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`THALIDOMIDE: Emerging Role in Cancer Medicine
`
`Paul Richardson, Teru Hideshima, and Kenneth Anderson
`Jerome Lipper Myeloma Center, Division of Hematologic 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
`n Abstract Thalidomide—removed from widespread clinical 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 management of 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, clinical trials have confirmed benefit in relapsed disease, and
`the role of thalidomide in treating newly diagnosed patients is currently under study. 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 event in the history of drug development 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 of activity has fostered its application
`in a variety of disease states (Table 1) (3–5). Because of its teratogenic effects,
`thalidomide is now used under strict 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
`
`Autoimmune diseases
`
`Dermatologic diseases
`Other disorders
`
`Solid tumors (e.g., brain, breast, renal cell carcinoma);
`hematologic malignancies (e.g., multiple myeloma)
`HIV/AIDS and related 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
`Behcet’s syndrome; prurigo nodularis; pyoderma gangrenosum
`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 shown to inhibit angiogenesis in-
`duced by basic fibroblast growth factor (fl-FGF) in a rabbit cornea micropocket as-
`say and by vascular endothelial growth factor (VEGF) in a murine model of corneal
`vascularization (6, 7). In human studies, the drug appears to undergo activation
`to metabolites with antiangiogenic activity (8). Because of these antiangiogenic
`properties, thalidomide is 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 antimyeloma activity.
`Its other potential actions include modulation of adhesion molecules, inhibition
`of tumor necrosis alpha factor (TNF-fi), downregulation of lymphocyte surface
`molecules, lowering of CD4:CD8 peripheral lymphocyte ratios, and direct effects
`on myeloma cells themselves (10, 14–18).
`This chapter presents a comprehensive review of the pharmacology of thalido-
`mide, a description of preclinical studies in multiple myeloma to illustrate the
`drug’s complex putative mechanisms of action, and a description of 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-
`Nfphthalimidogglutarimide [C13 O4 N2 H9] and its gram molecular weight is
`258.2 (19).
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`Figure 1 Structures of thalidomide and its potent ana-
`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 effects, 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 thus efforts at formulating only the R isomer have 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
`poor solubility 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 confounded the definition of a dose-response effect against
`human cancer.
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`TABLE 2 Single dose pharmacokinetic parameters of thalidomide in humans (27)
`
`Mean apparent
`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
`
`tmax
`
`3.3*
`
`4.4*
`3.4
`
`5.8
`4.4
`
`t1=2 (L)
`
`Vd (L)
`
`6.5
`
`18.3
`5.7
`
`4.1
`8.7
`
`66.9
`
`165.8
`78.2
`
`53.0
`120.7
`
`Abbreviations: tmax, time to reach maximum concentrations; t1=2, elimination half-life; Vd, volume of distribution;
`HIV, human immunodeficiency virus.
`*Median value.
`
`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 (tmax) in a mean of »4 h (23–25, 29).
`
`Distribution
`Animal studies have demonstrated a wide distribution of thalidomide throughout
`most tissues and organs (28). It is present in semen following oral administration
`in rabbits, but it is not known whether the drug is present in human semen (19, 30).
`Human pharmacokinetic studies to date also indicate that thalidomide has a large
`apparent volume of distribution (Vd) (24–26). Further, studies in elderly prostate
`cancer patients suggest variability in Vd, 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 isoenzymes in rats, recent
`evaluation of single- or multiple-dose pharmacokinetic parameters of oral thalido-
`mide at 200 mg daily in healthy human volunteers has indicated that thalidomide
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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
`nonabsorbed portion 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 measured at 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-
`mide is oral hormonal contraceptives, which showed no significant interaction. An-
`imal studies suggest that thalidomide enhances the 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’Amato et al., while evaluating thalidomide’s mechanism of teratogenicity, found
`that it exhibited antiangiogenic properties (6, 7). They postulated that thalido-
`mide inhibited angiogenesis by interrupting processes induced by fl-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 thalidomide is that it selectively inhibits TNF-fi
`production while leaving the patient’s immune system otherwise intact (38). This
`has led to its application in various disorders characterized by abnormal TNF-fi
`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-fi inhibition is unclear, but
`it does appear to differ from other TNF-fi inhibitors such as pentoxyfylline and
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`dexamethasone (39, 40). One mechanism postulated is that thalidomide inhibits
`TNF-fi synthesis by accelerating degradation of TNF-fi mRNA, resulting in a
`significant but incomplete suppression of TNF-fi protein production (39, 41). Of
`particular interest is the recent demonstration that thalidomide decreases the bind-
`ing activity of NF-•B which in turn controls activation of the TNF-fi gene (42).
`It has also been postulated that thalidomide’s effect on angiogenesis may be
`through TNF-fi inhibition, since TNF-fi has proangiogenic effects (6). However,
`the absence of a demonstrable TNF-fi effect in experimental models of angiogen-
`esis, coupled with the inability of strong TNF-fi inhibitors to directly influence
`angiogenesis, suggests that thalidomide’s antiangiogenic activity is not related to
`TNF-fi 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-fi 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
`Common side effects 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 thalidomide is 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 when the drug is combined with steroids and particularly
`
`TABLE 3 Clinical adverse events reported during thalidomide use (27)
`
`Neurologic
`
`Gastrointestinal
`
`Dermatologic
`
`Miscellaneous
`
`Sedation
`Dizziness
`Mood changes
`Headaches
`
`Constipation
`Nausea
`Increased appetite
`
`Exfoliative/erythrodermic
`Cutaneous reactions
`Brittle fingernails
`Pruritis
`
`Xerostomia
`Weight gain
`Edema of face/limbs
`Reduction in thyroid
`hormone secretion
`Hypotension
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`with anthracycline-based chemotherapy, causing early cessation of one study ex-
`ploring a combination of 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
`coronary disease taking multiple blood-pressure-lowering medications along with
`thalidomide (27).
`It is well-known that thalidomide must not be used during pregnancy, and rec-
`ommended contraceptive methods must be used by both men and women of child-
`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 cancer patients, thalidomide should be used with caution when there 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 I and II
`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 was initially used to treat multiple myeloma because of
`its antiangiogenic effects, the mechanism of its antimyeloma activity 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 myeloma cell 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 myeloma cell growth
`and survival (59–61) and confer drug resistance (62); importantly, thalidomide
`modulates adhesive interactions (14) and thereby may alter tumor cell growth,
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`Figure 2 Possible effect of thalidomide on myeloma cells’ and bone marrow stro-
`mal cells’ (BMSCs’) microenvironment in vivo. (A) Thalidomide directly inhibits
`myeloma cell growth. (B) Thalidomide inhibits myeloma cell adhesion to BMSCs.
`(C) Thalidomide blocks IL-6, TNF-fi, and IL-1fl secretion from BMSCs. (D) Thalido-
`mide blocks the ability of VEGF and fl-FGF to stimulate neovascularization of bone
`marrow. (E) Thalidomide induces IL-2 and INF-(cid:176) secretion from T cells.
`
`survival, and drug resistance. Third, cytokines secreted into the bone marrow mi-
`croenvironment by myeloma cells and/or BMSCs, such as IL-6, IL-1fl, IL-10,
`and TNF-fi may augment myeloma cell growth and survival (61, 63), and thalido-
`mide may alter their secretion and bioactivity (64). Fourth, thalidomide decreases
`the secretion of VEGF, IL-6 (65), and flFGF by myeloma and/or BMSCs.
`Since IL-6 is known to promote myeloma cell growth and survival, and VEGF
`has been demonstrated to induce myeloma cell 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 myeloma cells (18). IMiDs induce growth
`inhibition of myeloma cells in a dose-dependent fashion, with an IC50 as low as
`0.1–1 „M. IMiDs are effective against myeloma cell 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 3b), a potent myeloma growth and antiapop-
`totic factor. IMiDs also showed significant activity against drug-resistant patient
`myeloma cells (Figure 4). The observed clinical activity of thalidomide in patients
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`Figure 3 Effect of Dex and IL-6 on response of multiple myeloma cells to thalido-
`mide and the IMiDs. (a) MM.1S cells were cultured with 1.0 „M thalidomide,
`IMiD1, IMiD2, or IMiD3 in control media alone (⁄) or with 0.001 ( ), 0.01 ( ), and
`0.1 „M (¥) Dex. (b) MM.1S cells were cultured in control media alone and with 0.1
`and 1.0 „M thalidomide, IMiD1, IMiD2, or IMiD3 either in the presence (⁄) or ab-
`sence (¥) of IL-6 (50 ng/ml). In each case, 3H-TdR uptake was measured during the
`last 8 h of48-h cultures. Values represent the mean ( § SD) 3H-TdR (cpm) of triplicate
`cultures.
`
`with myeloma that is refractory to conventional therapies (69), coupled with our
`in vivo studies, suggests that thalidomide can overcome resistance to conventional
`treatments. In addition, our laboratory work suggests that dexamethasone can add
`to the antiproliferative effect of thalidomide and its analogs in vitro, suggesting
`potential utility of coupling these agents in novel therapeutics.
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`Figure 4 Effect of thalidomide and its analogs on DNA synthesis of patient multi-
`ple myeloma cells cultured with control media (⁄), or with 0.1 „M ( ), 1.0 „M ( ),
`10 „M ( ), and 100 „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) 3H-TdR (cpm) of triplicate cultures.
`
`The mechanism of growth inhibition induced by thalidomide and its analogs is
`not totally understood. IMiDs, and to a lesser extent thalidomide, induce apopto-
`sis of myeloma MM.1S cells, as evidenced both by increased 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 mechanism of 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 DNA synthesis triggered by IL-6 even in the presence of these drugs. In
`contrast, in Hs Sultan cells and AS cells, derived from a multiple myeloma patient,
`(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 myeloma cells 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
`myeloma cells. Conversely, the frequent regrowth of progressive myeloma noted
`
`Figure 5 Effect of thalidomide and its analogs on p21 expression in multiple mye-
`loma (MM) cell lines and patient cells. (a) 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 „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-fi-tubulin Ab.
`(b) MM.1S, 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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`Figure 6 Effect of thalidomide and its analogs on growth and apoptotic signaling
`in myeloma cell lines, MM.1S and MM.1R cells. (a) MM.1S cells were cultured for
`48 h in media, with 50 „M of PD98059, and with 10 „M 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. (b) MM.1S and MM.1R cells were treated with
`thalidomide (100 „M), IMiD 1 (100 „M), or Dex (10 „M) and harvested at 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 myeloma cell 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 was also found
`in dexamethasone-resistant, IMiDs-sensitive MM.1R myeloma cells.
`Recent studies have also demonstrated that myeloma cell lines and patient
`myeloma cells produce TNF-fi (63). TNF-fi in turn triggers IL-6 secretion from
`BMSCs in a dose-dependent fashion; importantly, TNF-fi is a more potent
`stimulus of IL-6 secretion than TGF-fl1 and VEGF (Figure 7). Remarkably, TNF-fi
`also induces NF-•B activation and expression of LFA-1, ICAM-1, VCAM-1,
`and MUC-1 on myeloma cell lines, as well as ICAM-1 and VCAM-1 on BMSCs.
`
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`Figure 7 TNF-fi secretion from IM-9 multiple myeloma (MM) cells stimulates IL-6
`secretion from bone marrow stromal cells (BMSCs). (a) IM-9 MM cells, BMSCs, and both
`together were cultured for 24 h (⁄) and 48 h (¥). (b) IM-9 myeloma cells, BMSCs, and
`both together were cultured for 48 h. (c) BMSCs were cultured for 48 h in the presence of
`media alone, VEGF (10 ng/ml), TNF-fi (10 ng/ml), or TGF-fl1 (10 ng/ml). (d ) Two multi-
`
`ple myeloma patient BMSCs (¥, x) were cultured for 24 h with TNF-fi (0.0001–10 ng/ml).
`TNF-fi (a) or IL-6 (b, c, d ) levels were measured in culture supernatants by ELISA. Values
`represent the mean (§ SD) of triplicate cultures.
`
`As a result, adherence of myeloma cells to BMSCs is significantly upregulated
`by TNF-fi–related induction of these adhesion molecules. Adherence of myeloma
`cells to BMSCs induces drug resistance of myeloma cells, induces IL-6 secre-
`tion in BMSCs, and activates p44/42 MAPK in myeloma cells, thereby promoting
`tumor cell growth. Because thalidomide and its analogs are potent inhibitors of
`TNF-fi 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-fi 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-fi induces adhesion molecules on myeloma
`
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`cells and BMSCs, increasing IL-6 secretion and the binding of myeloma cells to
`BMSCs; conversely, blockade of TNF-fi–induced NF-•B activation inhibits these
`sequelae. These studies confirm a central role for TNF-fi in the growth and sur-
`vival of myeloma cells in the bone marrow milieu and suggest the utility of novel
`therapeutics targeting TNF-fi in multiple myeloma.
`Keifer et al. recently reported that thalidomide inhibited NF-•B activation trig-
`gered by TNF-fi in Jurkat T cells (74). This inhibitory effect of thalidomide on
`NF-•B activation was mediated by suppression of I•B kinase (IKK) activity.
`Phosphorylation of I•Bfi, inhibitory molecule of NF-•B, by IKK is essential for
`degradation of I•Bfi. Since adherence of myeloma cells to BMSCs induces IL-6
`secretion from BMSCs via NF-•B activation (60), and TNF-fi induces adhesion
`molecules on both myeloma cells and BMSCs also via NF-•B activation, NF-•B
`may be a novel therapeutic target.
`A recent study demonstrated that thalidomide and IMiDs augment natural
`killer (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-(cid:176) and IL-12 secre-
`tion. Treatment of patient peripheral blood mononuclear cells with thalidomide
`or IMiDs triggered increased lysis of autologous myeloma cells. Furthermore, pa-
`¡
`C
`CD56
`cells in response to thalidomide/IMiDs
`tients showed an increase in CD3
`therapy. Lentzch et al. (76) demonstrated that thalidomide and IMiDs inhibited
`microvessel density in murine transplanted Hs Sultan cells, resulting in longer
`survival of IMiD-treated mice than of nontreated mice. These findings strongly
`support the antiangiogenic effect of thalidomide and IMiDs in vivo.
`In summary, extensive preclinical studies in multiple myeloma provide a com-
`pelling basis for the development and testing of thalidomide and the IMiDs in a
`new treatment paradigm to target both the tumor cell and the microenvironment,
`overcome classical drug resistance, and improve outcome in this presently incur-
`able disease.
`
`CLINICAL STUDIES IN MULTIPLE MYELOMA
`
`Despite recent advances in treatment, including transplantation, myeloma remains
`incurable and more effective therapies are clearly needed (77, 78). The develop-
`ment of resistance to chemotherapy and radiation, characteristic of myeloma, has
`spurred research in new biologically derived treatment strategies. The strategy of
`antiangiogenesis is based on observations that hematologic malignancies, such as
`multiple myeloma, are associated with intense neovascularization of bone marrow
`and thus may be angiogenesis-dependent (79, 80). However, although angiogene-
`sis appears well-established as a key component in the growth, progression, and
`metastatic spread of solid tumors (81), its role in hematologic malignancy remains
`to be defined, and only relatively recently has clinical investigation suggested its
`potential in this setting (67, 69, 80, 82–84).
`
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`In 1994, Vacca and colleagues reported a high correlation between the extent
`of bone marrow angiogenesis and the labeling index (LI) of marrow plasma cells
`and disease activity in patients with multiple myeloma (80). Subsequent stud-
`ies have confirmed extensive bone marrow vascularization in multiple myeloma
`(67, 69, 83, 84) and have associated poor prognosis with both elevated levels of an-
`giogenic cytokines, such as fl-FGF and VEGF, and increased bone marrow levels
`of mast cells, which secrete a variety of angiogenic factors (80, 83–85). More-
`over, recent reports have shown increased bone marrow angiogenesis in acute
`lymphoblastic leukemia (ALL) in children (79). Collectively, these findings have
`provided the rationale for the use of antiangiogenic drugs in the treatment of mul-
`tiple myeloma and other hematologic malignancies.
`Thalidomide therapy for advanced refractory myeloma began after an encour-
`aging initial experience in two patients at the University of Arkansas prompted
`a large phase II study, which assessed the efficacy and toxicity of single-agent
`thalidomide in relapsed and refractory multiple myeloma (69). The primary end-
`point of this trial was paraprotein response. Additional