever (87). Of
the 17 patients with MDM2 gene ampli-
fication, 1 patient had a confirmed partial
response and 14 had stable disease (87).
RG7112 is currently in Phase I studies for pa-
tients with advanced solid tumor or leukemia
and in combination trial in patients with
AML (http://clinicaltrials.gov). RO5503781
(RG7388, Roche) from a different chemical
class is another orally bioavailable MDM2
inhibitor [2] in phase I clinical trials in patients
with advanced malignancies (monotherapy)
or with AML (monotherapy or in combination
with cytarabine) (http://clinicaltrials.gov).
MI-219 and MI-77301 (University of
Michigan/Ascenta/Sanofi). MI-219 contains
a spiro-oxindole core structure and was discov-
ered using a structure-based de novo design
strategy to mimic the key p53 binding residues
by the Wang laboratory at the University of
Michigan. It binds to MDM2 with a high affin-
ity and demonstrates a very high specificity over
MDMX and other proteins (88). MI-219 has a
well-defined mechanism of action as a potent
and specific MDM2 inhibitor in vitro and in
vivo and demonstrates potent on-target antitu-
mor activity and excellent tolerability in animal
models (88). Further chemical modifications of
 ME65CH20-Wang ARI 11 October 2013 11:20
MI-219 by the Wang laboratory to improve its
binding affinity to MDM2, on-target cellular
activity, and pharmacokinetics led to the discov-
ery of MI-77301 (SAR405838), which has been
advanced into clinical development by Sanofi.
CGM097 (Novartis). CGM097 is another
oral MDM2 inhibitor entering human clinical
trials in early 2013 (http://clinicaltrial.gov),
although no information is disclosed about its
chemical structure and in vitro and in vivo
characterizations.
Challenges and Future Directions
Annu. Rev. Med. 2014.65. Downloaded from www.annualreviews.org
by Grand Valley State University on 11/13/13. For personal use only.
Targeting MDM2 to reactivate p53 offers excit-
ing prospects in cancer treatment. This notion
is supported by promising preclinical results.
As with other targeted drugs, additional studies
are needed to thoroughly investigate the poten-
tial side effects of and emergence of drug resis-
tance to MDM2 inhibitors in clinically relevant
settings.
Potential side effects. Several studies have
shown that unleashing p53 from MDM2 sup-
pression could have detrimental effects on
the normal tissues. For example, deletion of
MDM2 in mice is embryonic lethal, which can
be rescued by concurrent deletion of p53 (89,
90). Deletion of MDM2 in specific types of
cells, such as neuronal progenitors and car-
diomyocytes, also leads to p53-dependent em-
bryonic lethality (91–93). Mice with mutations
at S21 and T23 of p53 to evade MDM2 bind-
ing exhibit constitutive p53 activation and seg-
mental progeria that is correlated with the de-
pletion of adult stem cells in multiple tissues,
including bone marrow, brain and testes (94).
Further, MDM2 heterozygous mice are sensi-
tive to low-dose irradiation treatment (95). All
these observations raise the question whether
activation of p53 by MDM2 inhibitors will be
toxic to normal tissues, especially when under
stresses. Preclinical studies have demonstrated
good tolerability of Nutlin-3 and MI-219 in
mice with no manifest signs of toxicity (83,
88). Moreover, Nutlin-3 synergizes with the
Changes may still occur before final publication online and in print
DNA-damaging agent topotecan in preclini-
cal retinoblastoma models without any obvious
side effects in mice (96), implying that MDM2
inhibitors may not be toxic to normal tissues
even when combined with genotoxic therapies.
These observations suggest that pharmacolog-
ical inhibition of MDM2 activity does not fully
phenocopy those of MDM2 gene deletion in
terms of the toxicity to normal tissues.
One critical difference between activation of
p53 via genetic deletion of MDM2 and pharma-
cological activation of p53 by MDM2 inhibitors
is the presence of high levels of MDM2 in tis-
sues where p53 is activated for the pharmaco-
logical approach due to the feedback loop of
p53-MDM2 regulation (83, 88). The induction
of MDM2 accumulation by MDM2 inhibitors
may achieve attenuated p53 activation that is
sufficient for strong antitumor activity with-
out causing toxicity in normal tissues. Indeed,
phase I data for RG7112 from patients with ad-
vanced cancer demonstrate a good tolerability
with neutropenia being the major toxicity, sug-
gesting that activation of p53 in the bone mar-
row by MDM2 inhibitors may be responsible
for its on-target toxicity.
MDMX-mediated drug resistance. Al-
though MDM2 inhibitors such as nutlins and
MI-219 efficiently induce cell cycle arrest
in all tumor cell lines with WT p53 status,
they induce apoptosis in only a subset of
WT p53 cancer lines (84, 88). MDMX,
which is not efficiently antagonized by nutlins
or MI-219, has been proposed as a major
determinant for drug resistance (88, 97).
Indeed, knockdown of MDMX by siRNAs
sensitizes WT p53 cancer cells to nutlin- or
MI-219-induced apoptosis (88, 98). Therefore,
combinations of selective MDM2 inhibitors
with agents that can directly or indirectly target
MDMX may achieve synergy. It is recently
reported that Hsp90 inhibitor 17AAG can
destabilize MDMX protein and potentiates
nutlin-induced p53 transcription of PUMA.
Consequently, 17AAG synergizes with nutlin,
in part through inhibiting MDMX and AKT
signaling, to induce apoptosis in WT p53
www.annualreviews.org • Apoptosis-Based Cancer Therapies 20.11
 ME65CH20-Wang ARI 11 October 2013 11:20

cell lines and their xenografts in mice (99).
Dual inhibitors of MDM2 and MDMX or
MDMX-selective inhibitors are also under
intensive development, which is beyond the
scope of this review.
Because the activity of MDM2 inhibitors de-
pends on activation of WT p53, MDM2 in-
hibitors may induce p53 mutation, which would
render tumor cells resistant. In fact, treatment
of p53 WT SJSA-1 cells with Nutlin-3 in vitro
leads to the selection for p53 mutations and
subsequent drug resistance (100), although this
observation has yet to be confirmed in vivo.
Nevertheless, drug-induced p53 mutation by
MDM2 inhibitors should be evaluated in clini-
Annu. Rev. Med. 2014.65. Downloaded from www.annualreviews.org
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hibitors targeting the IAP proteins (cIAP1/2
and XIAP), and inhibitors targeting MDM2
to reactivate p53. In each case, several com-
pounds have been advanced into clinical de-
velopment. Evidence of on-target activity has
been observed in patients for each class of com-
pounds with a manageable toxicity profile for
these apoptosis-inducing agents, and objective
clinical responses have been demonstrated. Per-
haps not surprisingly, these molecularly tar-
geted agents are effective only as single agents in
a subset of human cancer models in preclinical
evaluation. Accordingly, development of pre-
dictive biomarkers will be critical for their suc-
cessful development as monotherapies. In ad-

cal trials. dition, because it is expected that these new

CONCLUDING REMARKS
Over the past decade, major advancements
have been made in the development of small
molecule inhibitors to target the apoptosis
pathways. In this review, we focus on three
different classes of compounds: inhibitors tar-
geting the antiapoptotic BCL-2 proteins, in-
compounds will be combined with standard of
care (chemotherapy and radiation therapy) in
most cases, development of rational combina-
tion strategies will be essential. Moreover, in-
depth understanding of de novo or acquired
resistant mechanisms is needed to develop
strategies to combat clinical resistance that has
been observed for all molecularly targeted an-
ticancer drugs.

DISCLOSURE STATEMENT
S.W. is a consultant and owns stock in Ascenta and Ascentage Pharma, which have licensed IAP,
MDM2, or BCL-2/BCL-XL inhibitors from the University of Michigan for clinical development,
and receives royalty from the University of Michigan on these inventions.

ACKNOWLEDGMENTS
We thank the past and present members of the Wang laboratory for their contributions to the
discovery of small molecule inhibitors targeting BCL-2, IAP, and MDM2 and our development
partners (Ascenta, Sanofi, DebioPharma, and Ascentage) for clinical development of these new
agents. Funding from the National Cancer Institute, National Institutes of Health, the Department
of Defense, the Breast Cancer Research Foundation, the Prostate Cancer Foundation, the Susan
G. Komen Foundation, the Leukemia and Lymphoma Society, Ascenta, Sanofi, and Ascentage
Pharma is greatly appreciated. The authors apologize to the many authors whose works have not
been cited owing to space limitations or the narrow scope of this review.

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