Thalidomide-Analogue Biology: Immunological, Molecular and Epigenetic Targets in Cancer Therapy

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 4,5 a range of biological effects and putative molecular targets. nodosum leprosum (ENL) reawakened interest in the drug. 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 6,7 8 9 sia (MDS) and multiple myeloma (MM). Despite an initially ulceration, Behcet’s syndrome, Crohn’s disease, cutaneous 10 infamous track record that spans 450 years of human exposure, lupus 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

1Gene 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; 4Haematology 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: [email protected] 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 likewise attenuated in tandem with TNFa levels by IMiDs.21 N O Thalidomide pre-treatment also abrogates LPS-induced septic 22 NH shock in animal models of gram-negative septicemia. The in vitro correlate is that thalidomide suppresses the release of TNFa from O O human monocytes in response to mycobacterial antigens or LPS.23 thalidomide (Thalomid) TNFa suppression occurs downstream of receptor activation and transcriptional events by acceleration of TNFa mRNA O degradation,24 a mechanism that is specific to thalidomide analogues (Figure 3a). In addition to enhancing the degradation N O TNFa mRNA, the IMiDs were recently implicated as having a wider NH role in translational regulation through downregulation of the 25 NH2 O eukaryotic elongation initiation factor (eIF)-4E. As the availability lenalidomide (Revlimid, IMiD3, CC5013) of eIF4E is a rate limiting step in the recruitment of mRNAs- bearing complex 50-untranslated regions to the ribosome, IMiDs NH2 O may preferentially downregulate the translation of a range of proteins with ‘fragile’ 50-untranslated regions. The translational N O modulation of transcription factors upstream of pro-inflammatory NH cascades is important in IMiD-related suppression of inflammation. Cyclooxygenase (COX)-2 is a key regulator of arachidonic acid O O metabolism and rate-limits prostaglandin production. LPS- pomalidomide (Actimid, IMiD1, CC4047) stimulated COX-2 expression is transcriptionally regulated by 26,27 Figure 1. Chemical structures of the ‘IMiDs’, thalidomide and its CCAAT/enhancer-binding protein isoforms that are subject to amino-phthaloyl-substituted analogues. 50-cap-dependent translation in association with eIF4E.25 Therefore, the effects of IMiDs on eIF4E-regulated transcription (Figure 1). These thalidomide analogues have an established anti- factors are likely to directly regulate the expression of neoplastic role, and will be the focus of this review. 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 IMID CHEMISTRY AND DEVELOPMENT have demonstrable anti-cancer activities including anti-angiogenic and anti-metastatic effects.28 COX-2 inhibitors have efficacy in Thalidomide (a-(N-phthalimido)glutarimide) is a synthetic chemoprophylaxis of familial colorectal polyposis syndromes,29 glutamic acid derivative. Thalidomide is a chiral molecule; this is but have not shown significant benefit when added to biologically important as the S( À ) and R( þ ) enantiomers mediate 13 chemotherapy for established COX-2 overexpressing epithelial different activities. However, segregation of biological effects by tumors.30 Myeloma cells harboring oncogenic RAS mutations also administration of pure enantiomer is not feasible, as rapid 14,15 show selective upregulation of COX-2 expression, and in these physiological inter-conversion occurs in vivo. The cells COX-2 inhibition abrogates the protective effects of bone second generation IMiDs CC-5013 (Revlimid, IMiD3, hereafter marrow stromal cells on myeloma following chemotherapy lenalidomide) and CC-4047 (Actimid, IMiD1, hereafter treatment.31 There is some clinical evidence that COX-2 selective pomalidomide), are amino-phthaloyl-substituted thalidomide inhibition has activity in MM,32 indicating that the anti- analogues developed on the basis of augmented TNFa inflammatory effects of IMiDs may be relevant in this disease. 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 (2) Costimulation of T-cells efficacy and toxicity profiles. Although TNFa suppression is an 33 34 18 Correlative pre-clinical and clinical evidence suggests that anti-inflammatory mechanism of IMiDs in ENL, TNFa inhibition is IMiD-mediated T-cell stimulation might augment anti-tumor thought to be less relevant in other diseases such as MM (where 19 immunity. The IMiDs modulate cytokine expression in a cell-type TNFa-directed therapies do not have efficacy ). Thus, the notion and context-dependent manner to either suppress or stimulate that second generation IMiDs are generally more potent than the release of selected interleukins. In contrast to the suppression thalidomide is an oversimplification of thalidomide-analogue of LPS-induced monocyte inflammatory cytokine release, IMiDs biology. Although thalidomide analogues share important costimulate the release of IL-2 and interferon (IFN)-g from immunomodulatory hallmarks (Figure 2), they differ in toxicity T lymphocytes undergoing CD3 ligation, mitogenic stimulation profiles and clinical efficacy between different disease types. or in response to dendritic cell presented super- or allo-antigens Moreover, thalidomide resistant myeloma can be rescued by (Figure 3b).35,36 This results in T-cell proliferation, T-helper-1 treatment by second generation IMiDs, potentially indicating 33 20 subset skewing and immuno-adjuvant anti-tumor effects divergent mechanistic activities within the same disease. 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 IMMUNOLOGICAL HALLMARKS OF IMID ACTIVITY Moreover, lenalidomide induces tyrosine-phosphorylation of (1) Suppression of LPS-induced monocyte TNFa production and CD28, facilitating the recruitment and activation of upstream associated anti-inflammatory activity nodes in intracellular signaling cascades including The IMiDs possess clinically relevant anti-inflammatory activity. phosphatidylinositol-3-kinase (PI3K) and nuclear factor (NF)- ENL is an immune complication of Mycobacterium leprae infection kB.36,37 As neither CD28 nor the IMiDs possess intrinsic kinase manifesting with fever, skin lesions and multi-organ dysfunction activity, an indirect mechanism of CD28 phosphorylation, such as because of antibody complex deposition, complement activation recruitment of other signaling proteins including protein and cytokine dysregulation.5 Patients with ENL have increased kinase-C (PKC), is postulated. Pomalidomide costimulation cellular and circulating levels of TNFa that rapidly reduce with activates PKC and PI3K to enhance nuclear translocation of thalidomide therapy.18 Inflammatory complications of other nuclear factor of activated T-cells (NFAT)-2 and activated protein

Oncogene (2013) 4191 – 4202 & 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 O O Second primary malignancy Limb deformities (phocomelia) Venous thromboembolic disease NH Cereblon binding Peripheral neuropathy N O Oxidative stress 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 TNFα IL2 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- OTHER DEFINING FEATURES OF THALIDOMIDE ANALOGUES quence of the immunostimulatory activity of IMiDs on T-cells. The (1) Anti-angiogenic properties secretion of IL-2 and IFN-g by CD4 T-cells following IMiD exposure Inflammatory cytokine networks and angiogenic pathways are augments NK-cell mediated killing against both (allogeneic) inherently linked, as neo-vascularization is a consequence of the leukaemic cell lines and autologous MM cells in vitro.38,40 The inflammatory response. Angiogenesis is important in tumor importance of T-cell help in IMiD responses is highlighted by the growth and metastasis. Experiments initially designed to dissect suppressive effects of calcineurin inhibitors and corticosteroids on thalidomide’s teratogenic potential subsequently demonstrated NK-cell cytotoxicity assays.37,40,41 In vivo administration of IMiDs it’s anti-angiogenic properties.43 Thus, suppression of angio- expands circulating NK-cell populations and synergizes with anti- genesis linked IMiD cytokine modulation and anti-cancer CD20 antibody therapy against B-cell lymphoma xenografts in an activity. However, in contrast to the anti-inflammatory and NK-cell-dependent fashion.42 This is potentially explained by the immunomodulatory properties that are conserved across observation that lenalidomide augments antibody-dependent cell primate and rodent species’, anti-angiogenic effects appear to cytotoxicity against rituximab (anti-CD20) treated CD20 þ tumor be relatively species specific. As the teratogenic effects of

& 2013 Macmillan Publishers Limited Oncogene (2013) 4191 – 4202 Molecular IMiD targets J Shortt et al 4194 Notwithstanding the putative role of interspecies metabolism in VEGFR FGFR IMiD-mediated suppression of vascularization, thalidomide and its analogues have demonstrable activity against several key

PI3K angiogenesis regulators. In vivo suppression of pro-angiogenic Ceramide factors including IL-6, TNFa, VEGF, NF-kB and prostaglandin synthesis is predicted to indirectly downregulate angiogenesis.12,26,47 At the cellular level, the IMiDs downregulate NFκB WNT AKT paracrine production of VEGF and IL-6 by both bone marrow 48 u stroma and MM cells, alone or in co-culture. VEGF-induced u DDB1 u angiogenesis is modulated by intracellular pro-survival signaling Apoptosis 44,49 CRBN pathways and sphingolipid metabolism. Thalidomide activates Cul4 O H H O neutral sphingomyelinase to generate ceramide in human u Oxidative stress u umbilical vein endothelial cells resulting in both decreased u CRBN 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 44 Teratogenesis to VEGF and bFGF. Here, reduced endothelial cell migration correlated with downregulation of AKT phosphorylation, Figure 4. Overlapping mechanisms of teratogenesis and indicating that lenalidomide might uncouple the permissive anti-angiogenesis conveyed by thalidomide targets. The anti- effects of VEGF-receptor ligation from intracellular signaling angiogenic and teratogenic pathways modulated by thalidomide pathways. Together, these results indicate that the IMiDs are inherently linked. Thalidomide downregulates VEGF receptor (VEGFR) and fibroblast growth factor receptor (FGFR) signaling by modulate angiogenesis at multiple levels including systemic several mechanisms, including generation of ceramide and down- cytokine networks, local tumor/stroma interactions, surface regulation of effector pathways downstream of receptor signaling. expression of VEGF receptors and intracellular signaling Anti-apoptotic effectors antagonized in this fashion include AKT, pathways thereof. WNT and NF-kB. Concurrently, thalidomide induces oxidative stress (pro-apoptotic) and binds cereblon (CRBN) to inhibit the E3 (2) Teratogenicity ubiquitin–ligase activity of the CRBN/DDB-1/Cul4 complex. Direct ubiquitination (u) targets of this complex outside of its Intense scientific activity has been dedicated to determining the auto-ubiquitination function are not known. Together these molecular mechanisms of thalidomide teratogenicity.50 From a pro-apoptotic, anti-angiogenic, oxidative and CRBN-mediated translational perspective, a non-teratogenic yet efficacious IMiD effects combine to induce teratogenicity. 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 thalidomide show similar species specificity, it is likely that activities of IMiDs are separable from teratogenic effects remains a thalidomide’s anti-angiogenic capacity and teratogenicity are point of conjecture. Proposed embryotoxic mechanisms include also linked (Figure 4). 51 53–55 43 effects on angiogenesis, intracellular signaling, gene D’Amato et al. first described anti-angiogenic activity of orally regulation56 and the induction of oxidative stress.57 For an administered thalidomide and a related teratogenic analogue, excellent synopsis that attempts to unify existing conceptual EM-12 that was apparent in experimental systems using rabbit but frameworks, the reader is directed to a recent review by Ito et al.50 not chicken cells. Later, the same investigators demonstrated As mechanisms of thalidomide teratogenicity have been intraperitoneal, but not oral administration of thalidomide extensively reviewed elsewhere, they will only be covered here inhibited angiogenesis in bFGF and vascular endothelial growth 13 in brief: factor (VEGF)-driven cornea micro-pocket assays in the mouse. Conversely, orally administered lenalidomide reduced basic (a) Angiogenesis. Thalidomide’s anti-angiogenic effects provide fibroblast growth factor (bFGF)-induced angiogenesis in a rat insight into the predilection for limb deformities in its teratogenic 44 mesenteric window assay. The observation that route of phenotype. Developing limb buds comprise an immature vessel administration (oral versus intraperitoneal) resulted in divergent network that is hypersensitive to thalidomide’s anti-angiogenic anti-angiogenic effects with species specificity supported the effects.51 Thalidomide induces loss of newly formed vessels at notion that in vivo IMiD metabolism modulates anti-angiogenic critical stages in limb development before the onset of effects. This hypothesis was further substantiated using in vitro rat malformations.51 Changes in vasculature appear to precede aorta and human endothelial cell culture systems that are changes in gene expression, cytostatic effects and cell death in 45 insensitive to the presence of non-metabolized thalidomide. the developing limb. This suggests defects in vascular Here, supplementation of thalidomide with human or rabbit (but development are an upstream event in teratogenicity. not rat) hepatic microsomes resulted in reduced microvessel formation and endothelial cell growth. In a humanized myeloma (b) Oxidative stress. There is much evidence linking thalidomide xenograft model, co-engraftment of human fetal liver cells into a teratogenicity to oxidative stress. Bioactivation of thalidomide severe combined immunodeficient mouse augmented the anti- induces glutathione depletion and DNA-oxidation consistent with myeloma activity of thalidomide.46 Myeloma xenografts from the capacity to generate reactive oxygen species.57 Interestingly, thalidomide-treated, human fetal liver bearing mice showed the threshold for thalidomide-induced free-radical generation and reduced microvessel density compared with non-fetal liver embryonic DNA-oxidation is much lower in rabbits than mice, transplanted hosts. It was concluded that human hepatic corresponding with greater sensitivity to dysmorphogenesis. Pre- metabolism was required for thalidomide’s anti-myeloma activity treatment of rabbits with the free-radical spin trapping agent, in a mouse. However, this interpretation requires caution as alpha-phenyl-N-t-butylnitrone abolishes thalidomide-induced thalidomide metabolites were not assayed, the immunological DNA-oxidation and teratogenicity.57 The induction of oxidative effects of fetal liver grafts (which contain hematopoietic stem stress in developing limb buds (potentially compounded by tissue cells) were not accounted for, and thalidomide was administered hypoxia) induces alterations pro-survival signaling pathways parenterally rather than orally. including NF-kB,58 bone morphogenic protein/Dickkopf-1/Wnt54

Oncogene (2013) 4191 – 4202 & 2013 Macmillan Publishers Limited Molecular IMiD targets J Shortt et al 4195 and PI3K/AKT55 culminating in downregulation of FGF and inhibition to species-speciﬁc oxidative stress were elucidated, this apoptosis induction (Figure 4). might ﬁnally provide a unifying model (Figure 4).

(c) Dysfunction of the cereblon/DNA-binding protein 1 ubiquitin– ligase complex. Cereblon is a ubiquitously expressed protein that EVIDENCE FOR GENETIC AND EPIGENETIC EFFECTS OF IMIDS was identified as a candidate gene in a mild form of autosomal Although not mutagenic,66 thalidomide intercalates DNA.67,68 recessive, non-syndromal mental retardation.59,60 Cereblon binds Specifically, S( À ) thalidomide is predicted to intercalate into the voltage-gated chloride channels in the retina61 and calcium- major groove of the DNA-double helix structure at purine sites activated potassium channels in the brain.62 It also modulates (with greater affinity for guanine over adenine) where its AMP-activated protein kinase activity.63 In autosomal recessive, glutarimide moiety oriented in a 30-direction.56 The preferential non-syndromal mental retardation, a truncating cereblon point affinity of thalidomide for guanine nucleotides potentially explains mutation interrupts C-terminal myristoylation and phosphory- differential regulation of genes bearing GC-rich gene promoters. lation sequences, potentially disrupting intracellular localization The transcription factor Sp1 binds GC-boxes within promoter and signaling activity, respectively.59,60 regions and docks within the major groove of DNA that would be Cereblon is required for the E3 ubiquitin–ligase activity of a occupied by intercalated thalidomide.69,70 Candidate genes multi-protein complex containing DNA-binding protein 1 and regulated by Sp1 include insulin-like growth factor and its Cullin 4 (Figure 4). Thalidomide was recently demonstrated to receptor, insulin receptor substrate-1, FGF-2, FGF receptors and directly modulate FGF-mediated angiogenesis via a cereblon- several integrins. As these have a central roles in angiogenesis and dependent pathway.53 Using thalidomide to ‘pull down’ potential embryo development, modulation of gene expression via drug targets from crude cell extracts, Ito et al.53 identified cereblon intercalation at GC-rich promoters has been proposed as a as a thalidomide-binding protein. The antagonism of cereblon by mechanism for thalidomide teratogenesis (reviewed in Stephens thalidomide was subsequently shown to be necessary but et al.56). insufficient to induce teratogenicity in Zebrafish and chick The DNA-intercalation model provides a conceptual framework embryos. In the Zebrafish model, thalidomide-induced terato- for how thalidomide might reduce the efficiency of transcription genicity could be phenocopied by cereblon knockdown and at GC-rich promoters. However, emerging evidence suggests the rescued by overexpression of a thalidomide-insensitive cereblon IMiDs epigenetically promote the transactivation of genes at the mutant protein.53 Thalidomide exposure or cereblon knockdown same GC-rich loci. Lenalidomide and pomalidomide induce resulted in reduced FGF expression in the apical ectodermal ridge, growth arrest in selected myeloma and B-cell lymphoma cell providing a mechanistic link between thalidomide, cereblon and lines. This correlates with the induction of the cell cycle regulator FGF-mediated angiogenesis in teratogenicity. p21WAF-1 and concomitant inhibition of cyclin-dependent kinases, 71 Although the characterization of cereblon as a thalidomide- hypo-phosphorylation of Rb and G0–G1 arrest. Pomalidomide target provided much insight into teratogenic mechanisms, the rapidly upregulates CDKN1A mRNA expression via p53- role of complementary pathways remains undefined. In the independent recruitment of Sp1 to a proximal GC-rich region in human, cereblon mutations causing autosomal recessive, non- the CDKN1A promoter (Figure 5a).72 Sp1 recruitment occurs in the syndromal mental retardation do not associate with limb absence of a change in absolute Sp1 levels and is facilitated by the defects.60 Conversely, persons suffering from thalidomide- acetylation of histone H3 on lysines 9 and 14. As the IMiDs do not induced dysmelia do not demonstrate intellectual disability.1 As inhibit histone deacetylases, an indirect mechanism whereby the protein structure of cereblon is highly conserved, thalidomide recruitment of the histone demethylase, LSD-1 to H3K9 was should not discriminate between rodent and human (or Zebrafish) suggested.72 In this model, the acetylation of H3K9/14 is co- protein.53 Thus, the well described associations between species- regulated by LSD-1 demethylation at the same loci. specific thalidomide metabolism and angiogenic effects are not Epigenetic mechanisms are also implicated in IMiD-related well explained by the cereblon-model of teratogenicity. Another modulation of cytokine expression (Figure 5b).73 Cytokine caveat to the interpretation of Zebrafish modeling is the receptor ligation activates intracellular signaling through the supraphysiological thalidomide dosing required to induce anti- JAK/STAT pathway. STAT target genes include the suppressor of angiogenic and teratogenic phenotypes. Myeloma patients cytokine signaling (SOCS) genes that attenuate JAK/STAT receive 50–200 mg/day of thalidomide. A dose-escalation study activity.74 SOCS1 is a negative regulator of IFN-g, IL-2 and IL-6. in elderly prostate cancer patients recorded maximum steady- Hypermethylation of the SOCS1 promoter is reported in myeloma state plasma concentrations of 11 mg/ml (B40 mM) in persons patients, leading to augmented expression of IL-6, a potent receiving 1.2 g of thalidomide daily.64 However, Ito et al.53 plasma cell growth factor.75 Pomalidomide upregulated myeloma reported dysmorphogenesis with 400 mM of thalidomide applied cell SOCS1 expression concomitant with demethylation of its 73 directly to dechorionated embryos, diluted from a 400 mM DMSO silenced promoter region. Demethylation of the SOCS1 promoter stock solution. As thalidomide is relatively insoluble, repeated by pomalidomide or 5-azacitidine correlated with increased cycles of heating (to 65 1C) and agitation were required to myeloma cell sensitivity to subsequent lysis by cytotoxic solubilize thalidomide, introducing the potential for pre-analytical T lymphocytes. Interestingly, the epigenetic modulation of drug modification. Others have reported Zebrafish embryos to be SOCS1 was tumor specific, as the promoter methylation was much less sensitive to thalidomide application. For example, unchanged in effector lymphocyte populations.73 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 ACTIVITY IN MDS WITH DEL 5Q investigators used similar methodology, the reasons for this MDS refers to a group of clonal bone marrow stem-cell disorders 10-fold discrepancy in potency are unclear. In either case, the characterized by ineffective hematopoiesis and potential trans- concentrations used are much higher than those measured in formation to acute myeloid leukemia. MDS with associated human subjects. deletion of the long arm of chromosome 5 (del 5q) is an MDS Although it is apparent that cereblon is a potentially important subtype characterized by refractory macrocytic anemia, well IMiD target, there are still many unanswered questions regarding preserved thrombopoiesis and low bone marrow blasts.76 Unlike mechanisms of thalidomide embryotoxicity. In particular, the other MDS, 5q- syndrome has a high response rate to grossly divergent interspecies sensitivity to teratogenesis is lenalidomide, including cytogenetic remissions and improved unresolved. If a pathway linking thalidomide-related cereblon erythropoiesis.77 The clinical phenotype of MDS with del 5q is

& 2013 Macmillan Publishers Limited Oncogene (2013) 4191 – 4202 Molecular IMiD targets J Shortt et al 4196 megakaryopoiesis to co-operate with RPS14 deletion in the Sp1 induction of anemia while promoting platelet production. The predominant clinical response to lenalidomide in MDS p21 is improved erythropoiesis with a signiﬁcant proportion of 77 Me Me Ac Sp1 patients achieving independence from red cell transfusions. CDKN1A CDKN1A This is associated with cytogenetic improvement, but occurs at the Me LSD-1 Ac expense of thrombocytopenia and neutropenia. Despite detailed insight into the molecular mechanism of anemia and Me Me thrombocytosis in MDS with del 5q, the mechanism of

Me 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 85 IL6 upregulation of only one CDR gene, SPARC. SPARC regulates interactions with the extracellular matrix, mediating antiproliferative and anti-angiogenic effects in common with IMiD- related activity. An alternative hypothesis is that lenalidomide JAK further downregulates the function of haploinsufficient proteins coded in the CDR by post-transcriptional and post-translational STAT SOCS1 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 BCL2 phosphorylation residues on cell cycle regulators targeted by STAT Cdc25C and PP2A.86 This correlates with potent inhibition IL6 target genes of recombinant human Cdc25C phosphatase activity by 86 SOCS1 lenalidomide in the nanomolar concentration range in vitro. Me Me Lenalidomide’s effects on PP2A activity appear to be indirect, as SOCS1 SOCS1 Me they cannot be demonstrated on purified protein in the same assays. As Cdc25C and PP2A regulate the cell cycle, antagonism by Figure 5. Examples of epigenetic modulation of gene targets by lenalidomide explains the selective induction of growth arrest and IMiDs. (a) Genes bearing GC-rich promoter regions are preferentially apoptosis in del 5q cells compared with their normal counterparts. targeted by IMiDs either by direct DNA intercalation (not shown) or The erythroid phenotype of MDS with del 5q is p53 alterations in methylation status (Me) and histone acetylation (Ac). dependent.82 Moreover, neoplastic escape from lenalidomide The transcription factor Sp1 is excluded from a GC-rich region of the in vivo is associated with selection for sub-clones with mutant CDKN1A promoter by methylation. Recruitment of the demethylase 87 LSD-1 allows histone H3 acetylation at lysines 9 and 14, opening p53. Ribosomal biogenesis occurs in the nucleolus, which also the promoter to transcription of the cell cycle inhibitor p21WAF-1. provides a sink for the sequestration of the p53 ubiquitin ligase, (b) SOCS1 feedback suppresses JAK/STAT signaling in response to MDM2 (Figure 6a). Nucleolar stress due to a range of causes cytokine receptor ligation including IL-6. In myeloma cells, the including ribosomal protein deficiency results in the dispersion of SOCS1 promoter is silenced by hypermethylation, amplifying the free RPs that are able sequester MDM2 independently of its co- transcription of IL-6 regulated pro-survival effectors (for example, regulator ARF. The sequestration of MDM2 allows stabilization of BCL2). IMiD treatment demethylates the SOCS1 promoter, restoring p53. Lenalidomide might therefore attenuate the severity of SOCS1 expression and dampening pathophysiological JAK/STAT dyserythropoiesis indirectly by either reducing ribosomal stress or signaling. promoting p53 degradation. The suppressive effects of lenalidomide 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 specifically mapped to loss of a common deleted region (CDR) on 5q deletion by antagonising PP2A-enhanced ubiquitin-mediated chromosome 5.78 This CDR provides a list of 40 genes implicated clearance of MDM2.88 Thus, it is possible that lenalidomide in the biology of 5q- syndrome and therefore a template for the treatment reduces ribosomal stress and the p53 response thereof discovery of lenalidomide-responsive elements. In a seminal to ameliorate dyserythropoiesis (Figure 6a). The observation that paper, Ebert et al.79 used an RNA-mediated interference lenalidomide can destabilize p53 is clinically significant, as p53 approach to interrogate the CDR. They identified ribosomal suppression is a highly oncogenic effect. IMiD-mediated p53 protein (RP) S14 haploinsufficiency as a key determinate of the suppression provides a mechanistic explanation for the recently erythroid differentiation defect in MDS with del 5q. This correlated published observation that prolonged exposure to lenalidomide is well with prior descriptions of heterozygous germline mutations associated with the development of second cancers (discussed for several other RPs in patients with Diamond–Blackfan anemia, a further below).89–92 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 ACTIVITY IN MM AND MATURE B-CELL NEOPLASMS RP haploinsufficiency to induce anemia.82,83 Although RP MM is a malignancy of immunoglobulin producing plasma cells haploinsufficiency contributes to the p53-dependent anemia of associated with systemic toxicities including renal failure, anemia, MDS with del 5q, other genes are implicated in the generation immunoparesis and destructive skeletal lesions.93 The mechanism of dysplastic megakaryocytic hyperplasia and thrombocytosis. of action of IMiDs in MM is complex and likely reflects the In particular, loss of microRNA-145 in the CDR results in increased sum-total of a range of independent targets including direct expression of the transcription factor Fli-1 (ref. 84). Fli-1 toxicity to MM cells, disruption of the protective bone marrow overexpression causes a shift from erythropoiesis to microenvironment and augmentation of host-mediated anti-

Oncogene (2013) 4191 – 4202 & 2013 Macmillan Publishers Limited Molecular IMiD targets J Shortt et al 4197

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

& 2013 Macmillan Publishers Limited Oncogene (2013) 4191 – 4202 Molecular IMiD targets J Shortt et al 4198 of conventional therapeutics.101,102 Elaborate co-culture systems The contribution of T-cells to immune-mediated control of MM have defined both direct interactions with bone marrow stromal in vivo has been a difficult question to address, with conflicting cells and humoral components of cytokine networks as essential reports characterizing both impaired/exhausted and activated to MM viability.101,103–105 In particular, osteoclastogenic cytokines clonal expansions of MM-specific T-cells in MM patients.126–129 of the TNF-family and IL-6 are implicated in myeloma cell growth There is agreement, however, on the responsiveness of T-cells to and skeletal destruction.102,106 Paracrine and autocrine interleukin immunomodulatory agents. The stimulatory effect of IMiDs on networks combine with cell adhesion molecules to activate T-cell function is driven by PI3K-mediated activation of nuclear intracellular pro-survival pathways including PI3K/AKT, NF-kB factor of activated T-cells-2.37,130 This results in increased and RAS/MAPK.107,108 Myeloma cell adherence to bone marrow production of IL-2 and NK-cell stimulation.37,40 Further to this, stroma also upregulates VEGF secretion to promote IMiDs appear to polarize T-cells towards a Th-1 phenotype angiogenesis.48 By downregulating cytoprotective cytokines (for through costimulation, resulting in the production of IFN-g and example, IL-1b, IL-6, IL-10 and VEGF), the IMiDs may disrupt suppression of IL-10.131,132 The first study to show protective anti- protection by the microenvironment, sensitizing myeloma to tumor immunity by IMiDs in conjunction with whole-tumor cell concurrent cytotoxic therapies.109 vaccination was demonstrated in a CT-26 colon mouse model.33 As conventional therapies are initially successful in debulking a More recently, Noonan et al.34 showed single-agent lenalidomide high proportion of the myeloma burden, the bone marrow niche therapy in MM patients augmented endogenous MM-specific CD8 is postulated to rescue clonogenic myeloma stem cells ultimately T-cell responses.34 Both Th-17 and regulatory T-cells have a responsible for disease relapse.110 Although the literature on the pathogenic role in MM, with higher T-regulatory cells conferring identification of myeloma stem cells are somewhat nebulous and poorer outcomes in MM patients.133,134 The ability of IMiDs to without consensus, studies have eluded to several populations of reduce Th-17 cells, and inhibit the generation and function of myeloma cells that have clonogenic capacity. These include the Foxp3 T-regulatory cells may result in the mediation of T-cell CD138 À CD27 þ cells that are lenalidomide, dexamethasone, responses against MM.34,135 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 CLINICAL TOXICITIES PROVIDE MECHANISTIC INSIGHT INTO clonogenic properties in NOD/SCID mice.113 ‘Side-population IMID BIOLOGY 114 cells’ defined by the capacity to efflux Hoechst 3342 stain, The differing side effect profiles of thalidomide and its IMiD have also been identified as a source of stem-cell like derivatives highlight important biological distinctions between chemoresistant cancer-initiating cells. The proliferation and these closely related compounds not merely explained by issues survival of MM side-population cells is augmented by bone of potency. Elucidation of the mechanisms responsible for marrow stroma through pathways that are suppressible by divergent clinical toxicities could potentially inform the biology thalidomide and lenalidomide.110 Lenalidomide also reduces the 110 of tumor-specific responses to the respective analogues. The clonogenic potential of myeloma side-population cells. This major cumulative dose-limiting toxicity of thalidomide is periph- indicates that suppression of the myeloma ‘stem-cell’ may be an eral neuropathy136 with minimal bone marrow suppression. important facet to IMiD activity, particularly in the prevention of Although lenalidomide is more potent than thalidomide (for relapse from minimal residual disease states (for example, example, in TNFa suppression assays), it does not cause following autologous stem-cell transplantation). neuropathy.89 Rather, lenalidomide suppresses normal hematopoiesis resulting in neutropenia and thrombocytopenia. Pomalidomide also causes neutropenia, with a lower incidence (c) Immunological disease control of neuropathy than thalidomide.137 All of the IMiDs are Hematological malignancies, particularly MM are characterized by prothrombotic, with prophylactic anticoagulant therapy often progressive immune cell dysregulation, of which T- and NK-cell administered concurrent to the IMiDs to prevent venous suppression is well described.115–117 Lenalidomide’s ability to 37,38,40 thromboembolic disease. The greatest emerging concern with potentiate T- and NK-cell activation has resulted in chronic lenalidomide exposure is a measurable increase in the exploratory studies elucidating the role of IMiDs in the incidence of second primary malignancies.89–92 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 (a) Peripheral neuropathy largely governed by NKG2D, with a lesser role for DNAM-1 and Neuropathy occurs in most patients receiving chronic thalidomide NKp46.118 Interestingly, IMiDs do not appear to alter the therapy with an actuarial incidence of over 70% at 12 months.136 expression of these receptors to confer functional changes in The etiology of thalidomide-induced neuropathy is multifactorial, NK-cell activity.40,118,119 IMiDs upregulate CD16 expression on NK as patients often have pre-existing nerve damage and many cells to increase antibody-dependent cell cytotoxicity against receive concurrent neurotoxic therapies. In particular, the MM.120,121 It is becoming evident that IMiDs are able to decrease combination of thalidomide with bortezomib is associated with the activation threshold of NK-cell cytotoxicity through a high risk of nerve damage.138 However, the potent new- modulation of inhibitory receptor interactions. MM-induced generation proteasome inhibitor carfilzomib is not neuropathic, expression of the programmed cell death receptor 1 inhibits NK- indicating that the neurotoxicity of bortezomib may be ‘off target’ cell cytotoxicity.122 Treatment with lenalidomide reduces inhibition of other serine proteases.139 HtrA2/Omi was identified programmed cell death receptor 1 ligand on MM cells and thus as a non-proteasomal bortezomib target that is spared by sensitizes MM cells to lysis by autologous NK cells.122 Killer-cell carfilzomib and protects neurons from oxidative stress.139 Thus, Immunoglobulin-like receptors (KIR) are well described as the thalidomide-induced free-radical generation57 and bortezomib- major functional inhibitory receptors on NK cells.123 As MM cells induced depletion of neuroprotective serine proteases (for express high levels of the KIR ligand, HLA class I, it is likely that this example, HtrA2/Omi) could combine to cause the mitochondrial interaction contributes to NK-cell dysfunction in vivo.124,125 IMiDs toxicity observed in neuropathic states. Indeed, genomic profiling are able to reduce the expression of KIR on NK cells.119 identified a single-nucleotide polymorphism of glutathione Furthermore, a recent study demonstrated the therapeutic S-transferase theta-1 (GSTT1) as predictive of neuropathy in a efficacy of combination IMiD and anti-KIR blocking therapy in a cohort of myeloma patients receiving thalidomide.140 As GSTT1 is mouse model of MM.125 a cellular regulator of reactive oxygen species, this further

Oncogene (2013) 4191 – 4202 & 2013 Macmillan Publishers Limited Molecular IMiD targets J Shortt et al 4199 implicated thalidomide-induced oxidative stress in the etiology of CONCLUSIONS nerve damage. Despite their simple chemical structures, thalidomide and its analogues exhibit pleotropic immunomodulatory and anti-neoplastic effects. This conveys remarkable clinical efficacy in clearly (b) Hematological toxicities defined disease entities, specifically granulomatous inflammation, Neutropenia and the risk of sepsis is a major dose-limiting toxicity mature B-cell neoplasms and MDS with del 5q. The molecular of lenalidomide. Lenalidomide treatment is also associated with mechanisms of IMiD activity are likely to be both divergent and þ impaired stem-cell mobilization for autologous CD34 collection complementary across these distinct diseases. In particular the 141 before transplantation in MM. This is despite the observation combined ability to modulate multiple targets from the level of þ that IMiDs induce the expansion of CD34 bone marrow the epigenome to post-translational protein modulations and 142 progenitors in vitro. However, the transcriptional changes that systemic cytokine networks highlights the complexity of IMiD þ evoke CD34 proliferation in vitro are also associated with a biology. Unfortunately, the beneficial properties of IMiDs may be 143 differentiation block within the granulocytic lineage. This inseparable from serious clinical toxicities including teratogenicity, apparent ‘maturation arrest’ is associated with downregulation secondary malignancy, myelosuppression and nerve-damage, of the transcription factor PU.1, and results in a relative excess ostensibly because they are mediated by shared molecular of promyelocytes. Promyelocytes are rich in prothrombotic targets. It is hoped that the next chapter in the ‘thalidomide mediators including cathepsin G, a platelet agonist implicated in saga’1 will be the identification of new targets that can be the pathogenesis of disseminated intravascular coagulation therapeutically manipulated by novel non-IMiD pharmaceuticals 144 complicating acute promyelocytic leukemia. Therefore, to derive clinical benefit without toxicity. 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 CONFLICT OF INTEREST COX-2 has been clearly implicated in adverse thrombotic events in The authors declare no conflict of interest. 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 REFERENCES withdrawal release a range of pro-coagulant mediators.44,146 A targeted genetic screen of myeloma patients identified single- 1 Mellin GW, Katzenstein M. The saga of thalidomide. Neuropathy to embryopathy, with case reports of congenital anomalies. NEnglJMed1962; 267: 1184–1192. nucleotide polymorphisms associated with thalidomide- 2 McBride WG. Thalidomide and congenital abnormalities. Lancet 1961; 278: 1358. associated thrombosis in genes and pathways important in drug 3 Lenz W, Pfeiffer RA, Kosenow W, Hayman DJ. Thalidomide and congenital 146 metabolism, DNA-repair and cytokine regulation. The authors abnormalities. 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