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`Copyright ©) 2002 by Annual Reviews. All rights reserved
`
`THALIDOMIDE: Emerging Role in Cancer Medicine
`
`Paul Richardson, Teru Hideshima, and Kenneth Anderson
`Jerome Lipper Myeloma Center, Division ofHematologic Oncology, Department of
`Adult Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston,
`Massachusetts 02115; e-mail: paul_richardson@dfci.harvard.edu
`
`Key Words
`
`immunomodulation, antiangiogenesis, myeloma
`
`@ Abstract Thalidomide—removed from widespreadclinical use by 1962 because
`of severe teratogenicity—has antiangiogenic and immunomodulatory effects, includ-
`ing the inhibition of tumor necrosis alpha factor. It has now returned to practice as
`an effective oral agent in the managementof various disease states including ery-
`thema nodosum leprosum, for which it was approved by the U.S. Food and Drug
`Administration in 1998, and more recently certain malignancies, including multiple
`myeloma. Although thalidomide’s mechanism of action remains incompletely under-
`stood, considerable insight has been generated by extensive preclinical studies in mul-
`tiple myeloma. Moreover,clinicaltrials have confirmedbenefit in relapsed disease, and
`the role of thalidomide in treating newly diagnosedpatients is currently understudy.Its
`use in other tumors is under evaluation, with promise in renal cell carcinoma,prostate
`cancer, glioma, and Kaposi’s sarcoma. Activity has also been demonstrated in chronic
`graft-versus-host disease and in symptom relief as part of palliative care.
`
`INTRODUCTION
`
`A tragic cvent in the history of drug dcvclopment occurred with the over-the-
`counter marketing of thalidomide in Europe during the late 1950s for the treat-
`ment of pregnancy-associated morning sickness. As early as 1961, reports of
`teratogenicity and dysmyelia (stunted limb growth) associated with thalidomide
`use prompted its subsequent withdrawal (1,2). The return of thalidomide as a
`therapy in certain conditions stems from its broad array of pharmacoimmuno-
`logic effects (3). This rehabilitation was reflected by its approval in 1998 by the
`U.S. Food and Drug Administration for the short-term treatment of cutaneous
`manifestations of moderate to severe erythema nodosum leprosum (ENL), along
`with its use as maintenance therapy to prevent and suppress the cutaneous man-
`ifestations of ENL recurrence (3). Thalidomide has since became a treatment of
`choice for ENL, and its wide spectrum ofactivity has fostered its application
`in a variety of disease states (Table 1) (3-5). Because ofits teratogenic effects,
`thalidomide is now used understrict guidelines to prevent fetal exposure to the
`drug (4).
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`TABLE 1
`
`Potential therapeutic uses of thalidomide currently under investigation
`
`Cancer and related conditions
`
`Infectious diseases
`
`Autoimmunediseases
`
`Solid tumors(e.g., brain, breast, renal cell carcinoma);
`hematologic malignancies (¢.g., multiple myeloma)
`HIV/AIDSandrelated conditions;
`aphthous ulcerations; wasting syndrome;
`mycobacterial infections(e.g., tuberculosis)
`
`Discoid and systemic lupus erythematosus;
`chronic graft-versus-host disease; inflammatory
`bowel disease; rheumatoid arthritis; multiple sclerosis
`
`Dermatologic diseases
`
`Behcet’s syndrome; prurigo nodularis,; pyoderma gangrenosum
`
`Other disorders
`
`Sarcoidosis; diabetic retinopathy; macular degeneration
`
`In the field of medical oncology, the discovery of thalidomide’s antiangiogenic
`properties has coincided with the emerging importance of angiogenesis in tumor
`growth and progression. Thalidomide has been shownto inhibit angiogenesis in-
`duced bybasic fibroblast growthfactor (8-FGF)in a rabbit cornea micropocketas-
`say and by vascular endothelial growth factor (VEGF) ina murine modelof corneal
`vascularization (6,7). In human studies, the drug appears to undergo activation
`to metabolites with antiangiogenic activity (8). Because of these antiangiogenic
`properties, thalidomideis currently undergoing evaluation in the treatment of var-
`ious solid tumors, multiple myeloma, and other hematologic malignancies (9-13).
`Results in multiple myeloma are particularly promising, although thalidomide’s
`antiangiogenic effects are believed to be only part of its antimyelomaactivity.
`Its other potential actions include modulation of adhesion molecules, inhibition
`of tumornecrosis alpha factor (TNF-a), downregulation of lymphocyte surface
`molecules, lowering of CD4:CD8 peripheral lymphocyteratios, and direct effects
`on myelomacells themselves (10, 14-18).
`This chapter presents a comprehensive review of the pharmacology ofthalido-
`mide, a description of preclinical studies in multiple myelomato illustrate the
`drug’s complex putative mechanismsof action, and a descriptionof clinical stud-
`ies in multiple myeloma. Studies in other hematologic malignancies are also ad-
`dressed, as is the status of research in solid tumors and in other cancer-related
`applications.
`
`PHARMACOLOGY
`
`Thalidomide is a derivative of glutamic acid and is pharmacologically classi-
`fied as an immunomodulatory agent (19). Structurally, thalidomide contains two
`amide rings and a single chiral center (Figure 1); its full chemical name is alpha-
`N{phthalimido }glutarimide [C73 O4 N2 H9] and its gram molecular weight is
`258.2 (19).
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`THALIDOMIDE
`
`631
`
`Thalidomide
`
`At
`
`Oo
`
`N
`
`o&-(N-phthalimido) glutarimide
`
`° A
`
`nalogs (also known as IMiDs)
`
`CisdS.
`yt
`
`° c
`
`Structures ofthalidomide and its potent ana-
`Figure 1
`logues, immunomodulatory drugs (IMiDs).
`
`The currently available formulation is a nonpolar racemic mixture present as
`the optically active S and R isomers at physiologic pH, which can effectively
`cross cell membranes (19, 20). The S isomer has been linked to thalidomide’s
`teratogenic cffects, whereas the R isomer appears to be primarily responsible for
`its sedative properties (7, 20, 21). The isomers rapidly interconvert at physiologic
`pH in vivo, and thusefforts at formulating only the R isomerhave failed to obviate
`the teratogenic potential of thalidomide (20, 22).
`
`Pharmacokinetics
`
`Pharmacokinetic analysis of thalidomide in humans has been limited by the ab-
`sence of a suitable intravenous formulation, owing to the drug’s instability and
`poorsolubility in water. The pharmacokinetics of thalidomide have therefore been
`determined only from animal studies and in humans receiving the oral therapy.
`Single-dose thalidomide trials have been conducted in healthy volunteers, patients
`with HIV infection, and patients with hormone-refractory prostate cancer (23-27).
`As Table 2 shows, the pharmacokinetics appear highly variable. Moreover, in
`patients with HIV infection, dose adjustments based on both ideal body weight and
`body surface did not affect this variability (23,24). As a result of this variability,
`the pharmacokinetic properties of thalidomide in humans have not been well char-
`acterized, and this has confoundedthe definition of a dose-responseeffect against
`human cancer.
`
`
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`TABLE 2
`
`Single dose pharmacokinetic parameters of thalidomide in humans (27)
`
`Meanapparent
`pharmacokinetic parameters
`
`Population
`
`Elderly patients with
`hormone-refractory
`prostate cancer
`Patients with HIV
`infection
`
`Healthy female volunteers
`
`Healthy male volunteers
`
`Dose
`
`200 mg
`
`800 mg
`300 mg
`
`200 mg
`
`200 mg
`
`tnax
`
`3.3"
`
`44°
`3.4
`
`5.8
`
`44
`
`ty2(L)
`
`6.5
`
`18.3
`5.7
`
`41
`
`8.7
`
`“a(L)
`
`66.9
`
`165.8
`78.2
`
`53.0
`
`120.7
`
`Abbreviations: fax, time to reach maximum concentrations; t, ., elimination half-life; V4, volume of distribution,
`HIV, human immunodeficiencyvirus.
`*
`Medianvalue.
`
`Absorption
`
`When oral thalidomide at a dose of 100 mg/kg has been administered in animal
`studies, maximum serum concentrations were reached within 4 h (28). Absorption
`was apparently independent of the administered doses and slower than drug elim-
`ination. Recent studies in humans show a similar pattern; thalidomide at 200 mg
`per dose reaches peak concentration (tn) in a mean of ~4 h (23-25, 29).
`
`Distribution
`
`Animal studies have demonstrated a wide distribution of thalidomide throughout
`mosttissues and organs (28). It is present in semen following oral administration
`in rabbits, but it is not known whetherthe drug is present in human semen (19, 30).
`Human pharmacokinetic studies to date also indicate that thalidomide hasa large
`apparent volumeofdistribution (3) (24-26). Further, studies in elderly prostate
`cancer patients suggest variability in 13, possibly due to alterations in absorption
`and plasma protein binding (24).
`
`Metabolism
`
`Thalidomide undergoes rapid and spontaneous nonenzymatic hydrolytic cleavage
`at physiologic pH to generate up to 50 metabolites, of which five are considered
`primary metabolites (8, 20, 22, 28,31). Research efforts to better characterize the
`biologic properties of the specific metabolites have been complicated by their in-
`stability and rapid degradation under physiologic conditions (32). Whereas in vitro
`studies suggest thalidomide induces cytochrome P-450 isoenzymesin rats, recent
`evaluation of single- or multiple-dose pharmacokinetic parameters of oral thalido-
`mide at 200 mgdaily in healthy human volunteers has indicated that thalidomide
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`633
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`does not inhibit or induce its own metabolism over a 21-day period in humans,
`and thus very little metabolism of thalidomide is thought to occur via the hepatic
`cytochrome P-450 system (26, 33, 34).
`
`Excretion
`
`Thalidomide appears to be rapidly excreted in urine as its metabolites, with the
`nonabsorbcdportion of the drug excreted unchanged in feces. However, clearance
`is primarily nonrenal; mean terminal half-lives of the R and S isomers in healthy
`male human volunteers were measuredat 4.6 and 4.8 h, respectively (23, 28, 29).
`In a study report of urinary excretion data for a single dose (200 mg daily), the
`elimination half-life was ~8 h, with minimal drug excretion over a 24-h period
`(23). Both single and multiple dosing of thalidomide in older prostate cancer
`patients revealed a significantly longer half-life at a higher dose (1200 mg daily)
`than at a lower dose (200 mg daily) (24). Conversely, no effect of increased age
`on elimination half-life was identified in the age range of 55-80 years (24). Thus,
`the effects of renal or hepatic dysfunction on the clearance of thalidomide remain
`unclear, and additional studies are needed to better characterize age-related or
`physiologic effects on drug clearance.
`
`Drug Interactions
`
`The only drug that has been systematically evaluated for interaction with thalido-
`mideis oral hormonal contraceptives, which showednosignificant interaction. An-
`imal studies suggest that thalidomide enhancesthe sedative effects of barbituates
`and alcohol as well as the catatonic effects of chlorpromazine and reserpine. Cen-
`tral nervous system stimulants (including methamphetamine and methylphenidate)
`appear to counteract the depressant effects of thalidomide (35).
`
`Potential Antitumor Effects
`
`D’ Amatoetal., while evaluating thalidomide’s mechanism ofteratogenicity, found
`that it exhibited antiangiogenic properties (6,7). They postulated that thalido-
`mide inhibited angiogenesis by interrupting processes induced by 8-FGF and/or
`VEGF (6, 7,36). Further in vitro studies suggested that the antiangiogenic effect
`of thalidomide was due to specific metabolites and not the parent compound (37).
`Another important property of thalidomideis that it selectively inhibits TNF-a
`production while leaving the patient’s immune system otherwise intact (38). This
`has led to its application in various disorders characterized by abnormal TNF-a
`activity, including ENL, mycobacterium tuberculosis infection, graft-versus-host
`disease, rheumatoid arthritis, systemic lupus erythematosis, multiple sclerosis,
`Crohn’s disease, cancer- and HIV-related cachexia, diabetes mellitus, and endo-
`toxic shock.
`
`The exact mechanism of thalidomide-induced TNF-a inhibition is unclear, but
`it does appear to differ from other TNF-a inhibitors such as pentoxyfylline and
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`dexamethasone (39, 40). One mechanism postulated is that thalidomide inhibits
`TNF-a synthesis by accelerating degradation of TNF-~ mRNA,resulting in a
`significant but incomplete suppression of TNF-a protein production (39, 41). Of
`particular interest is the recent demonstration that thalidomide decreases the bind-
`ing activity of NF-«B whichin tum controls activation of the TNF-a gene (42).
`It has also been postulated that thalidomide’s effect on angiogenesis may be
`through TNF-e inhibition, since TNF-a has proangiogenic effects (6). However,
`the absence of a demonstrable TNF-a effect in experimental models of angiogen-
`esis, coupled with the inability of strong TNF-a inhibitors to directly influence
`angiogenesis, suggests that thalidomide’s antiangiogenic activity is not related to
`TNF-a inhibition alone (6, 7).
`
`Immunomodulatory Effects
`
`Results of studies evaluating the effects of thalidomide on lymphocytes have been
`inconsistent (43-45). Emerging evidence suggests that thalidomide does not di-
`rectly suppress lymphocyte proliferation (14). However, differential effects on
`T cell stimulation, shifts in T cell responses, and inhibition of proliferation of
`already stimulated lymphocytes have been shown (17,44, 46-48). Modification
`of surface adhesion molecule on leukocytes, inhibition of neutrophil chemotaxis,
`and effects of cytokines other than TNF-a have been demonstrated, including
`the inhibition of interleukin (IL)-12 production, enhanced synthesis of IL-2, and
`inhibition of IL-6 (14, 16, 38, 49-51).
`
`Adverse Effects
`
`Commonside cffects reported during treatment with thalidomide are summarized
`in Table 3 (27). Sedation and constipation appear to be the most common adverse
`effects in cancer patients (9, 13,36). The most serious adverse effect associated
`with thalidomideis peripheral neuropathy. There may also be an increased inci-
`dence of thromboembolic events, but this appears to be rare when the drug is used
`as a single agent. However, recent reports have shown thromboembolic complica-
`tions to be more frequent whenthe drug is combined withsteroids and particularly
`
`TABLE 3 Clinical adverse events reported during thalidomide use (27)
`
`Neurologic
`
`Gastrointestinal
`
`Dermatologic
`
`Miscellaneous
`
`
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`Sedation Exfoliative/erythrodermic§XerostomiaConstipation
`
`
`
`Dizziness
`
`Nausea
`
`Cutaneous reactions
`
`Weight gain
`
`
`
`
`
`Mood changes Increased appetite—_Brittle fingernails Edemaofface/limbs
`
`Headaches
`
`Pruritis
`
`Reduction in thyroid
`hormonesecretion
`
`Hypotension
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`with anthracycline-based chemotherapy, causing early cessation of one study ex-
`ploring a combinationof thalidomide with doxil and dexamethasone(52, 53). Pos-
`sible cardiovascular effects of thalidomide include bradycardia and hypotension.
`The risk of adverse cardiovascular events appears greater in older patients with
`coronarydisease taking multiple blood-pressure-lowering medications along with
`thalidomide (27).
`It is well-knownthat thalidomide must not be used during pregnancy, and rec-
`ommended contraceptive methods must be used by both men and womenofchild-
`bearing potential. The System for Thalidomide Education and Prescribing Safety
`(STEPS), implemented to ensure the safe distribution of thalidomide, requires a
`patient’s compliance with contraception guidelines and mandatory surveillance
`procedures. In addition,all healthcare providers who plan to prescribe and/or dis-
`pense thalidomide must be registered with the program (4).
`Thalidomide-induced peripheral neuropathy commonly presents with numb-
`ness of the toes and feet, muscle cramps, weakness, signs of pyramidal tract in-
`volvement, and carpal tunnel syndrome (5, 19). Clinical improvement typically
`occurs upon prompt discontinuation of the drug, but long-standing sensory loss
`has been documented (54-57). Drug-related neuropathy is characterized as asym-
`metric, painful, peripheral paresthesia with sensory loss (5, 54-57). The risk of
`developing peripheral neuropathy during thalidomide treatment increases when
`high cumulative doses are administered and especially in the elderly (19).
`In cancerpatients, thalidomide should be used with caution whenthere is a prior
`history of neuropathy and when used in combination with other agents known to
`have neurotoxic effects (27). However, whether the incidence of thalidomide-
`induced peripheral neuropathy is increased in cancer patients with a history of
`vinca alkaloid use remains unknown. Safety data from current phase T and IT
`clinical trials of thalidomide in the treatment of solid tumors, multiple myeloma,
`and hematologic malignancies suggest that peripheral neuropathy occurs in 10%-—
`30% of patients (10, 12, 13, 58).
`
`PRECLINICAL STUDIES OF THALIDOMIDE
`
`AND ITS ANALOGS IN MULTIPLE MYELOMA
`
`Although thalidomide wasinitially used to treat multiple myeloma because of
`its antiangiogenic effects, the mechanism of its antimyelomaactivity appears to
`be more complex (Figure 2). Preclinical studies of thalidomide and its potent
`analogs (also known as immunomodulatory drugs, IMiDs) suggest that these drugs
`act against myeloma in several ways. First, there appears to be a direct effect on
`the myelomacell and/or bone marrow stromal cell, which inhibits tumor growth
`and survival. Second, adhesion of myeloma cells to bone marrow stromal cells
`(BMSCs) triggers secretion of cytokines, which augment myelomacell growth
`and survival (59-61) and confer drug resistance (62); importantly, thalidomide
`modulates adhesive interactions (14) and thereby may alter tumorcell growth,
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`Cc. Thalidomide
`
`MM cells ae
` IL-6
`
`
`Bone
`Thalidomid
`
`A. Thalidomidemm
`nal
`FIL-1B
`marrow
`
`Stromal
`
`
`
`
`marrow
`
`ssels
`
`
`TNFos
`
`oNIL-2
`ee (@Kalymphocytes 1
`
`D. Thalidomide
`
`(@)
`Soe
`CDB+ T cells
`NK. cells
`
`.
`:
`E. Thalidomide
`
`Figure 2 Possible effect of thalidomide on myelomacells’ and bone marrowstro-
`mal cells’ (BMSCs’) microenvironment in vivo. (4) Thalidomide directly mhibits
`myeloma cell growth. (B) Thalidomide inhibits myeloma cell adhesion to BMSCs.
`(C) Thalidomide blocks IL-6, TNF-a, and IL-1 secretion from BMSCs. (D) Thalido-
`mide blocks the ability of VEGF and 8-FGFto stimulate ncovascularization of bone
`marrow. (#) ‘halidomide induces IL-2 and INF-y secretion from ‘Icells.
`
`survival, and drug resistance. Third, cytokines secreted into the bone marrow mi-
`croenvironment by myeloma cells and/or BMSCs, such as IL-6, IL-1, IL-10,
`and TNF-a may augment myelomacell growth and survival (61, 63), and thalido-
`mide mayalter their secretion and bioactivity (64). Fourth, thalidomide decreases
`the secretion of VEGF,IL-6 (65), and BFGF by myeloma and/or BMSCs.
`Since IL-6 is known to promote myelomacell growth and survival, and VEGF
`has been demonstrated to induce myelomacell migration (66), thalidomide may
`directly block tumor cell growth and migration, as well as inhibiting bone mar-
`row angiogenesis (67, 68). Recent studies have demonstrated the direct antitumor
`activity of thalidomide and IMiDs on myelomacells (18). IMiDs induce growth
`inhibition of myelomacells in a dose-dependent fashion, with an ICs) as low as
`0.1-1 «M. IMiDs are effective against myelomacell lines resistant to conven-
`tional chemotherapeutic agents such as doxorubicin, melphalan, or mitoxantrone,
`as well as dexamethasone. Moreover, thalidomide and the IMiDs enhanced the
`antitumor activity of dexamethasone (Figure 3a); conversely, these effects were
`partially inhibited by IL-6 (Figure 34), a potent myeloma growth and antiapop-
`totic factor. IMiDs also showed significant activity against drug-resistant patient
`myelomacells (Figure 4). The observedclinicalactivity of thalidomidein patients
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`637
`
`0
`
`control
`
`Thal
`
`=
`IMiD1
`
`he
`IMiD2
`
`IMiD3
`
`
`
`
`
`
`
`5
`
`Bs
`23
`e
`oO
`
`0
`
`;
`54
`x3
`= 2
`oO 1
`Q
`
`0
`
`0
`
`1
`
`o
`IMiD1 (uM)
`
`Od
`IMiD3 (uM)
`
`1
`
`0
`
`1
`
`O.1
`Thal(uM))
`
`0.4
`IMiD2 (uM)
`
`1
`
`S.
`3
`Po
`oO
`
`1 0
`
`;
`S4
`x3
`22
`CO,
`0
`
`Figure 3. Effect of Dex and IT_-6 on response of multiple myeloma cells to thalido-
`mide and the IMiDs. (a) MM.15 cells were cultured with 1.0 «4M thalidomide,
`IMiD1, IMiD2, or IMiD3 in control media alone (©) or with 0.001 (&), 0.01 (&), and
`0.1 44M () Dex. (6) MM_1S cells were cultured in control media alone and with 0.1
`and 1.0 uM thalidomide, IMiD1, IMiD2, or IMiD3 eitherin the presence (C) orab-
`sence (m) of IL-6 (50 ng/ml). In each case, 7H-TdR uptake was measured during the
`
`last 8 h of 48-h cultures. Values represent the mean (+ SD) 7H-TdR (cpm)oftriplicate
`cultures.
`
`with myelomathatis refractory to conventional therapies (69), coupled with our
`in vivo studies, suggests that thalidomide can overcomeresistance to conventional
`treatments. In addition, our laboratory work suggests that dexamethasone can add
`to the antiproliferative effect of thalidomide andits analogs in vitro, suggesting
`potentialutility of coupling these agents in novel therapeutics.
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`Thal
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`IMiD1
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`IMi
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`iw}NO
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`= Oowo
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`CPM(x103)
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`A i] Fy CPM(x103)
`AICOSASSSSS
`RS
`MN2
`
`
`o-NMWALODNDOCO
`MEALEELPIAELELAEEEEELDTTTTTTTTTTT
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`Thal
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`IMiD‘1
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`IMiD2
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`IMiD3
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`Figure 4 Effect of thalidomide and its analogs on DNA synthesis of patient multi-
`ple myelomacells cultured with control media (6), or with 0.1 44M @®), 1.0 uM @),
`10 4M (@), and 100 2M (m) thalidomide, IMiD1, IMiD2, or IMiD3. In each case,
`3H-TdR uptake was measured during the last 8 h of 48-h cultures. Values represent the
`
`mean (+ SD) 7H-TdR (epm) oftriplicate cultures.
`
`The mechanism of growth inhibition induced by thalidomide andits analogs is
`nottotally understood. IMiDs,and to a lesser extent thalidomide, induce apopto-
`sis of myeloma MM.1S cells, as evidenced both byincreased sub-G1 cells on PI
`staining and by increased annexin V—positive cells. In these cells, which character-
`istically have wild-type p53, exposure to these agents (and dexamethasone) down-
`regulates p21, thereby facilitating G1-to-S transition and enhanced susceptibility
`to apoptosis. Moreover, p27 is also upregulated by treatment with both thalido-
`mide and IMiDs (T. Hideshima,et al., unpublished data). This profound apop-
`totic effect may correlate with the fact that complete responses to thalidomide are
`occasionally observed. The mechanismof thalidomide/IMiDs-induced apoptosis
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`is also not entirely understood. However, our preliminary data indicate the par-
`ticipation of caspase-8 activation in thalidomide/IMiDs-induced apoptosis. IL-6
`overcomes the downregulation of p21 induced by these agents, consistent with the
`increase in DNAsynthesis triggered by IL-6 even in the presence of these drugs. In
`contrast, in Hs Sultan cells and AScells, derived from a multiple myelomapatient,
`(which are both wild-type and mutant p53), the IMiDs and thalidomide induce
`p21 and related G1 growth arrest (Figure 5), thereby conferring protection from
`apoptosis, as has been observed in other systems (70, 71). Prior studies showed that
`p21 was constitutively expressed in the majority of myelomacells and inhibited
`proliferation in both a p53-dependent and -independent fashion (72).
`Previous reports that cells overexpressing p21 protein demonstrate chemore-
`sistance (73) further support the notion that a protective effect of G1 growth ar-
`rest is induced by thalidomide and IMiDs in Hs Sultan- and AS-patient-derived
`myelomacells. Conversely, the frequent regrowth of progressive myeloma noted
`
`A
`
`‘Cit.
`©
`=
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`8®
`@
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`§s
`1B:p21 ie aie ee ee& al
`
`iB: a-tubulin
`
`IB:p21
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`
`Hs Sultan-cell
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`(Bp21
`IB? o-tubulin
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`ete ct — a
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`MM patient cell
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`MM.1S
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`Hs Sultan
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`MM patient
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`we
`IP: p53
`IB: p53 gai
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`omt
`
`omt
`wt
`mt
`wt
`ne
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`Figure 5 Effect of thalidomide and tts analogs on p21 expression in multiple mye-
`loma (MM)cell lines and patient cells. (@) MM.1S cells were cultured with 10 ~M
`of thalidomide, IMiD1, IMiD2, and IMiD3 for 48 h. MM.1S cells were also cultured
`with IL-6 (50 ng/ml) alone and with IMiD1, 10 4£M Dex, and Dex plus IL-6. Cells
`were lysed, subjected to SDS PAGE, transferred to PVDF membrane, and blotted
`with anti-p21 Ab. The membrane was stripped and reprobed with anti-a-tubulin Ab.
`(b) MM.158,Hs Sultan, and patient MM cells were lysed and immunoprecipitated with
`wt-p53 and mt-p53 Ab,transferred to PVDF membrane, and blotted with anti-p53 Ab.
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`A
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`IL-6
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`non-stimulated
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`stimulated
`
`B
`g
`Bera e BSra
`@ 2 @4@ 4 2 22 4
`§
`FP S228 @2Be
`IB:phospho-MAPK sae
`IB:ERK2
`
`control
`—
`—
`5
`Qa
`Q
`x
`=
`Thal
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`=
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`8
`6
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`1B: PY. oi une test all en et
`IB:RAFTK iyloaAo)sil wa a .
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`MM.1S cell
`M.1R cell
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`Figure 6 Effect of thalidomide and its analogs on growth and apoptotic signaling
`in myelomacell lines, MM.1S and MM.1R cells. (@) MM.1S cells were cultured for
`48 h in media, with 50 uM of PD98059, and with 10 wM of IMiD1, IMiD2, or
`IMiD3. Cells were then triggered with 50 ng/ml of IL-6 for 10 min,lysed, transferred
`to PVDF membrane, and blotted with anti-phospho MAPK Ab. Blots were stripped
`and reprobed with anti-ERK2 Ab. (6) MM.1S and MM.1R cells were treated with
`thalidomide (100 4M), TMiD 1 (100 4M), or Dex (10 44M)and harvestedat 12 h. Total
`cell lysates were subjected to immunoprecipitation with anti-RAFTK Ab and analyzed
`by immunoblotting with anti-P-Tyr Ab or anti-RAFTK Ab.
`
`clinically upon discontinuation of thalidomide treatment may correlate with re-
`lease of drug-related G1 growth arrest. We have previously shown that related
`adhesion focal tyrosine kinase (RAFTK. Figure 6) mediated dexamethasone-
`induced myelomacell apoptosis. Thalidomide and IMiDs, like dexamethasone,
`induced tyrosine phosphorylation of RAFTK, suggesting a key role of RAFTK in
`thalidomide- and IMiDs-induced apoptosis. Activation of RAFTK wasalso found
`in dexamethasone-resistant, IMiDs-sensitive MM.1R myelomacells.
`Recent studies have also demonstrated that myelomacell lines and patient
`myeloma cells produce TNF-a (63). TNF-a in tum triggers IL-6 secretion from
`BMSCsin a dose-dependent fashion;
`importantly, TNF-a is a more potent
`stimulus of IL-6 secretion than TGF-f 1 and VEGF (Figure 7). Remarkably, TNF-a
`also induces NF-«B activation and expression of LFA-1, ICAM-1, VCAM-1,
`and MUC-1 on myelomacelllines, as well as ICAM-1 and VCAM-1 on BMSCs.
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`Cc
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`IM-9
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`BMSC
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`IM-9/BMSC
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`IM-9
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`BMSC
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`IM-9/BMSC
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`
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`IL-6(ng/ml)
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`
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`IL-6(ng/ml)=ONnnOo
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`= Oo
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`Sa
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`0
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`media
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`VEGF
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`NFO TGFB1
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`0
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`01
`0.00010.001 0.01
`TNF a (ng/ml)
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`1
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`10
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`ecretion from IM-9 multiple myeloma (MM) cells stimulates IL-6
`Figure 7 TNF-a s
`secretion from bone marrow stromal cells (BMSCs). (a) IM-9 MM cells, BMSCs, and both
`together were cultured for 24 h (CG) and 48 h (@). (6) IM-9 myeloma cells, BMSCs, and
`both together were cultured for 48 h. (¢) BMSCs were cultured for 48 h in the presence of
`media alone, VEGF (10 ng/ml), TNF-a (10 ng/ml), or TGF-61 (10 ng/ml). (7) Two multi-
`ple myelomapatient BMSCs (@, @) were cultured for 24 h with TNF-a (0.0001-10 ng/ml).
`TNF-« (a) or IL-6 (6
`,¢,d@) levels were measured in culture supernatants by ELISA. Values
`
`represent the mean (4
`t SD)oftriplicate cultures.
`
`As a result, adherence of myeloma cells to BMSCs is significantly upregulated
`by TNF-c-related induction of these adhesion molecules. Adherence of myeloma
`cells to BMSCsinduces drug resistance of myelomacells, induces IL-6 secre-
`tion in BMSCs,and activates p44/42 MAPKin myelomacells, thereby promoting
`tumorcell growth. Because thalidomide and its analogs are potent inhibitors of
`TNF-a production, they may not only act directly on myeloma cells but also
`act indirectly by inhibiting cytokine cross-talk in the myeloma/BMSC microen-
`vironment. In conclusion, TNF-a induces an increase in proliferation, as well as
`MAPK/ERK activation, in myeloma cells, and it induces IL-6 secretion, as well
`as NF-«B activation, in BMSCs. TNF-a induces adhesion molecules on myeloma
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`cells and BMSCs,increasing IL-6 secretion and the binding of myelomacells to
`BMSCs; conversely, blockade of TNF-c—induced NF-«B activation inhibits these
`sequelae. These studies confirm a central role for TNF-a in the growth and sur-
`vival of myelomacells in the bone marrowmilieu and suggest the utility of novel
`therapeutics targeting TNF-a in multiple myeloma.
`Keiferet al. recently reported that thalidomide inhibited NF-«B activationtrig-
`gered by TNF-a in Jurkat T cells (74). This inhibitory effect of thalidomide on
`NF-«B activation was mediated by suppression of IkB kinase (IKK) activity.
`Phosphorylation of IkBa, inhibitory molecule of NF-«B, by IKK is essential for
`degradation of I«Ba. Since adherence of myeloma cells to BMSCsinduces IL-6
`secretion from BMSCsvia NF-«B activation (60), and TNF-a induces adhesion
`molecules on both myeloma cells and BMSCsalso via NF-«B activation, NF-«B
`may be a novel therapeutic target.
`A recent study demonstrated that thalidomide and IMiDs augment natural
`laller (NK) cell cytotoxicity in multiple myeloma (75). This report showed that
`thalidomide and IMiDs do not induce T cell proliferation alone but act as co-
`stimulators to trigger proliferation of anti-CD3-stimulated T cells from multiple
`myeloma patients, accompanied by an increase in interferon-y and IL-12 secre-
`tion. Treatment of patient peripheral blood mononuclear cells with thalidomide
`or IMiDstriggered increasedlysis of autologous myelomacells. Furthermore, pa-
`tients showed an increase in CD3~ CD56*cells in responseto thalidomide/IMiDs
`therapy. Lentzchet al. (76) demonstrated that thalidomide and IMiDs inhibited
`microvessel density in murine transplanted Hs Sultan cells, resulting