Oncogene (2013) 32, 4191–4202

& 2013 Macmillan Publishers Limited All rights reserved 0950-9232/13

www.nature.com/onc

REVIEW
Thalidomide-analogue biology: immunological, molecular
and epigenetic targets in cancer therapy
J Shortt1,2,3, AK Hsu4 and RW Johnstone1,5

Thalidomide and its analogues (lenalidomide and pomalidomide) are small molecule glutamic acid derivatives of the
immunomodulatory drug (IMiD) class. In addition to the immuno-adjuvant and anti-inflammatory properties that define an IMiD,
the thalidomide analogues demonstrate an overlapping and diverse range of biological activities, including anti-angiogenic,
teratogenic and epigenetic effects. Importantly, the IMiDs possess anti-cancer activity with selectivity for molecularly defined
subgroups of hematological malignancies, specifically mature B-cell neoplasms and myelodysplasia with deletion of chromosome
5q. Emerging insight into the pathophysiological drivers of these IMiD-responsive disease states can now be synthesized using
previously disclosed IMiD activities and recently discovered thalidomide targets to build unifying models of IMiD mechanism of
action. Attention to mechanisms of IMiD-induced clinical toxicities, in particular the recently identified association of lenalidomide
with second primary malignancies, provides an additional tool for determination of drug mechanism. This review seeks to define
the molecular IMiD targets and biological outputs that underpin their anti-neoplastic activity. It is anticipated that elucidation of
important IMiD targets will allow the rational development of new-generation therapeutics with the potential to separate
thalidomide-analogue efficacy from clinical toxicity.

Oncogene (2013) 32, 4191–4202; doi:10.1038/onc.2012.599; published online 14 January 2013

Keywords: immune modulation; therapeutics; hematological malignancies

INTRODUCTION widespread withdrawal of thalidomide from clinical use in 1961.1–3

Thalidomide analogues are immunomodulatory drugs (IMiDs) with The serendipitous discovery of thalidomide’s efficacy in erythema
a range of biological effects and putative molecular targets. nodosum leprosum (ENL) reawakened interest in the drug.4,5
Thalidomide itself has clinical efficacy as an anti-inflammatory and Emerging insight into thalidomide’s anti-inflammatory and anti-
in a distinct cohort of neoplastic diseases including myelodyspla- angiogenic activity led to clinical trials in AIDS-related aphthous
sia (MDS) and multiple myeloma (MM). Despite an initially ulceration,6,7 Behcet’s syndrome,8 Crohn’s disease,9 cutaneous
infamous track record that spans 450 years of human exposure, lupus10 and various malignancies. The greatest testament to
thalidomide’s precise mechanisms of action remain undefined. thalidomide’s efficacy was that despite its teratogenicity it was re-
The diverse biological properties of thalidomide analogues instated as an FDA-approved treatment for ENL and MM (in 1998
indicate that they might possess divergent mechanistic activities and 2006, respectively, both available at http://www.fda.gov; last
in different diseases. However, thalidomide analogues are not accessed 28/05/2012).
indiscriminate anti-neoplastic agents. Rather, their clinical
specificity for a relatively narrow range of clearly definable
malignancies suggests a molecularly targeted mechanism of DEFINITION OF IMID
action, intricately linked to the biology of these susceptible The term IMiD refers to IMiDs licensed by Celgene Corporation
disease states. (Summit, NJ, USA) for the treatment of several inflammatory and
neoplastic diseases.11 The IMiDs were initially defined by their
capacity to inhibit lipopolysaccharide (LPS)-induced monocyte
HISTORICAL NOTE tumor necrosis factor (TNF)-a secretion, and further stratified by
It is speculated that thalidomide was tested as an antidote to their relative effects on other cytokines. Thus ‘class I’ IMiDs were
neurotoxins during World War II. This is consistent with characterized as broad inhibitors of LPS-induced inflammatory
thalidomide’s initial clinical indication as a sedative with anti- cytokines (for example, TNFa, interleukin (IL)-1b, IL-6 and IL-12),
emetic properties.1 Thalidomide was subsequently prescribed for whereas ‘class II’ compounds (so-called SelCIDS) showed
anxiety- and nausea-related conditions, including ‘morning selectivity for TNFa suppression.12 The SelCIDS are structurally
sickness’ to pregnant women in the 1950s.1 The ensuing diverse compounds unified by their capacity to antagonize
tragedy of birth defects including characteristic limb deformities phosphodiesterase-4. Class I (non-phosphodiesterase-4 inhibitory)
(dysmelia; stunted limb growth) is well documented and led to the IMiDs comprise thalidomide and its amino-substituted analogues
1
Gene Regulation Laboratory, Cancer Therapeutics Program, Peter MacCallum Cancer Centre, Locked Bag 1, A’Beckett St, Melbourne, Victoria, Australia; 2Department of Clinical
Haematology, Monash Medical Centre, Clayton, Victoria, Australia; 3Department of Medicine, Southern Clinical School, Monash University, Clayton, Victoria, Australia;
4
Haematology Immunology Translational Research Laboratory, Cancer Immunology Program, Peter MacCallum Cancer Centre, Locked Bag 1, A’Beckett St, Melbourne, Victoria,
Australia and 5Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, Victoria, Australia. Correspondence: Professor RW Johnstone, The Peter
MacCallum Cancer Institute, Cancer Therapeutics Program, St Andrews Place, East Melbourne 3002, Victoria, Australia.
E-mail: ricky.johnstone@petermac.org
Received 17 September 2012; revised 4 November 2012; accepted 5 November 2012; published online 14 January 2013
 Molecular IMiD targets
J Shortt et al
4192
O Mycobacterial infections such as tuberculous meningitis are
N O
NH
O O
thalidomide (Thalomid)
O degradation,24 a mechanism that is specific to thalidomide
N O
NH
NH2 O eukaryotic elongation initiation factor (eIF)-4E.25 As the availability
lenalidomide (Revlimid, IMiD3, CC5013)
NH2 O may preferentially downregulate the translation of a range of
N O
NH
O
O
pomalidomide (Actimid, IMiD1, CC4047)
Figure 1. Chemical structures of the ‘IMiDs’, thalidomide and its
amino-phthaloyl-substituted analogues.
(Figure 1). These thalidomide analogues have an established anti-
neoplastic role, and will be the focus of this review.
IMID CHEMISTRY AND DEVELOPMENT
Thalidomide (a-(N-phthalimido)glutarimide) is a synthetic
glutamic acid derivative. Thalidomide is a chiral molecule; this is
biologically important as the S(  ) and R( þ ) enantiomers mediate
different activities.13 However, segregation of biological effects by
administration of pure enantiomer is not feasible, as rapid
physiological inter-conversion occurs in vivo.14,15 The
second generation IMiDs CC-5013 (Revlimid, IMiD3, hereafter
lenalidomide) and CC-4047 (Actimid, IMiD1, hereafter
pomalidomide), are amino-phthaloyl-substituted thalidomide
analogues developed on the basis of augmented TNFa
inhibition (Figure 1).16,17 In this respect, second generation IMiDs
are up to 50 000 times more potent than thalidomide in vitro12
and subsequent clinical development has disclosed divergent
efficacy and toxicity profiles. Although TNFa suppression is an
anti-inflammatory mechanism of IMiDs in ENL,18 TNFa inhibition is
thought to be less relevant in other diseases such as MM (where
TNFa-directed therapies do not have efficacy19). Thus, the notion
that second generation IMiDs are generally more potent than
thalidomide is an oversimplification of thalidomide-analogue
biology. Although thalidomide analogues share important
immunomodulatory hallmarks (Figure 2), they differ in toxicity
profiles and clinical efficacy between different disease types.
Moreover, thalidomide resistant myeloma can be rescued by
treatment by second generation IMiDs, potentially indicating
divergent mechanistic activities within the same disease.20
IMMUNOLOGICAL HALLMARKS OF IMID ACTIVITY
(1) Suppression of LPS-induced monocyte TNFa production and
associated anti-inflammatory activity
The IMiDs possess clinically relevant anti-inflammatory activity.
ENL is an immune complication of Mycobacterium leprae infection
manifesting with fever, skin lesions and multi-organ dysfunction
because of antibody complex deposition, complement activation
and cytokine dysregulation.5 Patients with ENL have increased
cellular and circulating levels of TNFa that rapidly reduce with
thalidomide therapy.18 Inflammatory complications of other
Oncogene (2013) 4191 – 4202
likewise attenuated in tandem with TNFa levels by IMiDs.21
Thalidomide pre-treatment also abrogates LPS-induced septic
shock in animal models of gram-negative septicemia.22 The in vitro
correlate is that thalidomide suppresses the release of TNFa from
human monocytes in response to mycobacterial antigens or LPS.23
TNFa suppression occurs downstream of receptor activation
and transcriptional events by acceleration of TNFa mRNA
analogues (Figure 3a). In addition to enhancing the degradation
TNFa mRNA, the IMiDs were recently implicated as having a wider
role in translational regulation through downregulation of the
of eIF4E is a rate limiting step in the recruitment of mRNAs-
bearing complex 50 -untranslated regions to the ribosome, IMiDs
proteins with ‘fragile’ 50 -untranslated regions. The translational
modulation of transcription factors upstream of pro-inflammatory
cascades is important in IMiD-related suppression of inflammation.
Cyclooxygenase (COX)-2 is a key regulator of arachidonic acid
metabolism and rate-limits prostaglandin production. LPS-
stimulated COX-2 expression is transcriptionally regulated by
CCAAT/enhancer-binding protein isoforms26,27 that are subject to
50 -cap-dependent translation in association with eIF4E.25
Therefore, the effects of IMiDs on eIF4E-regulated transcription
factors are likely to directly regulate the expression of
pro-inflammatory gene loci, including COX-2.
The role of thalidomide’s anti-inflammatory targets in cancer
therapy is uncertain. Non-steroidal COX-2 enzymatic inhibitors
have demonstrable anti-cancer activities including anti-angiogenic
and anti-metastatic effects.28 COX-2 inhibitors have efficacy in
chemoprophylaxis of familial colorectal polyposis syndromes,29
but have not shown significant benefit when added to
chemotherapy for established COX-2 overexpressing epithelial
tumors.30 Myeloma cells harboring oncogenic RAS mutations also
show selective upregulation of COX-2 expression, and in these
cells COX-2 inhibition abrogates the protective effects of bone
marrow stromal cells on myeloma following chemotherapy
treatment.31 There is some clinical evidence that COX-2 selective
inhibition has activity in MM,32 indicating that the anti-
inflammatory effects of IMiDs may be relevant in this disease.
(2) Costimulation of T-cells
Correlative pre-clinical33 and clinical34 evidence suggests that
IMiD-mediated T-cell stimulation might augment anti-tumor
immunity. The IMiDs modulate cytokine expression in a cell-type
and context-dependent manner to either suppress or stimulate
the release of selected interleukins. In contrast to the suppression
of LPS-induced monocyte inflammatory cytokine release, IMiDs
costimulate the release of IL-2 and interferon (IFN)-g from
T lymphocytes undergoing CD3 ligation, mitogenic stimulation
or in response to dendritic cell presented super- or allo-antigens
(Figure 3b).35,36 This results in T-cell proliferation, T-helper-1
subset skewing and immuno-adjuvant anti-tumor effects33
including augmentation of cytotoxic effector functions.37,38 T-cell
costimulation by lenalidomide is only partially inhibited by
CTLA-4-Ig blockade of the B7/CD28 costimulatory axis.36
Moreover, lenalidomide induces tyrosine-phosphorylation of
CD28, facilitating the recruitment and activation of upstream
nodes in intracellular signaling cascades including
phosphatidylinositol-3-kinase (PI3K) and nuclear factor (NF)-
kB.36,37 As neither CD28 nor the IMiDs possess intrinsic kinase
activity, an indirect mechanism of CD28 phosphorylation, such as
recruitment of other signaling proteins including protein
kinase-C (PKC), is postulated. Pomalidomide costimulation
activates PKC and PI3K to enhance nuclear translocation of
nuclear factor of activated T-cells (NFAT)-2 and activated protein
& 2013 Macmillan Publishers Limited
 Molecular IMiD targets
J Shortt et al
4193
Immunomodulatory
Anti-neoplastic
Anti-inflammatory (TNFα suppression)
Multiple myeloma
Co-stimulation of T-cells
Mature B-cell lymphomas
Augmentation of NK-cell effector function
Myelodysplasia with del 5q
T-helper subset skewing (TH1 response)

Teratogenic Clinical toxicities

Interspecies specificity Second primary malignancy
Limb deformities (phocomelia) O O
Venous thromboembolic disease
Cereblon binding NH
Peripheral neuropathy
Oxidative stress N O
Bone marrow suppression

Anti-angiogenic Epigenetic
VEGF expression DNA intercalation
FGF signaling Promoter acetylation / demethylation
Sphingolipid metabolism

Figure 2. The pleotropic effects of thalidomide analogues.

LPS

TLR

MYD88
TCR
AKT CD28
p
PKC
PI3K

mTOR

eIF4E 5’ cap-dependent translation

AP1 nuclear translocation

TNFα
NFAT2 NFAT2 IL2
NFκB AP1 IL2
TNFα

Figure 3. Mechanisms of thalidomide-analogue immunomodulatory activity. (a) Suppression of LPS-induced TNFa secretion: Activation of
Toll-like receptors (TLR) by LPS activates NF-kB signaling downstream of MYD88. Transactivation of the TNFa promoter by NF-kB results in
TNFa mRNA expression. Thalidomide accelerates the degradation of TNFa mRNA, while reducing the availability of eIF4E for 50 -cap-dependent
translation of the same TNFa mRNA transcripts. (b) Costimulation of T-cells: in conjunction with T-cell receptor (TCR) ligation IMiD exposure
induces phosphorylation (p) of the CD28 costimulatory receptor, possibly by recruitment of PKC. Downstream activation of signaling
pathways, including PI3K results in nuclear translocation of nuclear factor of activated T-cells (NFAT)-2 and activated protein (AP-1) to the IL-2
promoter and increased IL-2 secretion.

(AP)-1 resulting in increased DNA-binding at the IL-2 cells. Clinical responses to thalidomide in myeloma patients
promoter.37,39 Accordingly, PI3K inhibitors can reduce IL-2 likewise correspond with increasing numbers of peripheral NK
secretion in response to IMiD costimulation.37 cells.38

(3) Augmentation of cytotoxic T- and NK-cell effector functions

The augmentation of cytotoxic effector cells is a direct conse-
quence of the immunostimulatory activity of IMiDs on T-cells. The
secretion of IL-2 and IFN-g by CD4 T-cells following IMiD exposure
augments NK-cell mediated killing against both (allogeneic)
leukaemic cell lines and autologous MM cells in vitro.38,40 The
importance of T-cell help in IMiD responses is highlighted by the
suppressive effects of calcineurin inhibitors and corticosteroids on
NK-cell cytotoxicity assays.37,40,41 In vivo administration of IMiDs
expands circulating NK-cell populations and synergizes with anti-
CD20 antibody therapy against B-cell lymphoma xenografts in an
NK-cell-dependent fashion.42 This is potentially explained by the
observation that lenalidomide augments antibody-dependent cell
cytotoxicity against rituximab (anti-CD20) treated CD20 þ tumor
OTHER DEFINING FEATURES OF THALIDOMIDE ANALOGUES
(1) Anti-angiogenic properties
Inflammatory cytokine networks and angiogenic pathways are
inherently linked, as neo-vascularization is a consequence of the
inflammatory response. Angiogenesis is important in tumor
growth and metastasis. Experiments initially designed to dissect
thalidomide’s teratogenic potential subsequently demonstrated
it’s anti-angiogenic properties.43 Thus, suppression of angio-
genesis linked IMiD cytokine modulation and anti-cancer
activity. However, in contrast to the anti-inflammatory and
immunomodulatory properties that are conserved across
primate and rodent species’, anti-angiogenic effects appear to
be relatively species specific. As the teratogenic effects of

& 2013 Macmillan Publishers Limited Oncogene (2013) 4191 – 4202

 Molecular IMiD targets
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VEGFR FGFR
PI3K
Ceramide
NFκB WNT AKT
u stroma and MM cells, alone or in co-culture.48 VEGF-induced
u
Apoptosis
u
DDB1
CRBN
H O H O
u
Oxidative stress u umbilical vein endothelial cells resulting in both decreased
u CRBN
???
Teratogenesis
Figure 4. Overlapping mechanisms of teratogenesis and
anti-angiogenesis conveyed by thalidomide targets. The anti-
angiogenic and teratogenic pathways modulated by thalidomide
are inherently linked. Thalidomide downregulates VEGF receptor
(VEGFR) and fibroblast growth factor receptor (FGFR) signaling by
several mechanisms, including generation of ceramide and down-
regulation of effector pathways downstream of receptor signaling.
Anti-apoptotic effectors antagonized in this fashion include AKT,
WNT and NF-kB. Concurrently, thalidomide induces oxidative stress
(pro-apoptotic) and binds cereblon (CRBN) to inhibit the E3
ubiquitin–ligase activity of the CRBN/DDB-1/Cul4 complex. Direct
ubiquitination (u) targets of this complex outside of its
auto-ubiquitination function are not known. Together these
pro-apoptotic, anti-angiogenic, oxidative and CRBN-mediated
effects combine to induce teratogenicity.
thalidomide show similar species specificity, it is likely that
thalidomide’s anti-angiogenic capacity and teratogenicity are
also linked (Figure 4).
D’Amato et al.43 first described anti-angiogenic activity of orally
administered thalidomide and a related teratogenic analogue,
EM-12 that was apparent in experimental systems using rabbit but
not chicken cells. Later, the same investigators demonstrated
intraperitoneal, but not oral administration of thalidomide
inhibited angiogenesis in bFGF and vascular endothelial growth
factor (VEGF)-driven cornea micro-pocket assays in the mouse.13
Conversely, orally administered lenalidomide reduced basic
fibroblast growth factor (bFGF)-induced angiogenesis in a rat
mesenteric window assay.44 The observation that route of
administration (oral versus intraperitoneal) resulted in divergent
anti-angiogenic effects with species specificity supported the
notion that in vivo IMiD metabolism modulates anti-angiogenic
effects. This hypothesis was further substantiated using in vitro rat
aorta and human endothelial cell culture systems that are
insensitive to the presence of non-metabolized thalidomide.45
Here, supplementation of thalidomide with human or rabbit (but
not rat) hepatic microsomes resulted in reduced microvessel
formation and endothelial cell growth. In a humanized myeloma
xenograft model, co-engraftment of human fetal liver cells into a
severe combined immunodeficient mouse augmented the anti-
myeloma activity of thalidomide.46 Myeloma xenografts from
thalidomide-treated, human fetal liver bearing mice showed
reduced microvessel density compared with non-fetal liver
transplanted hosts. It was concluded that human hepatic
metabolism was required for thalidomide’s anti-myeloma activity
in a mouse. However, this interpretation requires caution as
thalidomide metabolites were not assayed, the immunological
effects of fetal liver grafts (which contain hematopoietic stem
cells) were not accounted for, and thalidomide was administered
parenterally rather than orally.
Oncogene (2013) 4191 – 4202
Notwithstanding the putative role of interspecies metabolism in
IMiD-mediated suppression of vascularization, thalidomide and its
analogues have demonstrable activity against several key
angiogenesis regulators. In vivo suppression of pro-angiogenic
factors including IL-6, TNFa, VEGF, NF-kB and prostaglandin
synthesis is predicted to indirectly downregulate angiogen-
esis.12,26,47 At the cellular level, the IMiDs downregulate
paracrine production of VEGF and IL-6 by both bone marrow
angiogenesis is modulated by intracellular pro-survival signaling
Cul4
pathways and sphingolipid metabolism.44,49 Thalidomide activates
neutral sphingomyelinase to generate ceramide in human
cell growth and reduced expression of the VEGF receptors,
binding
neurophilin-1 and FLK-1 (Figure 4).49 Lenalidomide downregulates
the migratory response of human umbilical vein endothelial cells
to VEGF and bFGF.44 Here, reduced endothelial cell migration
correlated with downregulation of AKT phosphorylation,
indicating that lenalidomide might uncouple the permissive
effects of VEGF-receptor ligation from intracellular signaling
pathways. Together, these results indicate that the IMiDs
modulate angiogenesis at multiple levels including systemic
cytokine networks, local tumor/stroma interactions, surface
expression of VEGF receptors and intracellular signaling
pathways thereof.
(2) Teratogenicity
Intense scientific activity has been dedicated to determining the
molecular mechanisms of thalidomide teratogenicity.50 From a
translational perspective, a non-teratogenic yet efficacious IMiD
would be highly desirable. Indeed the thalidomide enantiomers,
metabolites and analogues vary in their teratogenic potential51
and the anti-angiogenic and immunomodulatory properties of
IMiDs appear to be separable.52 Whether the anti-neoplastic
activities of IMiDs are separable from teratogenic effects remains a
point of conjecture. Proposed embryotoxic mechanisms include
effects on angiogenesis,51 intracellular signaling,53–55 gene
regulation56 and the induction of oxidative stress.57 For an
excellent synopsis that attempts to unify existing conceptual
frameworks, the reader is directed to a recent review by Ito et al.50
As mechanisms of thalidomide teratogenicity have been
extensively reviewed elsewhere, they will only be covered here
in brief:
(a) Angiogenesis. Thalidomide’s anti-angiogenic effects provide
insight into the predilection for limb deformities in its teratogenic
phenotype. Developing limb buds comprise an immature vessel
network that is hypersensitive to thalidomide’s anti-angiogenic
effects.51 Thalidomide induces loss of newly formed vessels at
critical stages in limb development before the onset of
malformations.51 Changes in vasculature appear to precede
changes in gene expression, cytostatic effects and cell death in
the developing limb. This suggests defects in vascular
development are an upstream event in teratogenicity.
(b) Oxidative stress. There is much evidence linking thalidomide
teratogenicity to oxidative stress. Bioactivation of thalidomide
induces glutathione depletion and DNA-oxidation consistent with
the capacity to generate reactive oxygen species.57 Interestingly,
the threshold for thalidomide-induced free-radical generation and
embryonic DNA-oxidation is much lower in rabbits than mice,
corresponding with greater sensitivity to dysmorphogenesis. Pre-
treatment of rabbits with the free-radical spin trapping agent,
alpha-phenyl-N-t-butylnitrone abolishes thalidomide-induced
DNA-oxidation and teratogenicity.57 The induction of oxidative
stress in developing limb buds (potentially compounded by tissue
hypoxia) induces alterations pro-survival signaling pathways
including NF-kB,58 bone morphogenic protein/Dickkopf-1/Wnt54
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and PI3K/AKT55 culminating in downregulation of FGF and
apoptosis induction (Figure 4).
(c) Dysfunction of the cereblon/DNA-binding protein 1 ubiquitin–
ligase complex. Cereblon is a ubiquitously expressed protein that
was identified as a candidate gene in a mild form of autosomal
recessive, non-syndromal mental retardation.59,60 Cereblon binds
voltage-gated chloride channels in the retina61 and calcium-
activated potassium channels in the brain.62 It also modulates
AMP-activated protein kinase activity.63 In autosomal recessive,
non-syndromal mental retardation, a truncating cereblon point
mutation interrupts C-terminal myristoylation and phosphory-
lation sequences, potentially disrupting intracellular localization
and signaling activity, respectively.59,60
Cereblon is required for the E3 ubiquitin–ligase activity of a
multi-protein complex containing DNA-binding protein 1 and
Cullin 4 (Figure 4). Thalidomide was recently demonstrated to
directly modulate FGF-mediated angiogenesis via a cereblon-
dependent pathway.53 Using thalidomide to ‘pull down’ potential
drug targets from crude cell extracts, Ito et al.53 identified cereblon
as a thalidomide-binding protein. The antagonism of cereblon by
thalidomide was subsequently shown to be necessary but
insufficient to induce teratogenicity in Zebrafish and chick
embryos. In the Zebrafish model, thalidomide-induced terato-
genicity could be phenocopied by cereblon knockdown and
rescued by overexpression of a thalidomide-insensitive cereblon
mutant protein.53 Thalidomide exposure or cereblon knockdown
resulted in reduced FGF expression in the apical ectodermal ridge,
providing a mechanistic link between thalidomide, cereblon and
FGF-mediated angiogenesis in teratogenicity.
Although the characterization of cereblon as a thalidomide-
target provided much insight into teratogenic mechanisms, the
role of complementary pathways remains undefined. In the
human, cereblon mutations causing autosomal recessive, non-
syndromal mental retardation do not associate with limb
defects.60 Conversely, persons suffering from thalidomide-
induced dysmelia do not demonstrate intellectual disability.1 As
the protein structure of cereblon is highly conserved, thalidomide
should not discriminate between rodent and human (or Zebrafish)
protein.53 Thus, the well described associations between species-
specific thalidomide metabolism and angiogenic effects are not
well explained by the cereblon-model of teratogenicity. Another
caveat to the interpretation of Zebrafish modeling is the
supraphysiological thalidomide dosing required to induce anti-
angiogenic and teratogenic phenotypes. Myeloma patients
receive 50–200 mg/day of thalidomide. A dose-escalation study
in elderly prostate cancer patients recorded maximum steady-
state plasma concentrations of 11 mg/ml (B40 mM) in persons
receiving 1.2 g of thalidomide daily.64 However, Ito et al.53
reported dysmorphogenesis with 400 mM of thalidomide applied
directly to dechorionated embryos, diluted from a 400 mM DMSO
stock solution. As thalidomide is relatively insoluble, repeated
cycles of heating (to 65 1C) and agitation were required to
solubilize thalidomide, introducing the potential for pre-analytical
drug modification. Others have reported Zebrafish embryos to be
much less sensitive to thalidomide application. For example,
Cheng et al.65 reported that thalidomide concentrations of
2 mg/ml (B7–8 mM) were the minimum requirement to induce
vascular deformities in transgenic fli1a: EGFP Zebrafish. As both
investigators used similar methodology, the reasons for this
10-fold discrepancy in potency are unclear. In either case, the
concentrations used are much higher than those measured in
human subjects.
Although it is apparent that cereblon is a potentially important
IMiD target, there are still many unanswered questions regarding
mechanisms of thalidomide embryotoxicity. In particular, the
grossly divergent interspecies sensitivity to teratogenesis is
unresolved. If a pathway linking thalidomide-related cereblon
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Molecular IMiD targets
J Shortt et al
4195
inhibition to species-specific oxidative stress were elucidated, this
might finally provide a unifying model (Figure 4).
EVIDENCE FOR GENETIC AND EPIGENETIC EFFECTS OF IMIDS
Although not mutagenic,66 thalidomide intercalates DNA.67,68
Specifically, S(  ) thalidomide is predicted to intercalate into the
major groove of the DNA-double helix structure at purine sites
(with greater affinity for guanine over adenine) where its
glutarimide moiety oriented in a 30 -direction.56 The preferential
affinity of thalidomide for guanine nucleotides potentially explains
differential regulation of genes bearing GC-rich gene promoters.
The transcription factor Sp1 binds GC-boxes within promoter
regions and docks within the major groove of DNA that would be
occupied by intercalated thalidomide.69,70 Candidate genes
regulated by Sp1 include insulin-like growth factor and its
receptor, insulin receptor substrate-1, FGF-2, FGF receptors and
several integrins. As these have a central roles in angiogenesis and
embryo development, modulation of gene expression via
intercalation at GC-rich promoters has been proposed as a
mechanism for thalidomide teratogenesis (reviewed in Stephens
et al.56).
The DNA-intercalation model provides a conceptual framework
for how thalidomide might reduce the efficiency of transcription
at GC-rich promoters. However, emerging evidence suggests the
IMiDs epigenetically promote the transactivation of genes at the
same GC-rich loci. Lenalidomide and pomalidomide induce
growth arrest in selected myeloma and B-cell lymphoma cell
lines. This correlates with the induction of the cell cycle regulator
p21WAF-1 and concomitant inhibition of cyclin-dependent kinases,
hypo-phosphorylation of Rb and G0–G1 arrest.71 Pomalidomide
rapidly upregulates CDKN1A mRNA expression via p53-
independent recruitment of Sp1 to a proximal GC-rich region in
the CDKN1A promoter (Figure 5a).72 Sp1 recruitment occurs in the
absence of a change in absolute Sp1 levels and is facilitated by the
acetylation of histone H3 on lysines 9 and 14. As the IMiDs do not
inhibit histone deacetylases, an indirect mechanism whereby
recruitment of the histone demethylase, LSD-1 to H3K9 was
suggested.72 In this model, the acetylation of H3K9/14 is co-
regulated by LSD-1 demethylation at the same loci.
Epigenetic mechanisms are also implicated in IMiD-related
modulation of cytokine expression (Figure 5b).73 Cytokine
receptor ligation activates intracellular signaling through the
JAK/STAT pathway. STAT target genes include the suppressor of
cytokine signaling (SOCS) genes that attenuate JAK/STAT
activity.74 SOCS1 is a negative regulator of IFN-g, IL-2 and IL-6.
Hypermethylation of the SOCS1 promoter is reported in myeloma
patients, leading to augmented expression of IL-6, a potent
plasma cell growth factor.75 Pomalidomide upregulated myeloma
cell SOCS1 expression concomitant with demethylation of its
silenced promoter region.73 Demethylation of the SOCS1 promoter
by pomalidomide or 5-azacitidine correlated with increased
myeloma cell sensitivity to subsequent lysis by cytotoxic
T lymphocytes. Interestingly, the epigenetic modulation of
SOCS1 was tumor specific, as the promoter methylation was
unchanged in effector lymphocyte populations.73
ACTIVITY IN MDS WITH DEL 5Q
MDS refers to a group of clonal bone marrow stem-cell disorders
characterized by ineffective hematopoiesis and potential trans-
formation to acute myeloid leukemia. MDS with associated
deletion of the long arm of chromosome 5 (del 5q) is an MDS
subtype characterized by refractory macrocytic anemia, well
preserved thrombopoiesis and low bone marrow blasts.76 Unlike
other MDS, 5q- syndrome has a high response rate to
lenalidomide, including cytogenetic remissions and improved
erythropoiesis.77 The clinical phenotype of MDS with del 5q is
Oncogene (2013) 4191 – 4202
 Molecular IMiD targets
4196
Sp1
Me Me Ac Sp1
CDKN1A
Me
LSD-1 Ac
Me Me
Me
IL6
JAK
STAT SOCS1
BCL2
STAT
IL6 target genes
Me Me
SOCS1
Me
Figure 5. Examples of epigenetic modulation of gene targets by
IMiDs. (a) Genes bearing GC-rich promoter regions are preferentially
targeted by IMiDs either by direct DNA intercalation (not shown) or
alterations in methylation status (Me) and histone acetylation (Ac).
The transcription factor Sp1 is excluded from a GC-rich region of the
CDKN1A promoter by methylation. Recruitment of the demethylase
LSD-1 allows histone H3 acetylation at lysines 9 and 14, opening
the promoter to transcription of the cell cycle inhibitor p21WAF-1.
(b) SOCS1 feedback suppresses JAK/STAT signaling in response to
cytokine receptor ligation including IL-6. In myeloma cells, the
SOCS1 promoter is silenced by hypermethylation, amplifying the
transcription of IL-6 regulated pro-survival effectors (for example,
BCL2). IMiD treatment demethylates the SOCS1 promoter, restoring
SOCS1 expression and dampening pathophysiological JAK/STAT
signaling.
specifically mapped to loss of a common deleted region (CDR) on
chromosome 5.78 This CDR provides a list of 40 genes implicated
in the biology of 5q- syndrome and therefore a template for the
discovery of lenalidomide-responsive elements. In a seminal
paper, Ebert et al.79 used an RNA-mediated interference
approach to interrogate the CDR. They identified ribosomal
protein (RP) S14 haploinsufficiency as a key determinate of the
erythroid differentiation defect in MDS with del 5q. This correlated
well with prior descriptions of heterozygous germline mutations
for several other RPs in patients with Diamond–Blackfan anemia, a
congenital bone marrow failure syndrome.80,81 The erythroid
defect conveyed by RPS14 loss is p53 dependent, indicating that
activation of the p53 tumor suppressor pathway is necessary for
RP haploinsufficiency to induce anemia.82,83 Although RP
haploinsufficiency contributes to the p53-dependent anemia of
MDS with del 5q, other genes are implicated in the generation
of dysplastic megakaryocytic hyperplasia and thrombocytosis.
In particular, loss of microRNA-145 in the CDR results in increased
expression of the transcription factor Fli-1 (ref. 84). Fli-1
overexpression causes a shift from erythropoiesis to
Oncogene (2013) 4191 – 4202
J Shortt et al
megakaryopoiesis to co-operate with RPS14 deletion in the
induction of anemia while promoting platelet production.
The predominant clinical response to lenalidomide in MDS
p21 is improved erythropoiesis with a significant proportion of
patients achieving independence from red cell transfusions.77
CDKN1A This is associated with cytogenetic improvement, but occurs at the
expense of thrombocytopenia and neutropenia. Despite detailed
insight into the molecular mechanism of anemia and
thrombocytosis in MDS with del 5q, the mechanism of
lenalidomide activity remains something of an enigma as RPS14
is not an IMiD-responsive gene.85 Gene-expression profiling of
lenalidomide-treated del 5q erythroblasts revealed significant
upregulation of only one CDR gene, SPARC.85 SPARC regulates
interactions with the extracellular matrix, mediating anti-
proliferative and anti-angiogenic effects in common with IMiD-
related activity. An alternative hypothesis is that lenalidomide
further downregulates the function of haploinsufficient proteins
coded in the CDR by post-transcriptional and post-translational
mechanisms. This might explain selective toxicity for del 5q cells
over normal cells with a diploid gene complement at the same
locus. For example, del 5q cells are haplodeficient for the protein
phosphatases Cdc25C and PP2A and the expression of
these genes is lower in MDS with del 5q than with other
MDS.86 Lenalidomide increases the retention of inhibitory
phosphorylation residues on cell cycle regulators targeted by
Cdc25C and PP2A.86 This correlates with potent inhibition
of recombinant human Cdc25C phosphatase activity by
SOCS1 lenalidomide in the nanomolar concentration range in vitro.86
Lenalidomide’s effects on PP2A activity appear to be indirect, as
SOCS1
they cannot be demonstrated on purified protein in the same
assays. As Cdc25C and PP2A regulate the cell cycle, antagonism by
lenalidomide explains the selective induction of growth arrest and
apoptosis in del 5q cells compared with their normal counterparts.
The erythroid phenotype of MDS with del 5q is p53
dependent.82 Moreover, neoplastic escape from lenalidomide
in vivo is associated with selection for sub-clones with mutant
p53.87 Ribosomal biogenesis occurs in the nucleolus, which also
provides a sink for the sequestration of the p53 ubiquitin ligase,
MDM2 (Figure 6a). Nucleolar stress due to a range of causes
including ribosomal protein deficiency results in the dispersion of
free RPs that are able sequester MDM2 independently of its co-
regulator ARF. The sequestration of MDM2 allows stabilization of
p53. Lenalidomide might therefore attenuate the severity of
dyserythropoiesis indirectly by either reducing ribosomal stress or
promoting p53 degradation. The suppressive effects of lenalido-
mide on mRNA translation25 potentially reduces the biosynthetic
demands of a cell on haploinsufficient ribosome biogenesis.
Lenalidomide also promoted degradation of p53 in the context of
5q deletion by antagonising PP2A-enhanced ubiquitin-mediated
clearance of MDM2.88 Thus, it is possible that lenalidomide
treatment reduces ribosomal stress and the p53 response thereof
to ameliorate dyserythropoiesis (Figure 6a). The observation that
lenalidomide can destabilize p53 is clinically significant, as p53
suppression is a highly oncogenic effect. IMiD-mediated p53
suppression provides a mechanistic explanation for the recently
published observation that prolonged exposure to lenalidomide is
associated with the development of second cancers (discussed
further below).89–92
ACTIVITY IN MM AND MATURE B-CELL NEOPLASMS
MM is a malignancy of immunoglobulin producing plasma cells
associated with systemic toxicities including renal failure, anemia,
immunoparesis and destructive skeletal lesions.93 The mechanism
of action of IMiDs in MM is complex and likely reflects the
sum-total of a range of independent targets including direct
toxicity to MM cells, disruption of the protective bone marrow
microenvironment and augmentation of host-mediated anti-
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IL6
SPARC

AKT

Ineffective erythropoiesis SOCS1 PI3K

(macrocytic anemia) MYD88

mTOR

AKT

p p53
RP eIF4E PP2A NFκB
CRBN
mTOR

Ribosomal stress
(Haploinsuffcient RPS14) eIF4E

RP RP MDM2
RP
+
RP IRF-4 cMYC

Free ribosomal proteins

Figure 6. Cell intrinsic mechanisms of IMiD activity in specific disease states. (a) MDS with del 5q: haploinsufficiency for RPS14 induces
ribosomal stress and releases free ribosomal proteins to sequester the p53 ubiquitin ligase, MDM2. Activation of the p53 tumor suppressor
pathway causes ineffective erythropoiesis (macrocytic anemia). IMiD treatment may ameliorate ribosomal stress by antagonism of mTOR/
eIF4E-regulated protein translation while concurrently inhibiting PP2A activity to stabilize MDM2 levels and dampen the p53 response. In
addition, upregulation of SPARC expression has potential anti-proliferative effects through interactions with the tumor microenvironment.
RPS14, PP2A and SPARC are all genes in the 5q common deleted region. (b) Mature B-cell neoplasms (for example, MM and diffuse large B-cell
lymphoma of activated B-cell phenotype (DLBCL-ABC) subtype) show activation of pro-survival NF-kB and IRF-4 signaling through diverse
mechanisms including MYD88 mutation and IL-6 receptor signaling. IRF-4 and cMYC translation is rate limited by the availability of eIF4E; the
mutually permissive effects of IRF-4, cMYC and eIF4E form an oncogenic ‘feed-forward loop’. IMiD treatment disrupts this pathophysiological
network at multiple levels including upregulation of SOCS1, and antagonism of NF-kB dependent IRF-4/cMYC axis. Cereblon binding is
required for some of this cell intrinsic anti-neoplastic activity.

tumor immunity. Myeloma is a clinically and molecularly
heterogeneous disease.93,94 Despite this heterogeneity, IMiD-
responsiveness is a defining feature of the majority of MM at
presentation; a phenotype shared with selected other mature
‘post-germinal center’ B-cell malignancies.95 This potentially
indicates that the IMiDs target fundamental aspects of mature
B-cell biology.
(a) Myeloma cell autonomous effects
An intriguing property of the IMiDs is anti-tumor activity with
selectivity for neoplasms of mature B-cell origin. In rare instances
this is explained by 5q loss,71 however, a more complex molecular
interaction is implicated in the majority of cases. Mature B-cell
malignancies including MM and diffuse large B-cell lymphoma of
activated B-cell phenotype show dependence on constitutive
NF-kB signaling and upregulation of interferon regulatory factor
(IRF)-4 (Figure 6b).95 Mutually permissive ‘feed-forward loops’
linking IRF-4 to cMYC implicate the former as an ‘oncogene of
addiction’ in MM.96 MYD88 is an adaptor protein that mediates
interleukin and toll-like receptor signaling. Gain-of-function
MYD88 mutations and copy number amplification are observed
in more than half of diffuse large B-cell lymphoma of activated
B-cell phenotype and are associated with increased IRF-4
expression.97 Genomic profiling of MM revealed frequent
mutations of genes implicated in NF-kB signaling and IRF-4
itself.94 Thus, diverse oncogenic mechanism with the net effect of
NF-kB and IRF-4 activation link these IMiD-responsive diseases.
Recent evidence suggests that IMiDs can downregulate the
NF-kB/IRF-4 axis at multiple levels. In MM, antagonism of the
eIF4E translational axis decreases CCAAT/enhancer-binding pro-
teinb protein expression to impair transcription at the IRF-4
locus.25 In diffuse large B-cell lymphoma of activated B-cell
phenotype, lenalidomide relieves the suppressive effects of
constitutive MYD88 signaling on interferon-b by downregulating
IRF-4 (ref. 95). The ensuing lethal type-1 interferon response
mediates apoptosis through the extrinsic (death receptor)
pathway. Similarly, Mitsiades et al.98 correlated IMiD-induced
downregulation of NF-kB with activation of the extrinsic apoptotic
pathway and sensitization to death-receptor agonists including
TNF-related apoptosis related ligand in human myeloma cell lines.
Interestingly, the direct cytostatic and apoptotic effects of IMiDs
are less evident in lymphomas and myelomas after depletion of
cereblon, linking this mediator of teratogenicity to anti-tumor
effects.95,99,100 Acutely, shRNA-mediated cereblon knockdown
induces cytostatic and apoptotic effects in MM cell lines that
correlates with a reduction in IRF-4 expression.100 Those MM cells
surviving sustained cereblon knockdown are lenalidomide
resistant when compared with their parental counterparts.
Conversely, prolonged (4 to 6 months) in vitro treatment with
lenalidomide generates IMiD-resistant MM cell lines that show
reduced cereblon expression relative to non-lenalidomide treated
controls.99,100 The resistance phenotype is specific to IMiDs, as
lenalidomide-conditioned MM cell lines remained sensitive to
apoptosis induction by dexamethasone. Cereblon mRNA
expression was also reportedly reduced in primary patient MM
samples with clinically resistant disease following a period of
lenalidomide therapy.100 However, the effects of cereblon
knockdown did not entirely phenocopy IMiD treatment. For
example, gene expression profiling of OPM2 MM cells identified
2398 differentially regulated genes following lenalidomide
treatment, but only 123 were concordant with the cereblon
knockdown gene set of 768 genes.100 Thus, although it is apparent
that cereblon mediates some of the cell-intrinsic activity of IMiDs
in MM, complementary mechanisms and other molecular targets
are likely to be at play. This is consistent with observations in
teratogenicity, where cereblon binding is necessary but
insufficient to explain the teratogenic phenotype.53
(b) Disruption of the myeloma niche and supportive cytokine
networks
The bone marrow microenvironment provides a permissive niche
for MM growth and promotes resistance to the apoptotic effects

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of conventional therapeutics.101,102 Elaborate co-culture systems
have defined both direct interactions with bone marrow stromal
cells and humoral components of cytokine networks as essential
to MM viability.101,103–105 In particular, osteoclastogenic cytokines
of the TNF-family and IL-6 are implicated in myeloma cell growth
and skeletal destruction.102,106 Paracrine and autocrine interleukin
networks combine with cell adhesion molecules to activate
intracellular pro-survival pathways including PI3K/AKT, NF-kB
and RAS/MAPK.107,108 Myeloma cell adherence to bone marrow
stroma also upregulates VEGF secretion to promote
angiogenesis.48 By downregulating cytoprotective cytokines (for
example, IL-1b, IL-6, IL-10 and VEGF), the IMiDs may disrupt
protection by the microenvironment, sensitizing myeloma to
concurrent cytotoxic therapies.109
As conventional therapies are initially successful in debulking a
high proportion of the myeloma burden, the bone marrow niche
is postulated to rescue clonogenic myeloma stem cells ultimately
responsible for disease relapse.110 Although the literature on the
identification of myeloma stem cells are somewhat nebulous and
without consensus, studies have eluded to several populations of
myeloma cells that have clonogenic capacity. These include the
CD138  CD27 þ cells that are lenalidomide, dexamethasone,
cyclophosphamide and bortezomib resistant with a high level of
hedgehog signaling.111,112 Furthermore, a small population of
CD34 þ CD45low myeloma cells has been shown to have
clonogenic properties in NOD/SCID mice.113 ‘Side-population
cells’ defined by the capacity to efflux Hoechst 3342 stain,114
have also been identified as a source of stem-cell like
chemoresistant cancer-initiating cells. The proliferation and
survival of MM side-population cells is augmented by bone
marrow stroma through pathways that are suppressible by
thalidomide and lenalidomide.110 Lenalidomide also reduces the
clonogenic potential of myeloma side-population cells.110 This
indicates that suppression of the myeloma ‘stem-cell’ may be an
important facet to IMiD activity, particularly in the prevention of
relapse from minimal residual disease states (for example,
following autologous stem-cell transplantation).
(c) Immunological disease control
Hematological malignancies, particularly MM are characterized by
progressive immune cell dysregulation, of which T- and NK-cell
suppression is well described.115–117 Lenalidomide’s ability to
potentiate T- and NK-cell activation37,38,40 has resulted in
exploratory studies elucidating the role of IMiDs in the
immunological control of MM. NK-cell interaction with target
cells involves a complex set of activating and inhibitory receptors.
Activating receptors involved in MM cell recognition and lysis are
largely governed by NKG2D, with a lesser role for DNAM-1 and
NKp46.118 Interestingly, IMiDs do not appear to alter the
expression of these receptors to confer functional changes in
NK-cell activity.40,118,119 IMiDs upregulate CD16 expression on NK
cells to increase antibody-dependent cell cytotoxicity against
MM.120,121 It is becoming evident that IMiDs are able to decrease
the activation threshold of NK-cell cytotoxicity through
modulation of inhibitory receptor interactions. MM-induced
expression of the programmed cell death receptor 1 inhibits NK-
cell cytotoxicity.122 Treatment with lenalidomide reduces
programmed cell death receptor 1 ligand on MM cells and thus
sensitizes MM cells to lysis by autologous NK cells.122 Killer-cell
Immunoglobulin-like receptors (KIR) are well described as the
major functional inhibitory receptors on NK cells.123 As MM cells
express high levels of the KIR ligand, HLA class I, it is likely that this
interaction contributes to NK-cell dysfunction in vivo.124,125 IMiDs
are able to reduce the expression of KIR on NK cells.119
Furthermore, a recent study demonstrated the therapeutic
efficacy of combination IMiD and anti-KIR blocking therapy in a
mouse model of MM.125
Oncogene (2013) 4191 – 4202
The contribution of T-cells to immune-mediated control of MM
in vivo has been a difficult question to address, with conflicting
reports characterizing both impaired/exhausted and activated
clonal expansions of MM-specific T-cells in MM patients.126–129
There is agreement, however, on the responsiveness of T-cells to
immunomodulatory agents. The stimulatory effect of IMiDs on
T-cell function is driven by PI3K-mediated activation of nuclear
factor of activated T-cells-2.37,130 This results in increased
production of IL-2 and NK-cell stimulation.37,40 Further to this,
IMiDs appear to polarize T-cells towards a Th-1 phenotype
through costimulation, resulting in the production of IFN-g and
suppression of IL-10.131,132 The first study to show protective anti-
tumor immunity by IMiDs in conjunction with whole-tumor cell
vaccination was demonstrated in a CT-26 colon mouse model.33
More recently, Noonan et al.34 showed single-agent lenalidomide
therapy in MM patients augmented endogenous MM-specific CD8
T-cell responses.34 Both Th-17 and regulatory T-cells have a
pathogenic role in MM, with higher T-regulatory cells conferring
poorer outcomes in MM patients.133,134 The ability of IMiDs to
reduce Th-17 cells, and inhibit the generation and function of
Foxp3 T-regulatory cells may result in the mediation of T-cell
responses against MM.34,135
CLINICAL TOXICITIES PROVIDE MECHANISTIC INSIGHT INTO
IMID BIOLOGY
The differing side effect profiles of thalidomide and its IMiD
derivatives highlight important biological distinctions between
these closely related compounds not merely explained by issues
of potency. Elucidation of the mechanisms responsible for
divergent clinical toxicities could potentially inform the biology
of tumor-specific responses to the respective analogues. The
major cumulative dose-limiting toxicity of thalidomide is periph-
eral neuropathy136 with minimal bone marrow suppression.
Although lenalidomide is more potent than thalidomide (for
example, in TNFa suppression assays), it does not cause
neuropathy.89 Rather, lenalidomide suppresses normal hemato-
poiesis resulting in neutropenia and thrombocytopenia.
Pomalidomide also causes neutropenia, with a lower incidence
of neuropathy than thalidomide.137 All of the IMiDs are
prothrombotic, with prophylactic anticoagulant therapy often
administered concurrent to the IMiDs to prevent venous
thromboembolic disease. The greatest emerging concern with
chronic lenalidomide exposure is a measurable increase in the
incidence of second primary malignancies.89–92
(a) Peripheral neuropathy
Neuropathy occurs in most patients receiving chronic thalidomide
therapy with an actuarial incidence of over 70% at 12 months.136
The etiology of thalidomide-induced neuropathy is multifactorial,
as patients often have pre-existing nerve damage and many
receive concurrent neurotoxic therapies. In particular, the
combination of thalidomide with bortezomib is associated with
a high risk of nerve damage.138 However, the potent new-
generation proteasome inhibitor carfilzomib is not neuropathic,
indicating that the neurotoxicity of bortezomib may be ‘off target’
inhibition of other serine proteases.139 HtrA2/Omi was identified
as a non-proteasomal bortezomib target that is spared by
carfilzomib and protects neurons from oxidative stress.139 Thus,
thalidomide-induced free-radical generation57 and bortezomib-
induced depletion of neuroprotective serine proteases (for
example, HtrA2/Omi) could combine to cause the mitochondrial
toxicity observed in neuropathic states. Indeed, genomic profiling
identified a single-nucleotide polymorphism of glutathione
S-transferase theta-1 (GSTT1) as predictive of neuropathy in a
cohort of myeloma patients receiving thalidomide.140 As GSTT1 is
a cellular regulator of reactive oxygen species, this further
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implicated thalidomide-induced oxidative stress in the etiology of
nerve damage.
(b) Hematological toxicities
Neutropenia and the risk of sepsis is a major dose-limiting toxicity
of lenalidomide. Lenalidomide treatment is also associated with
impaired stem-cell mobilization for autologous CD34 þ collection
before transplantation in MM.141 This is despite the observation
that IMiDs induce the expansion of CD34 þ bone marrow
progenitors in vitro.142 However, the transcriptional changes that
evoke CD34 þ proliferation in vitro are also associated with a
differentiation block within the granulocytic lineage.143 This
apparent ‘maturation arrest’ is associated with downregulation
of the transcription factor PU.1, and results in a relative excess
of promyelocytes. Promyelocytes are rich in prothrombotic
mediators including cathepsin G, a platelet agonist implicated in
the pathogenesis of disseminated intravascular coagulation
complicating acute promyelocytic leukemia.144 Therefore,
IMiD-induced changes in granulocyte maturation may directly
contribute to an increased risk of venous thrombosis.143 Other
well-described IMiD activities are pro-coagulant. Suppression of
COX-2 has been clearly implicated in adverse thrombotic events in
patients treated for non-malignant inflammatory conditions.26,145
On-target suppression of angiogenesis is also pro-thrombotic, as
endothelial cells that become stressed in response to VEGF
withdrawal release a range of pro-coagulant mediators.44,146 A
targeted genetic screen of myeloma patients identified single-
nucleotide polymorphisms associated with thalidomide-
associated thrombosis in genes and pathways important in drug
metabolism, DNA-repair and cytokine regulation.146 The authors
suggested that susceptibility to IMiD-associated thrombosis
was secondary to rapid dissolution of myeloma in the context
of apoptosis induction by concurrent DNA-damaging stimuli.
The thrombogenicity of IMiDs is therefore multifactorial and
contributed to by underlying malignancy and associated
treatments.
(c) Lenalidomide and second primary malignancies
With the benefit of longer myeloma-related progression free
survival, patients receiving chronic lenalidomide maintenance
therapy have an increased risk of both hematopoietic and solid
organ cancers.89 Several major clinical studies identified an
increased incidence of second primary malignancies in MM
patients receiving chronic ‘maintenance’ lenalidomide.90–92 The
incidence of second cancers in these studies was as high as 7%
with a hazard ratio close to double that of placebo-treated
controls. Elucidation of the precise molecular oncogenic
mechanisms is confounded by prior or concurrent exposure to
genotoxic agents, in particular the alkylator melphalan. Second
primary cancers have not yet been associated with other IMiDs,
and lenalidomide does not appear to accelerate the progression
of MDS with del 5q to acute leukemia.89 However, as the ‘danger
signal’ from these large lenalidomide trials has only recently
emerged, mechanistic insights remain speculative. Lenalidomide
is not considered directly mutagenic, but could plausibly synergize
with DNA-damaging cytotoxics in oncogenesis. For example,
lenalidomide destabilizes p53,88 an important tumor suppressor in
the context of DNA-damage. This might also explain the lack
of an association between thalidomide and second cancers,
as it is less potent in this regard. However, all of the IMiDs bind
cereblon and dysfunction of the cereblon/DDB-1 ubiquitin–ligase
complex might also impair the DNA-damage response, as DDB-1
functions in nucleotide excision DNA-repair.147 Elucidation of the
capacity for IMiDs to potentiate mutagens requires urgent
scientific attention in order to inform the safe delivery of
IMiD/chemotherapy combinations in the clinic.
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CONCLUSIONS
Despite their simple chemical structures, thalidomide and its
analogues exhibit pleotropic immunomodulatory and anti-neo-
plastic effects. This conveys remarkable clinical efficacy in clearly
defined disease entities, specifically granulomatous inflammation,
mature B-cell neoplasms and MDS with del 5q. The molecular
mechanisms of IMiD activity are likely to be both divergent and
complementary across these distinct diseases. In particular the
combined ability to modulate multiple targets from the level of
the epigenome to post-translational protein modulations and
systemic cytokine networks highlights the complexity of IMiD
biology. Unfortunately, the beneficial properties of IMiDs may be
inseparable from serious clinical toxicities including teratogenicity,
secondary malignancy, myelosuppression and nerve-damage,
ostensibly because they are mediated by shared molecular
targets. It is hoped that the next chapter in the ‘thalidomide
saga’1 will be the identification of new targets that can be
therapeutically manipulated by novel non-IMiD pharmaceuticals
to derive clinical benefit without toxicity.
CONFLICT OF INTEREST
The authors declare no conflict of interest.
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