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Abstract

Although thalidomide (Thal) was initially used to treat multiple myeloma (MM) because of its known antiangiogenic effects, the mechanism of its anti-MM activity is unclear. These studies demonstrate clinical activity of Thal against MM that is refractory to conventional therapy and delineate mechanisms of anti-tumor activity of Thal and its potent analogs (immunomodulatory drugs [IMiDs]). Importantly, these agents 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 dexamethasone (Dex). Moreover, Thal and the IMiDs enhance the anti-MM activity of Dex and, conversely, are inhibited by interleukin 6. As for Dex, apoptotic signaling 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 microenvironment, overcome classical drug resistance, and achieve improved outcome in this presently incurable disease.

Introduction

Thalidomide (Thal) was originally used in Europe for the treatment of morning sickness in the 1950s but was withdrawn from the market in the 1960s because of reports of teratogenicity and phocomelia associated with its use.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, including cancer and related conditions, infectious diseases, autoimmune diseases, dermatologic diseases, and other disorders such as sarcoidosis, macular degeneration, and diabetic retinopathy.3 Recent reports of increased bone marrow (BM) angiogenesis in multiple myeloma (MM),4 5coupled with the known antiangiogenic properties of Thal,6provided the rationale for its use to treat MM.7Importantly, Thal induced clinical responses in 32% of MM patients whose disease was refractory to conventional and high-dose therapy,7suggesting that it can overcome drug resistance because of its alternative mechanisms of anti-MM activity. Besides alkylating agents and corticosteroids, Thal now, therefore, represents 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-1β, IL-10, and tumor necrosis factor (TNF)–α, may augment MM cell growth and survival,12and Thal may alter their secretion and bioactivity.15 Fourth, vascular endothelial 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 immunomodulatory effects, such as induction of a Th1 T-cell response with secretion of interferon gamma (IFN-γ) and IL-2.17Already 2 classes of Thal analogs have been reported, including phosphodiesterase 4 inhibitors that inhibit TNF-α 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-γ 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.

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 (Northwestern 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 μg/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% CD38+CD45RA) 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 −20°C until use. Drugs were diluted in culture medium (0.0001 to 100 μM) 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-α production and increase IL-10 production from lipopolysaccharide (LPS)–stimulated peripheral 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-γ secretion, but are not phosphodiesterase 4 inhibitors. The IMiDs also inhibit TNF-α, IL-1β, 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 × 104cells/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 with3H-TdR (0.5 μCi/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 μL 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 μL 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 × 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 μg/mL of RNAse (Sigma). Cells were stained with propidium iodide (PI; 5 μg/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 μmol/L of Thal or 1 μmol/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 × 106cells/mL) in binding buffer (10 mmol/L HEPES, pH 7.4, 140 mmol/L NaCl, 2.5 mmol/L CaCl2). MM cells (1 × 105) were incubated with annexin V-FITC (5 μL; Pharmingen, San Diego, CA) and PI (5 μg/mL) for 15 minutes at room temperature. Annexin V+PI− apoptotic cells were enumerated by using the Epics cell sorter (Coulter).

Immunoblotting

MM cells were cultured with 10 μmol/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 μg/mL leupeptin, and 5 μg/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 × 107) with the use of lysis buffer. Lysates were incubated with anti-mutant (mt) or wild-type (wt) p53 monoclonal Abs (Calbiochem, 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 chemiluminescence (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 μmol/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 μg/mL leupeptin, and 5 μg/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 electrophoresis in 7.5% SDS-PAGE gels, transferred to nitrocellulose paper, and analyzed by immunoblotting. The antigen-antibody complexes were visualized by chemiluminescence.

Statistical analyses

Statistical significance of differences observed in drug-treated versus control cultures was determined by using the Studentt test. The minimal level of significance wasP < .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 complete response (absence of monoclonal protein on immunofixation 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).

Table 1.

Response to thalidomide in multiple myeloma*

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 inhibited 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 μmol/L IMiD1, 0.1-1.0 μmol/L IMiD2, and 0.1-1.0 μmol/L IMiD3 (P < .001). Fifty percent inhibition of proliferation of Hs Sultan cells was noted at 0.1 μmol/L IMiD1, 1.0 μmol/L IMiD2, and 1.0 μmol/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 μmol/L) of Thal. No significant inhibition of DNA synthesis of U266 MM cells was noted in cultures with 0.001 to 100 μmol/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 μmol/L) for only 2 of the 4 SelCIDs (SelCIDs 1 and 3, Figure 1C). Further studies, therefore, focused on Thal and the IMiDs.

Fig. 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 μM) of Thal (♦), IMiD1 (▪), IMiD2 (●), and IMiD3 (▴). (C) MM.1S cells were cultured with increasing concentrations (12.5-100 μM) of SelCID1 (♦), SelCID2 (▪), SelCID3 (▴), and SelCID4 (●). In each case 3H-TdR uptake (left panels) or MTT cleavage (r

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