Plenary paper
`
`Thalidomide and its analogs overcome drug resistance of human multiple
`myeloma cells to conventional therapy
`Teru Hideshima, Dharminder Chauhan, Yoshihito Shima, Noopur Raje, Faith E. Davies, Yu-Tzu Tai, Steven P. Treon, Boris Lin,
`Robert L. Schlossman, Paul Richardson, George Muller, David I. Stirling, and Kenneth C. Anderson
`
`Although thalidomide (Thal) was ini-
`tially used to treat multiple myeloma
`(MM) because of its known antiangio-
`genic effects,
`the mechanism of
`its
`anti-MM activity is unclear. These stud-
`ies demonstrate clinical activity of Thal
`against MM that is refractory to conven-
`tional
`therapy and delineate mecha-
`nisms of anti-tumor activity of Thal and
`its potent analogs (immunomodulatory
`drugs [IMiDs]). Importantly, these agents
`Introduction
`
`act directly, by inducing apoptosis or
`G1 growth arrest, in MM cell lines and in
`patient MM cells that are resistant to
`melphalan, doxorubicin, and dexameth-
`asone (Dex). Moreover, Thal and the
`IMiDs enhance the anti-MM activity of
`Dex and, conversely, are inhibited by
`interleukin 6. As for Dex, apoptotic sig-
`naling triggered by Thal and the IMiDs is
`associated with activation of related
`adhesion focal tyrosine kinase. These
`
`studies establish the framework for the
`development and testing of Thal and the
`IMiDs in a new treatment paradigm to
`target both the tumor cell and the micro-
`environment, overcome classical drug
`resistance, and achieve improved out-
`come in this presently incurable dis-
`ease. (Blood. 2000;96:2943-2950)
`
`© 2000 by The American Society of Hematology
`
`Thalidomide (Thal) was originally used in Europe for the treatment
`of morning sickness in the 1950s but was withdrawn from the
`market in the 1960s because of reports of teratogenicity and
`phocomelia associated with its use. The renewed interest in Thal
`stems from its broad spectrum of pharmacologic and immunologic
`effects.1 Because of its immunomodulatory and antiangiogenic
`effects, it has been used to effectively treat erythema nodosum
`leprosum, an inflammatory manifestation of leprosy.2 Potential
`therapeutic applications span a wide spectrum of diseases, includ-
`ing cancer and related conditions, infectious diseases, autoimmune
`diseases, dermatologic diseases, and other disorders such as
`sarcoidosis, macular degeneration, and diabetic retinopathy.3 Re-
`cent reports of increased bone marrow (BM) angiogenesis in
`multiple myeloma (MM),4,5 coupled with the known antiangio-
`genic properties of Thal,6 provided the rationale for its use to treat
`MM.7 Importantly, Thal induced clinical responses in 32% of MM
`patients whose disease was refractory to conventional and high-
`dose therapy,7 suggesting that it can overcome drug resistance
`because of its alternative mechanisms of anti-MM activity. Besides
`alkylating agents and corticosteroids, Thal now, therefore, repre-
`sents the third distinct class of agents useful in the treatment
`of MM.
`Given its broad spectrum of activities, Thal may be acting
`against MM in several ways.8 First, Thal may have a direct effect
`on the MM cell and/or BM stromal cell to inhibit their growth and
`survival. For example, free radical–mediated oxidative DNA
`damage may play a role in the teratogenicity of Thal9 and may also
`have anti-tumor effects. Second, adhesion of MM cells to BM
`stromal cells both triggers secretion of cytokines that augment MM
`
`cell growth and survival10-12 and confers drug resistance13; Thal
`modulates adhesive interactions14 and, thereby, may alter tumor
`cell growth, survival, and drug resistance. Third, cytokines secreted
`into the BM microenvironment by MM and/or BM stromal cells,
`such as interleukin (IL)-6, IL-1b, IL-10, and tumor necrosis factor
`(TNF)–a, may augment MM cell growth and survival,12 and Thal
`may alter their secretion and bioactivity.15 Fourth, vascular endothe-
`lial growth factor (VEGF) and basic fibroblast growth factor 2
`(bFGF-2) are secreted by MM and/or BM stromal cells and may
`play a role both in tumor cell growth and survival, as well as BM
`angiogenesis.5,16 Given its known antiangiogenic activity,6 Thal
`may inhibit activity of VEGF, bFGF-2, and/or angiogenesis in
`MM. However, Singhal et al.7 observed no correlation of BM
`angiogenesis with response to Thal, suggesting that it may not
`be mediating anti-MM activity by its antiangiogenic effects.
`Finally, Thal may be acting against MM by its immunomodula-
`tory effects, such as induction of a Th1 T-cell response with
`secretion of interferon gamma (IFN-g) and IL-2.17 Already 2
`classes of Thal analogs have been reported, including phospho-
`diesterase 4 inhibitors that inhibit TNF-a but do not enhance
`T-cell activation (selected cytokine inhibitory drugs [SelCIDs])
`and others that are not phosphodiesterase 4 inhibitors but
`markedly stimulate T-cell proliferation as well as IL-2 and
`IFN-g production (immunomodulatory drugs [IMiDs]).15
`In this study, we have begun to characterize the mechanisms of
`activity of Thal and these analogs against human MM cells.
`Delineation of their mechanisms of action, as well as mechanisms
`of resistance to these agents, will both enhance understanding of
`MM disease pathogenesis and derive novel treatment strategies.
`
`From the Department of Adult Oncology, Dana-Farber Cancer Institute, and
`Department of Medicine, Harvard Medical School, Boston, MA; and Celgene
`Corporation, Warren, NJ.
`
`Submitted March 8, 2000; accepted June 28, 2000.
`
`Supported by National Institutes of Health grant PO1 78378 and the Doris Duke
`Distinguished Clinical Research Scientist Award (K.C.A.).
`
`Reprints: Kenneth C. Anderson, Dana-Farber Cancer Institute, 44 Binney St.,
`Boston, MA 02115; e-mail: kenneth anderson@dfci.harvard.edu.
`
`The publication costs of this article were defrayed in part by page charge
`payment. Therefore, and solely to indicate this fact, this article is hereby
`marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
`
`© 2000 by The American Society of Hematology
`
`BLOOD, 1 NOVEMBER 2000 z VOLUME 96, NUMBER 9
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`2943
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`

`2944
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`HIDESHIMA et al
`
`BLOOD, 1 NOVEMBER 2000 z VOLUME 96, NUMBER 9
`
`Materials and methods
`
`MM-derived cell lines and patient cells
`
`Dexamethasone (Dex)-sensitive (MM.1S) and Dex-resistant (MM.1R)
`human MM cell lines were kindly provided by Dr Steven Rosen (Northwest-
`ern University, Chicago, IL). Doxorubicin (Dox)-, mitoxantrone (Mit)-, and
`melphalan (Mel)-sensitive and -resistant RPMI-8226 human MM cells
`were kindly provided by Dr William Dalton (Moffitt Cancer Center, Tampa,
`FL). RPMI-8226 cells resistant to Dox, Mit, and Mel included Dox 6 and
`Dox 40 cells, MR20 cells, and LR5 cells, respectively. Hs Sultan human
`MM cells were obtained from the American Type Culture Collection
`(Rockville, MD). All MM cell lines were cultured in RPMI-1640 media
`(Sigma Chemical, St Louis, MO) that contained 10% fetal bovine serum, 2
`mmol/L L-glutamine (GIBCO, Grand Island, NY), 100 U/mL penicillin,
`and 100 mg/mL streptomycin (GIBCO). Drug-resistant cell lines were
`cultured with either Dox, Mit, Mel, or Dex to confirm their lack of drug
`sensitivity. MM patient cells (96% CD381CD45RA2) were purified from
`patient BM samples, as previously described.18
`
`Thal and analogs
`
`Thal and analogs (Celgene, Warren, NJ) were dissolved in DMSO (Sigma)
`and stored at 220°C until use. Drugs were diluted in culture medium
`(0.0001 to 100 mM) with , 0.1% DMSO immediately before use. The Thal
`analogs used in this study were 4 SelCIDs (SelCIDs 1, 2, 3, and 4), which
`are phosphodiesterase 4 inhibitors that inhibit TNF-a production and
`increase IL-10 production from lipopolysaccharide (LPS)–stimulated periph-
`eral blood mononuclear cells (PBMCs) but do not stimulate T-cell
`proliferation; and 3 IMiDs (IMiD1, IMiD2, and IMiD3), which do stimulate
`T-cell proliferation, as well as IL-2 and IFN-g secretion, but are not
`phosphodiesterase 4 inhibitors. The IMiDs also inhibit TNF-a, IL-1b, and
`IL-6 and greatly increase IL-10 production by LPS-stimulated PBMCs.15
`
`DNA synthesis
`
`DNA synthesis was measured as previously described.19 MM cells (3 3 104
`cells/well) were incubated in 96-well culture plates (Costar, Cambridge,
`MA) in the presence of media, Thal, SelCID1, SelCID2, SelCID3,
`SelCID4, IMiD1, IMiD2, IMiD3, and/or recombinant IL-6 (50 ng/mL)
`(Genetics Institute, Cambridge, MA) for 48 hours at 37°C. DNA synthesis
`was measured by [3H]-thymidine (3H-TdR; NEN Products, Boston, MA)
`uptake. Cells were pulsed with 3H-TdR (0.5 mCi/well) during the last 8
`hours of 48-hour cultures, harvested onto glass filters with an automatic cell
`harvester (Cambridge Technology, Cambridge, MA), and counted by using
`the LKB Betaplate scintillation counter (Wallac, Gaithersburg, MD). All
`experiments were performed in triplicate.
`Colorimetric assays were also performed to assay drug activity. Cells
`from 48-hour cultures were pulsed with 10 mL of 5 mg/mL 3-(4,5-
`dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide (MTT; Chemicon
`International Inc, Temecula, CA) to each well for 4 hours, followed by 100
`mL isopropanol
`that contained 0.04 HCl. Absorbance readings at a
`wavelength of 570 nm were taken on a spectrophotometer (Molecular
`Devices Corp., Sunnyvale, CA).
`
`Cell cycle analysis
`
`MM cells (1 3 106) cultured for 72 hours in media alone, Thal, IMiD1,
`IMiD2, and IMiD3 were harvested, washed with phosphate-buffered saline
`(PBS), fixed with 70% ethanol, and pretreated with 10 mg/mL of RNAse
`(Sigma). Cells were stained with propidium iodide (PI; 5 mg/mL; Sigma),
`and cell cycle profile was determined by using the program M software
`on an Epics flow cytometer (Coulter Immunology, Hialeah, FL), as in
`prior studies.20
`
`Detection of apoptosis
`
`In addition to identifying sub-G1 cells as described above, apoptosis
`was also confirmed by using annexin V staining. MM cells were cultured
`
`in media (0.01% DMSO) or with 10 mmol/L of Thal or 1 mmol/L IMiD1,
`IMiD2, and IMiD3 at 37°C for 72 hours, with addition of drugs at
`24-hour intervals. Cells were then washed twice with ice-cold PBS and
`resuspended (1 3 106 cells/mL) in binding buffer (10 mmol/L HEPES,
`pH 7.4, 140 mmol/L NaCl, 2.5 mmol/L CaCl2). MM cells (1 3 105) were
`incubated with annexin V-FITC (5 mL; Pharmingen, San Diego, CA) and
`PI (5 mg/mL) for 15 minutes at room temperature. Annexin V1PI2
`apoptotic cells were enumerated by using the Epics cell sorter (Coulter).
`
`Immunoblotting
`
`MM cells were cultured with 10 mmol/L of Thal, IMiD1, IMiD2, or
`IMiD3; harvested; washed; and lysed using lysis buffer: 50 mmol/L
`HEPES (pH 7.4), 150 mmol/L NaCl, 1% Triton-X 100, 30 mmol/L
`sodium pyrophosphate, 5 mmol/L EDTA, 2 mmol/L Na3VO4, 5 mmol/L
`NaF, 1 mmol/L phenylmethyl sulfonyl fluoride (PMSF), 5 mg/mL
`leupeptin, and 5 mg/mL aprotinin. For detection of p21, cell lysates
`were subjected to SDS-PAGE, transferred to polyvinylidene difluoride
`(PVDF) membrane, and immunoblotted with anti-p21 antibody
`(Ab; Santa Cruz Biotech, Santa Cruz, CA). The membrane was stripped
`and reprobed with anti–alpha tubulin Ab (Sigma) to ensure equivalent
`protein loading. For detection of p53, cell lysates were prepared from
`MM cells (2 3 107) with the use of lysis buffer. Lysates were incubated
`with anti-mutant (mt) or wild-type (wt) p53 monoclonal Abs (Calbio-
`chem, San Diego, CA) and then immunoprecipitated overnight with
`protein A Sepharose (Sepharose CL-4B; Pharmacia, Uppsala, Sweden).
`Immune complexes were analyzed by immunoblotting with horseradish
`peroxidase–conjugated anti-p53 Ab reactive with both mt and wt p53
`(Calbiochem).
`To characterize growth signaling, immunoblotting was also done
`with anti-phospho–specific MAPK Ab (New England Biolabs, Beverly,
`MA) in the presence or absence of IL-6 (Genetics Institute) and/or the
`MEK 1 inhibitor PD98059 (New England Biolabs), as in prior studies.21
`Antigen-antibody complexes were detected by using enhanced chemilu-
`minescence (Amersham, Arlington Heights, IL). Blots were stripped and
`reprobed with anti-ERK2 Ab (Santa Cruz Biotech) to ensure equivalent
`protein loading.
`To characterize apoptotic signaling, MM cells were cultured with 100
`mmol/L of Thal, IMiD1, IMiD2, or IMiD3; harvested; washed; and lysed in
`1 mL of lysis buffer (50 mmol/L Tris, pH 7.4, 150 mM NaCl, 1% NP-40, 5
`mmol/L EDTA, 2 mmol/L Na3VO4, 5 mmol/L NaF, 1 mmol/L PMSF, 5
`mg/mL leupeptin, and 5 mg/mL aprotinin), as in prior studies.22 Lysates
`were incubated with anti-related adhesion focal tyrosine kinase (RAFTK)
`Ab for 1 hour at 4°C and then for 45 minutes after the addition of protein
`G–agarose (Santa Cruz Biotech). Immune complexes were analyzed by
`immunoblotting with anti-P-Tyr Ab (RC20; Transduction Laboratories,
`Lexington, KY) or anti-RAFTK Abs. Proteins were separated by electro-
`phoresis in 7.5% SDS-PAGE gels, transferred to nitrocellulose paper, and
`analyzed by immunoblotting. The antigen-antibody complexes were visual-
`ized by chemiluminescence.
`
`Statistical analyses
`
`Statistical significance of differences observed in drug-treated versus
`control cultures was determined by using the Student t test. The minimal
`level of significance was P , .05.
`
`Results
`
`Treatment of MM patients with Thal
`
`Seventeen (39%) of 44 patients with MM treated at our institute
`responded to Thal (Table 1). This response included 6 men and 11
`women. These patients had received a median of 4 (1-9) prior
`treatment regimens, and 10 patients had a prior high-dose therapy
`and hematopoietic stem cell
`transplant. One patient achieved
`
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`BLOOD, 1 NOVEMBER 2000 z VOLUME 96, NUMBER 9
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`THALIDOMIDE OVERCOMES DRUG RESISTANCE OF MM
`
`2945
`
`Table 1. Response to thalidomide in multiple myeloma*
`
`Patient
`
`Sex†
`
`Prior
`therapies
`
`Prior
`stem cell
`transplant
`
`1
`2
`3
`4
`5
`6
`7
`8
`9
`10
`11§
`12§
`13§
`14§
`15
`16
`17
`
`M
`F
`F
`M
`F
`F
`M
`F
`F
`M
`F
`M
`F
`F
`F
`F
`M
`
`3
`5
`1
`6
`1
`5
`9
`5
`5
`5
`4
`4
`3
`2
`1
`1
`2
`
`Yes
`No
`Yes
`No
`No
`Yes
`Yes
`Yes
`No
`Yes
`Yes
`Yes
`Yes
`Yes
`No
`No
`No
`
`Maximum
`change
`M protein‡
`
`2 58%(PR)
`2 78%(PR)
`1 16%(SD)
`2 56%(PR)
`2 62%(PR)
`2 100%(CR)
`2 54%(PR)
`2 68%(PR)
`2 90%(PR)
`2 9%(SD)
`2 59%(PR)
`2 64%(PR)
`2 14%(SD)
`2 55%(PR)
`2 31%(SD)
`2 12%(SD)
`2 55%(PR)
`
`Duration of
`thalidomide
`therapy (mo)
`
`Maximum
`daily dose
`thalidomide
`
`8.5
`6.0
`6.5
`9.0
`5.5
`13
`10
`4.0
`7.5
`1.5
`5.5
`7.0
`4.5
`4.0
`6.0
`4.5
`6.0
`
`200 mg
`400 mg
`100 mg
`200 mg
`200 mg
`500 mg
`800 mg
`200 mg
`400 mg
`400 mg
`400 mg
`400 mg
`400 mg
`800 mg
`400 mg
`400 mg
`200 mg
`
`Current status
`(daily thalidomide dose)
`
`Continued response (200 mg)
`Continued response (400 mg)
`Continued response (100 mg)
`Continued response (200 mg)
`Continued response (50 mg)
`Continued response (50 mg)
`Progressed (800 mg)
`Continued response, discontinued
`Continued response (400 mg)
`Progressed
`Progressed
`Progressed
`Progressed
`Continued response (800 mg)
`Continued response (400 mg)
`Progressed
`Continued response (100 mg)
`
`*As of January 1, 2000.
`†Male (M) or female (F).
`‡Partial response (PR) is $ 50% decrease in M protein; complete response (CR) is absence of M protein on immunofixation and normal bone marrow biopsy; stable
`disease (SD) is # 50% decrease in M protein; progression is $ 25% increase in M protein or progressive clinical disease.
`§Also received decadron therapy.
`
`complete response (absence of monoclonal protein on immunofix-
`ation and normal BM biopsy), 11 patients achieved partial response
`(. 50% decrease in monoclonal protein), and 5 patients achieved
`stable disease (, 50% decrease in monoclonal protein). Patients
`received a median of 400 mg (range, 100-800 mg) maximum dose
`of daily Thal for a median of 6 months (range, 1.5-13 months). As
`of January 1, 2000, 11 patients have continued response at a median
`of 6 months (range, 4-13 months), and 6 patients have progressed at
`a median of 4.5 months (range, 1.5-10 months).
`
`Effect of Thal and analogs on DNA synthesis by MM cell lines
`and patient MM cells
`
`The effect of Thal and its analogs, including IMiD1, IMiD2,
`IMiD3, SelCID1, SelCID2, SelCID3, and SelCID4, on DNA
`synthesis of MM cell
`lines (MM.1S, Hs Sultan, U266, and
`RPMI-8226) was determined by measuring 3H-TdR uptake during
`the last 8 hours of 48-hour cultures, in the presence or absence of
`drug at various concentrations. IMiD1, IMiD2, and IMiD3 inhib-
`ited 3H-TdR uptake of MM.1S (Figure 1A) and Hs Sultan (Figure
`1B) cells in a dose-dependent fashion. Fifty percent inhibition of
`proliferation of MM.1S cells was noted at 0.01-0.1 mmol/L IMiD1,
`0.1-1.0 mmol/L IMiD2, and 0.1-1.0 mmol/L IMiD3 (P , .001).
`Fifty percent inhibition of proliferation of Hs Sultan cells was
`noted at 0.1 mmol/L IMiD1, 1.0 mmol/L IMiD2, and 1.0 mmol/L
`IMiD3 (P , .001). In contrast, only 15% and 20% inhibition in
`MM.1S and Sultan cells, respectively, were observed in cultures at
`even higher concentrations (100 mmol/L) of Thal. No significant
`inhibition of DNA synthesis of U266 MM cells was noted in
`cultures with 0.001 to 100 mmol/L Thal or these IMiDs (data not
`shown). The effects of these drugs on proliferation were confirmed
`by using MTT assays for MM.1S cells (Figure 1A) and Hs Sultan
`cells (Figure 1B). Although there was also a dose-dependent
`inhibition of proliferation of MM.1S cells by SelCIDs, 50%
`inhibition was observed only at high doses (100 mmol/L) for only 2
`of the 4 SelCIDs (SelCIDs 1 and 3, Figure 1C). Further studies,
`therefore, focused on Thal and the IMiDs.
`
`Figure 1. Effect of Thal and analogs on DNA synthesis of MM cell lines and
`patient cells. MM.1S (A) and Hs Sultan (B) cells were cultured with increasing
`concentrations (0.0001-100 mM) of Thal (r), IMiD1 (f), IMiD2 (F), and IMiD3 ((cid:140)). (C)
`MM.1S cells were cultured with increasing concentrations (12.5-100 mM) of SelCID1
`(r), SelCID2 (f), SelCID3 ((cid:140)), and SelCID4 (F). In each case 3H-TdR uptake (left
`panels) or MTT cleavage (right panels) were measured during the last 8 and 4 hours,
`respectively, of 48-hour cultures. Values represent the mean (6 SD) 3H-TdR (cpm) or
`absorbance of triplicate cultures.
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`2946
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`HIDESHIMA et al
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`BLOOD, 1 NOVEMBER 2000 z VOLUME 96, NUMBER 9
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`Effect of Thal and analogs in DNA synthesis of MM cells
`resistant to conventional therapy
`
`To examine whether there was cross-resistance between Thal and
`the IMiDs with conventional therapies, RPMI-8226 MM cells
`resistant to Dox (Dox6 and Dox40 cells), Mit (MR20 cells), or Mel
`(LR5 cells), and MM.1R cells resistant to Dex were similarly
`studied. Proliferation of Dox6 and Dox40, MR20, LR5, or MM1.R
`cells is unaffected by culture with 60 nmol/L and 400 nmol/L Dox,
`20 nmol/L Mit, 5 mmol/L Mel, and 1 mmol/L Dex, respectively
`(data not shown). Importantly, 3H-TdR uptake of Dox6, Dox40,
`MR20, or LR5 was inhibited in cultures with Thal and the IMiDs in
`a dose-dependent manner (1-100 mmol/L) versus media alone
`cultures (Figure 2A-D). For example, 10 mmol/L IMiD1 blocked
`proliferation of Dox6, Dox40, MR20, and LR5 cells by 20%, 33%,
`32%, and 21%, respectively (P , .001). The IMiDs similarly
`inhibited DNA synthesis of MM.1R cells in a dose-dependent
`fashion, with more than 50% inhibition at more than 1 mmol/L
`IMiD1 (P , .001; Figure 2E). These data suggest independent
`mechanisms of resistance to Dox, Mit, Mel, and Dex versus Thal
`and its analogs.
`
`Effect of Dex and IL-6 on response of MM cells
`to Thal and the ImiDs
`
`To determine whether the effects of Thal and the IMiDs are
`additive with conventional therapies, we next examined the effect
`
`of Dex (0.001-0.1 mmol/L) together with 1 mmol/L Thal or IMiDs
`on proliferation of Dex-sensitive MM.1S cells. As can be seen in
`Figure 3A, the IMiDs (1 mmol/L) significantly inhibited 3H-TdR
`uptake of MM.1S cells (60%-75% block, P , .01), and Dex
`(0.001-0.1 mmol/L) increased this inhibition in a dose-dependent
`fashion. For example, doses of 0.001 to 0.01 mmol/L Dex added to
`1 mmol/L IMiD1 increased the inhibition of proliferation by 35%
`relative to cultures with 1 mmol/L IMiD1 alone (P , .01). Given
`the additive effects of Dex and the IMiDs, as well as the known role
`of IL-6 as a growth factor and specific inhibitor of Dex-induced
`MM cell apoptosis,19,22,23 we also examined whether exogenous
`IL-6 could overcome the inhibition of DNA synthesis triggered by
`Thal and the IMiDs. Figure 3B demonstrates that IL-6 (50 ng/mL)
`triggers DNA synthesis of MM.1S cells in cultures with media
`alone, as well as in cultures with the IMiDs (0.1 and 1 mmol/L).
`
`Effect of Thal and analogs on DNA synthesis
`of patient MM cells
`
`The effect of Thal and the IMiDs on DNA synthesis of patient MM cells
`was next examined (Figure 4). As was true for MM.1S and Hs Sultan
`MM cell lines, 3H-TdR uptake of patients’ MM cells was also inhibited
`by IMiDs (0.1-100 mmol/L) in a dose-dependent fashion, whereas the
`inhibitory effect of Thal, even at 100 mmol/L, was not significant. Fifty
`percent inhibition of MM patient cells was observed at 100 mmol/L
`(Figure 4A) and 1 mmol/L (Figure 4B) IMiD1, respectively (P , .001).
`
`Figure 2. Effect of Thal and analogs on DNA synthe-
`sis of MM cells resistant to conventional therapy.
`Dox-resistant Dox6 (A) and Dox 40 (B), Mit-resistant
`(MR20; C), and Mel-resistant (LR5; D) cells were cultured
`with control media (M) or 1mmol/L (p), 10 mmol/L (z),
`100 mmol/L (f) of Thal, IMiD1, IMiD2, or IMiD3. Values
`represent the mean (6 SD) 3H-TdR (cpm) of triplicate
`cultures. (E) Dex-resistant MM.1R cells were cultured in
`control media (M) or with 0.1 mmol/L (p), 1 mmol/L ([z),
`10 mmol/L (o), or 100 mmol/L (f) of Thal, IMiD1, IMiD2, or
`IMiD3.
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`THALIDOMIDE OVERCOMES DRUG RESISTANCE OF MM
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`2947
`
`4% to 7%, respectively, under all culture conditions and was not
`increased by Thal or the IMiDs.
`
`Effect of Thal and analogs on p21 expression in MM cell lines
`and patient cells
`
`We next correlated these distinct biologic sequelae of Thal and the
`IMiDs with p21 status in MM.1S versus Hs Sultan and patient MM
`cells. As can be seen in Figure 6A, p21 expression was down-regulated
`by the IMiDs, as well as by Dex, in MM1.S cells; and IL-6 overcomes
`this inhibitory effect. In contrast, the IMiDs up-regulated p21 in Hs
`Sultan cells and patient MM cells. Immunoblotting with anti-tubulin Ab
`confirmed equivalent protein loading. Wt-p53 was recognized in
`MM.1S cells, whereas both wt- and mt-p53 were recognized in Hs
`Sultan cells and patient MM cells (Figure 6B). These studies further
`support the observation that Thal and the IMiDs can induce either
`apoptosis or G1 growth arrest in sensitive MM cells, and they are
`consistent with Thal and IMiD p53-mediated down-regulation of p21
`and susceptibility to p53-mediated apoptosis in MM.1S cells, in contrast
`to induction of p21 and growth arrest in Hs Sultan cells and patient
`MM cells, conferring protection from apoptosis.
`
`Effect of Thal and analogs on growth and apoptotic signaling
`in MM.1S and MM.1R cells
`
`We have previously characterized signaling cascades mediating
`MM cell growth and apoptosis, as well as the antiapoptotic effect of
`
`Figure 3. Effect of Dex and IL-6 on response of MM cells to Thal and the IMiDs.
`(A) MM.1S cells were cultured with 1.0 mM Thal, IMiD1, IMiD2, or IMiD3 in control
`media alone (M) or with 0.001 (p), 0.01 (z), and 0.1 mmol/L (f) Dex. (B) MM.1S cells
`were cultured in control media alone and with 0.1 and 1.0 mmol/L Thal, IMiD1, IMiD2,
`or IMiD3 either in the presence (M) or absence (f) of IL-6 (50 ng/mL). In each case,
`3H-TdR uptake was measured during the last 8 hours of 48-hour cultures. Values
`represent the mean (6 SD) 3H-TdR (cpm) of triplicate cultures.
`
`Effect of Thal and analogs on cell cycle profile of MM cell lines
`and patient MM cells
`
`To further analyze the mechanism of Thal- and IMiD-induced
`inhibition of DNA synthesis and to determine whether these drugs
`induced apoptosis of MM cells, we first examined the cell cycle
`profile of MM.1S, Hs Sultan cells, and patient MM cells cultured
`with media alone, Thal (10 mmol/L), or the IMiDs (1 mmol/L).
`Cells were harvested from 72-hour cultures and stained with PI. As
`shown in Figure 5A, all 3 IMiDs, and Thal to a lesser extent,
`increased sub-G1 MM.1S cells. Induction of apoptosis occurred at
`the dose-response curve noted for inhibition of proliferation.
`Twelve-hour cultures with Dex (10 mmol/L) served as a positive
`control for triggering increased sub-G1 cells. In contrast, no
`increase in sub-G1 cells was observed in cultures of Hs Sultan cells
`or of patient MM cells with Thal or the IMiDs. Importantly, Thal
`and the IMiDs induced G1 growth arrest in both Hs Sultan cells and
`in AS patient MM cells.
`To confirm these results, we performed annexin V staining of
`cells in these cultures. As can be seen in Figure 5B, the percentage
`of annexin V–positive cells in cultures of MM.1S cells with Thal,
`IMiD1, IMiD2, and IMiD3 was 32%, 55%, 51%, and 43%,
`respectively. Forty-six percent of annexin V staining was observed
`in cultures with Dex, whereas only 22% annexin V–positive cells
`were present in cultures with media alone. The percentage of
`annexin V–positive Hs Sultan cells and AS patient MM cells was
`
`Figure 4. Effect of Thal and analogs on DNA synthesis of patient MM cells. MM
`cells from patient 1 (A) and patient 2 (B) were cultured with control media (M) or with
`0.1 mmol/L (p), 1.0 mmol/L ([z), 10 mmol/L (o), and 100 mmol/L (f) Thal, IMiD1,
`IMiD2, or IMiD3. In each case, 3H-TdR uptake was measured during the last 8 hours
`of 48-hour cultures. Values represent the mean (6 SD) 3H-TdR (cpm) of triplicate
`cultures.
`
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`BLOOD, 1 NOVEMBER 2000 z VOLUME 96, NUMBER 9
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`Figure 5. Effect of Thal and analogs on cell cycle
`profile of MM cell lines and patient MM cells. (A)
`MM.1S cells, Hs Sultan cells, and patient MM cells were
`cultured with 10 mmol/L of Thal or 1 mmol/L of IMiD1,
`IMiD2, or IMiD3 for 72 hours. Cultures in media control
`alone served as a negative control and 18-hour cultures
`with 10 mmol/L Dex as positive controls. Cells were then
`stained with PI, and cell cycle profile was determined by
`flow cytometric analysis. (B) These MM.1S (f), Hs
`Sultan (p), and patient MM (M) cells were also stained
`with annexin V as an additional assay for apoptosis.
`
`IL-6.19,22-25 Because we have shown that IL-6–induced prolifera-
`tion is mediated by the ras-dependent mitogen-activated protein
`kinase (MAPK) cascade,19 we next examined the effect of Thal and
`the IMiDs on tyrosine phosphorylation of MAPK in IL-6–
`responsive MM.1S cells. Constitutive tyrosine phosphorylation
`of MAPK in MM.1S cells was down-regulated by the MEK1
`inhibitor PD98059 (50 mmol/L), which served as a positive control
`(Figure 6A), and to a lesser extent by the IMiDs (1 mmol/L; Figure
`7A) or Thal (10 mmol/L; data not shown). Treatment of MM.1S
`cells with IL-6 increased MAPK tyrosine phosphorylation, which
`was partially blocked by PD98059 but was unaffected by the
`IMiDs (Figure 7A) or Thal (data not shown). Stripping the blot
`and reprobing with anti-ERK2 Ab confirmed equivalent pro-
`tein loading.
`
`The observation that IL-6 can overcome the effects of Thal, the
`IMiDs, and Dex, coupled with our prior studies delineating
`signaling cascades mediating Dex-induced apoptosis and the
`protective effects of IL-6,22,23,25 suggested that RAFTK activation
`may be induced during apoptosis triggered by Thal and IMiDs.
`MM.1S and MM.1R cells were, therefore, next cultured with 1
`mmol/L Thal, IMiD1, IMiD2, or IMiD3 for 12 hours. Twelve-hour
`cultures with Dex (10 mmol/L) served as a positive control for
`activation of RAFTK. Total cell lysates were subjected to immuno-
`precipitation with anti-RAFTK Ab and analyzed by immunoblot-
`ting with anti-P-Tyr Ab or anti-RAFTK Ab. As can be seen in
`Figure 7B, Dex induced tyrosine phosphorylation of RAFTK in
`MM.1S cells but not in MM.1R cells. Importantly, IMiD1 induced
`RAFTK tyrosine phosphorylation in both MM.1S and MM.1R
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`THALIDOMIDE OVERCOMES DRUG RESISTANCE OF MM
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`cells, correlating with its effects on both Dex-sensitive and
`Dex-resistant MM cells.
`
`Discussion
`
`This study demonstrates for the first time a direct dose-dependent
`effect of Thal and these analogs on tumor cells. Thal has
`demonstrated clinical anti-MM activity at the University of Arkan-
`sas7 and in this study, and Thal at high concentrations (100 mmol/L)
`resulted in a modest (, 20%) inhibition of in vitro DNA synthesis
`of MM cells. SelCIDs also induced a dose-dependent inhibition of
`MM cells, but only 2 of 4 SelCIDs tested achieved 50% inhibition
`of proliferation, even at 100 mmol/L concentrations. Importantly,
`all 3 IMiDs tested achieved 50% inhibition of DNA synthesis at
`concentrations (0.1-1.0 mmol/L) corresponding to serum levels that
`are readily achievable, both confirming their direct action on tumor
`cells and suggesting their potential clinical utility. Moreover, the
`IMiDs inhibited the proliferation of Dox-, Mit-, and Mel-resistant
`MM cells by 20% to 35%, and of Dex-resistant MM cells by 50%.
`These in vitro effects correlate with the observed clinical activity of
`Thal
`in patients with MM that
`is refractory to conventional
`therapies, both at the University of Arkansas7 and reported in this
`study, and suggest their clinical utility to overcome drug resistance.
`Moreover, our studies further suggest that Dex can add to the
`antiproliferative effect of Thal and the IMiDs in vitro, suggesting
`the potential utility of coupling these agents therapeutically.
`Finally, our study also identified MM cells resistant to Thal and the
`analogs (U266 cells), which, therefore, can be used to study
`mechanisms of Thal resistance.
`Our studies demonstrate that Thal and the IMiDs are acting
`directly on MM cells, in the absence of accessory BM or T cells. It
`is also possible that these agents may be mediating their anti-MM
`effect by cytokines, given their known inhibitory effects on TNF-a,
`IL-1b, and IL-6.15 Our prior studies have characterized the growth
`
`Figure 6. Effect of Thal and analogs on p21 expression in MM cell lines and
`patient cells. (A) MM.1S cells were cultured with 10 mmol/L of Thal, IMiD1, IMiD2,
`and IMiD3 for 48 hours. MM.1S cells were also cultured with IL-6 (50 ng/mL) alone
`and with IMiD1, 10 mmol/L 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.1S, Hs Sultan,
`and patient MM cells were lysed and immunoprecipitated with wt-p53 and mt-p53
`Abs, transferred to PVDF membrane, and blotted with anti-p53 Ab.
`
`Figure 7. Effect of Thal and analogs on growth and apoptotic signaling in
`MM.1S and MM.1R cells. (A) MM.1S cells were cultured in media, with 50 mmol/L of
`PD98059 and with 10 mmol/L of IMiD1, IMiD2, or IMiD3 for 48 hours. Cells were then
`triggered with 50 ng/mL of IL-6 for 10 minutes, 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 Thal (100 mM), IMiD1
`(100 mmol/L), or Dex (10 mmol/L) and harvested at 12 hours. Total cell lysates were
`subjected to immunoprecipitation with anti-RAFTK Ab and analyzed by immunoblot-
`ting with anti-P-Tyr Ab or anti-RAFTK Ab.
`
`effects of IL-6 on human MM cells,12,26 and we, therefore, next
`determined the effect of exogenous IL-6 on drug activity. Our
`studies showed that IL-6 can overcome the effect of Thal and the
`IMiDs on MM cell lines and patient cells, suggesting that these
`novel drugs may, at least in part, be inhibiting IL-6 production. Our
`prior studies have further demonstrated that IL-6–induced prolifera-
`tion of MM cells is mediated through the MAPK cascade and that
`blockade of this pathway with either MAPK antisense oligonucleo-
`tide or the MEK1 inhibitor PD98059 can abrogate this re-
`sponse.19,21,24 The present study showed constitutive MAPK phos-
`phorylation in MM cells that is inhibited by PD98059 and, to a
`lesser extent, by the IMiDs. Importantly, IL-6–triggered MAPK
`tyrosine phosphorylation is also blocked by PD98059 but not by
`IMiDs. These studies, therefore, suggest that the IMiDs do not
`work only by directly inhibiting MAPK growth signaling and
`further support their potential activity in down-regulating IL-6
`production. In MM, IL-6 production in tumor cells can either be
`constitutive or induced, mediating autocrine tumor cell growth.26,27
`In addition, IL-6 is also produced by BM stromal cells in MM, a
`process that is up-regulated by tumor cell adhesion to BM stromal
`cells, with related tumor cell growth in a paracrine mechanism.10,11
`Our ongoing studies are, therefore, evaluating the effect of Thal and
`these analogs on IL-6 production in the BM microenvironment.
`Having shown the inhibitory effects of Thal and the IMiDs on
`3H-TdR uptake