Dihydroorotate Dehydrogenase Inhibitor A771726 (Leflunomide) Induces Apoptosis and Diminishes Proliferation of Multiple Myeloma Cells

Published OnlineFirst January 27, 2009; DOI: 10.1158/1535-7163.MCT-08-0664

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Dihydroorotate dehydrogenase inhibitor A771726 (leflunomide) induces apoptosis and diminishes proliferation of multiple myeloma cells

Philipp Baumann, Sonja Mandl-Weber, with the genotoxic agents melphalan, treosulfan, and Andreas Vo¨lkl, Christian Adam, Irmgard Bumeder, doxorubicin as well as with dexamethasone and bortezo- Fuat Oduncu, and Ralf Schmidmaier mib. Taken together, we show that inhibition of DHODH by A771726/leflunomide is effective in multiple myeloma. Department of Hematology and Oncology, Medizinische Considering the favorable toxicity profile and the great Klinik Innenstadt, Klinikum der Universita¨t Mu¨nchen, clinical experience with leflunomide in rheumatoid arthritis, Mu¨nchen, Germany this drug represents a potential new candidate for targeted therapy in multiple myeloma. [Mol Cancer Ther Abstract 2009;8(2):366–75] Multiple myeloma is still an incurable disease; therefore, new therapeutics are urgently needed. A771726 is the active metabolite of the immunosuppressive drug lefluno- Introduction mide, which is currently applied in the treatment of rheu- Multiple myeloma is an incurable disease accounting for matoid arthritis, BK virus nephropathy, and cytomegaly 1% to 2% of all neoplastic diseases (1) and it is viremia. Here, we show that dihydroorotate dehydroge- characterized by the accumulation of monoclonal plasma nase (DHODH) is commonly expressed in multiple myelo- cells in the bone marrow that produce a monoclonal ma cell lines and primary multiple myeloma cells. The immunoglobulin. In the last 15 years, treatment of multiple DHODH inhibitor A771726 inhibits cell growth in common myeloma has changed rapidly;despite high-dose chemo- myeloma cell lines at clinically achievable concentrations therapy accompanied by autologous stem cell support as in a time- and dose-dependent manner. Annexin V-FITC/ well as the introduction of new substances such as propidium iodide staining revealed induction of apoptosis bortezomib, thalidomide, and lenalidomide, multiple my- of multiple myeloma cell lines and primary multiple eloma cells become resistant to cytotoxic drugs and patients myeloma cells. The 5-bromo-2¶-deoxyuridine cell prolifer- eventually die of tumor progression (2–6). Deregulation of ation assay showed that inhibition of cell growth was several signaling pathways, such as the Akt/phosphatidy- partly due to inhibition of multiple myeloma cell prolifer- linositol 3-kinase, JAK/STAT pathway, and Ras/Raf/ ation. A771726 induced G1 cell cycle arrest via modula- extracellular signal-regulated kinase pathways, has been tion of cyclin D2 and pRbexpression. A771726 decreased described, contributing to drug resistance and proliferation phosphorylation of protein kinase B (Akt), p70S6K, and (7, 8). These findings prompt the search for new targets. eukaryotic translation initiation factor 4E-binding protein-1 Dihydroorotate dehydrogenase (DHODH) is the fourth as shown by Western blotting experiments. Furthermore, and rate-limiting enzyme in the synthesis pathway of we show that the stimulatory effect of conditioned pyrimidines (9). Unlike other enzymes of this pathway, this medium of HS-5 bone marrow stromal cells on mul- enzyme is not localized in the cytoplasm but in the inner tiple myeloma cell growth is completely abrogated mitochondrial membrane. Besides catalyzing the reaction by A771726. In addition, synergism studies revealed of dihydroorotate to orotate, it reduces ubiquinone in the synergistic and additive activity of A771726 together inner mitochondrial membrane, linking the pyrimidine pathway to the mitochondrial respiratory chain (10). On stimulation with mitogens, proliferating lymphocytes expand their pyrimidine pool by 8-fold and are dependent Received 7/16/09; revised 11/17/09; accepted 11/19/09; on DHODH (11). Because mitogens drive cell growth sur- published OnlineFirst 01/27/2009. vival and cell proliferation in multiple myeloma cells, this Grant support: European Hematology Association fellowship 2005/28 (R. Schmidmaier) and Fo¨rderungvon Forschungund Lehre of the LMU enzyme provides an attractive target in multiple myeloma. University of Munich (P. Baumann). Leflunomide (Arava) is a well-known immunosuppres- The costs of publication of this article were defrayed in part by the sive drug that has been approved for the treatment of payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to rheumatoid arthritis and has been used in >300,000 patients indicate this fact. worldwide (10). Besides rheumatoid arthritis, leflunomide Requests for reprints: Philipp Baumann, Department of Hematology and is also used as a virostatic drug because its activity against Oncology, Medizinische Klinik Innenstadt, Klinikum der Universita¨t cytomegalovirus and the BK virus has been shown (12–16). Mu¨nchen, Ziemssenstr. 1, 80336 Mu¨nchen, Germany. Phone: 49-89-5160-2237; Fax: 49-89-5160-4412. Leflunomide is rapidly converted in the gastrointestinal E-mail: [email protected] tract into the active metabolite A771726 (17), which inhibits Copyright C 2009 American Association for Cancer Research. DHODH (18–21). The high level of protein binding leads to doi:10.1158/1535-7163.MCT-08-0664 a plasma half-life of f15 days (17). The drug is eventually

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excreted into the urine as trifluoromethylaniline-oxanillic and doxorubicin were purchased from Sigma-Aldrich. acid. High drug concentrations up to 300 Amol/L can be Annexin V-FITC was purchased from Becton Dickinson. reached and side effects are rare even at these concen- Conjugated antibodies for flow cytometry raised against trations (16). CD11a, CD49d, CD54, CD126, and CD221 were obtained Inhibition of DHODH and tyrosine kinases using from Immunotech. leflunomide has never been elucidated in multiple myelo- Cell Growth Assay ma. In this study, we show that DHODH is expressed The WST-1 growth assay protocol was used as recom- in multiple myeloma cells and the active metabolite of mended by the manufacturer (Roche). Briefly, 4 Â 103 cells leflunomide A771726 shows strong anti-multiple myeloma per 100 AL were seeded to 96-well plates and incubated activity at clinically achievable concentrations. with the test compound for indicated periods. For the last 2 h of culture, cells were incubated with 10 AL WST-1. Absorbance at 440 nm was measured using a microplate Materials and Methods ELISA reader to detect metabolically intact cells (with a Cells reference wavelength of 680 nm). OPM-2, RPMI-8226, NCI-H929, and U266 cell lines and Analysis of Apoptosis and Cell Death the human bone marrow stromal cell line HS-5 were Cells were stained with propidium iodide and Annexin obtained from the American Type Culture Collection, V-FITC. Briefly, after two treatments with washing buffer grown in RPMI 1640 (Boehringer) containing 10% heat- [8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4,0.24gKH2PO4, and A inactivated FCS (Boehringer) in a humidified atmosphere 1LH2O (pH 7.2)], cells were resuspended in 400 L j Â 5 (37 C;5% CO 2), and seeded at a concentration of 1 10 Dulbecco’s PBS, and 100 AL of this cell suspension were cells/mL. Cells have been regularly tested for Mycoplasma incubated with 10 ALof50Ag/mL propidium iodide and and were free of this contamination. 5 AL Annexin V-FITC for 15 min at room temperature in Purification of Primary Multiple Myeloma Cells the dark. Cells were analyzed by flow cytometry (Coulter After informed consent was obtained from patients, EPICS XL-MCL;System II). mononuclear cells from bone marrow aspirates were Cell Cycle Analysis isolated using CD138 MACS beads (Miltenyi Biotech) Cellular DNA content was determined by flow cytom- according to the manufacturer’s instructions. Briefly, etry. Multiple myeloma cells were harvested and washed mononuclear cells were washed twice with chilled buffer twice with ice-cold PBS. After that, cells were resuspended (containing Dulbecco’s PBS, 0.5% FCS, and 2 mmol/L in 1 mL of 70% ethanol and fixed for 2 h at À20jC. Fixed EDTA) and resuspended in buffer. CD138 MACS beads cells were washed once with ice-cold PBS and resuspended were added to the cells and incubated for 15 min at 4jC. in 400 AL PBS and 40 ALof50Ag/mL propidium iodide were After that, cells were washed again, resuspended in 1 mL added to the cells. Cells were acquired by flow cytometry. buffer, and subjected to separation columns. After unla- Western Blot Analysis belled cells passed through the column, the separation Cells were washed three times in ice-cold PBS and lysed columns were removed from the separator and the labeled in a buffer containing 10 mmol/L Tris-HCl (pH 7.6), cells were flushed out of the separation columns. The 137 mmol/L NaCl, 1 mmol/L Na3VO4, 10 mmol/L NaF, separation procedure was repeated twice. After separation, 10 mmol/L EDTA, and 1% (v/v) Igepal CA-630 (NP-40) the purity of CD138 cells was examined by flow cytometry with the addition of 10 Ag/mL aprotinin, 10 Ag/mL using a CD138 PE-labeled antibody (BD Sciences). A purity leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride. of >90% CD138+ cells was accepted for further experiments. Extracted protein concentration was adjusted using a The Ethics Committee of the University of Munich colorimetric assay (Bio-Rad Protein Assay). Proteins were approved the study. subjected to SDS-PAGE and transferred to a polyvinylidene Reagents fluoride membrane (Millipore). The transfer buffer Melphalan, doxorubicin, and propidium iodide were contained 25 mmol/L Tris-HCl, 192 mmol/L glycine, and purchased from Calbiochem and WST-1 was purchased 20% (v/v) methanol. The membranes were blocked with from Roche. A771726 was bought from Calbiochem. TBS containing 5% dried milk and 0.05% Tween 20. After Polyclonal primary antibodies against pAkt1/2 (Thr308 washing four times with TBS with Tween 20, the and Ser473), mcl-1, bim-EL, p21, cyclin A, cyclin D1, cyclin membranes were incubated with appropriate primary D2, cyclin E, caspase-3, bcl-2, cdc25a, cdk2, cdk4, cdk6, bax, and secondary antibodies and visualized by autoradio- DHODH, and actin were obtained from Santa Cruz graphy using the ECL Western blotting detection system Biotechnology and phospho-p70S6K (Thr421/Ser424), phos- (GE Healthcare). pho-mammalian target of rapamycin (Ser2448), phospho-4E- Cell Proliferation Assay Using 5-Bromo-2 ¶-Deoxyuridine binding protein-1 (Thr70), p70S6K, cleaved caspase-3, pRb, Cell proliferation was determined using an ELISA-based bcl-xL, and mammalian target of rapamycin were obtained colorimetric assay [Cell Proliferation ELISA;5-bromo-2 ¶- from Cell Signaling. Secondary antibodies raised against deoxyuridine (BrdUrd)] from Roche Diagnostics following goat or rabbit epitopes were purchased from GE Health- the manufacturer’s instructions. Briefly, cells were seeded care. Bortezomib was purchased from Millennium Phar- in 96-well plates at a concentration of 1.5 Â 104 per well maceuticals, and melphalan, treosulfan, dexamethasone, in RPMI 1640 supplemented with 10% fetal bovine serum

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with or without the substances to be tested. After f54 h, myeloma cell lines OPM-2, RPMI-8226, NCI-H929, and BrdUrd labeling solution was added and the cells were U266 with clinically achievable concentrations of A771726 cultured for another 18 h in a humidified atmosphere for 48 and 96 h and determined cell growth by WST-1 j (37 C;5% CO 2). Then, the cells were centrifuged and the assay. The tetrazolium salt (WST-1) is changed to formazan supernatants were discarded. The cells were dried at 60jC by the succinate-tetrazolium reductase. The quantity of for 1 h. After fixation and DNA denaturation with FixDenat formazan dye is related directly with the number of for 30 min at room temperature, the cells were incubated metabolically active cells and was quantified by a multiwell with anti-BrdUrd-POD working solution for 90 min at photometer. Our experiments show that cell growth was room temperature. The anti-POD solution was removed totally abrogated on incubation with 200 Amol/L A771726 and the cells were washed three times with washing buffer. in all tested cells. The IC50 was quite lower in NCI-H929 Then, the tetramethylbenzidine substrate solution was and RPMI-8226 cells in comparison with OPM-2 and U266 added until color development was sufficient for detection. (Fig. 2A). Furthermore, we explored whether inhibition of The reaction was stopped with 1 mol/L H2SO4 and the cell growth was due to induction of apoptosis in multiple extinctions were measured within 5 min in a microplate myeloma cells and therefore incubated the four multiple reader at 450 nm (with a reference wavelength of 690 nm). myeloma cell lines with increasing concentrations of Isobologram Analysis A771726 for 72 h. After the incubation period, cells were Isobologram analysis was done using the Calcusyn stained with Annexin V-FITC and propidium iodide and software program (Biosoft, Ferguson). A combination index analyzed by flow cytometry. Our experiments show that (CI) < 0.9 indicates synergism, whereas CI = 0.9 to 1.1 apoptosis is moderately induced on incubation with 100 indicates additive effects. and 200 Amol/L (Fig. 2B). To confirm that apoptosis is Statistics induced by A7721726, we performed Western blotting Mean and SD from representative experiments are shown experiments and blotted caspase-3 and cleaved casp- in the figures. Data were confirmed by at least two ase-3. We observed that caspase-3 was cleaved after a independent experiments. The Wilcoxon test was used to 48 h incubation period at 200 Amol/L A771726 (Fig. 2C). compare different groups. P values < 0.05 were considered Furthermore, we analyzed primary multiple myeloma cells statistically significant. obtained from bone marrow aspirates from three patients. After purification with CD138 MACS, multiple myeloma A Results cells were incubated with 0 to 300 mol/L A771726 for 48 h and induction of apoptosis was determined by flow DHODHIsExpressedinMultipleMyelomaCellLines cytometry after staining with Annexin-V FITC and propi- and Primary Multiple Myeloma Cells dium iodide (Fig. 2D). As expected, apoptosis was induced Because no data regarding DHODH expression in in primary myeloma cells (75% at 200 Amol/L). multiple myeloma are available, we wanted to examine We conclude that A771726 strongly inhibits cell growth whether this enzyme is expressed in multiple myeloma cell of multiple myeloma cells and induces apoptosis at higher lines and primary multiple myeloma cells. We therefore concentrations. performed Western blotting experiments and blotted A771726 Inhibits Multiple Myeloma Cell Proliferation DHODH. Figure 1 shows that DHODH is expressed in all and Induces a G1Cell Cycle Arrest four multiple myeloma cell lines and primary cells Because induction of apoptosis could not fully explain obtained from bone marrow aspirates from multiple the reduction of cell growth, we asked whether A771726 myeloma patients. leads to inhibition of cell proliferation in multiple myeloma DHODH Inhibitor A771726 Inhibits Cell Growth and cells and performed a BrdUrd assay. We incubated OPM-2, Induces Apoptosis in Multiple Myeloma Cells NCI-H929, RPMI-8226, and U266 myeloma cells with We questioned whether A771726 affects cell growth in increasing concentrations of A771726 for 72 h. BrdUrd myeloma cells. We therefore incubated the human multiple was added 18 h before harvesting the cells. Figure 3A shows that myeloma cell proliferation is strongly reduced by the active metabolite of leflunomide. Strong (up to 94% reduction) and quite similar effects were seen in all four A tested cell lines with an IC50 between 50 and 100 mol/L. Because A771726 blocks proliferation of myeloma cells, we assumed a cell cycle arrest. OPM-2, NCI-H929, RPMI-8226, and U266 myeloma cells were incubated with increasing concentrations of leflunomide for 48 h. After the incubation Figure 1. DHODH is expressed in myeloma cell lines and primary multiple myeloma cells. The multiple myeloma cell lines OPM-2, RPMI- period, cells were harvested and further prepared for cell 8226, NCI-H929, and U266 were harvested and processed for Western cycle analysis. Figure 3B clearly shows that myeloma cells blottingas described in Materials and Methods. Primary myeloma cells accumulate in the G1 phase of the cell cycle. obtained from three bone marrow aspirates from myeloma patients were We further asked why A771726 induces a G cell cycle purified by CD138 MACS as described in Materials and Methods. Cells 1 were then lysed and directly subjected to SDS-PAGE, transferred to arrest. NCI-H929 and OPM-2 cells were incubated with membranes, and blotted with the indicated antibodies. 200 Amol/L leflunomide for up to 48 h and subjected to

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Figure 2. A771726 inhibits cell growth and induces apoptosis in multiple myeloma cells. A, multiple myeloma cell lines NCI-H929, OPM-2, RPMI-8226, and U266 were incubated with 0, 5, 10, 50, 100, and 200 Amol/L A771726 for 96 h and cell growth was determined by the WST-1 assay every 48 h. Mean and SD. Results from two independent experiments. B, NCI-H929, OPM-2, RPMI-8226, and U266 myeloma cells were incubated with 0, 5, 10, 50, 100, and 200 Amol/L A771726 for 72 h and induction of apoptosis was determined after Annexin V FITC/propidium iodide stainingby flow cytometry. Results from three independent experiments. *, P < 0.05 versus control. C, NCI-H929, OPM-2, RPMI-8226, and U266 myeloma cells were incubated with 0, 50, 100, or 200 Amol/L A771726 for 48 h, harvested, and further processed for Western blotting. Cells were then lysed and subjected to SDS-PAGE, transferred to membranes, and blotted with the antibody indicated. D, primary myeloma cells from three myeloma patients were purified usingCD138 MACS as described in Materials and Methods. Cells were then incubated with 0, 50, 100, 200, and 300 Amol/L A771726 for 48 h and induction of apoptosis was determined after stainingwith Annexin V-FITC and propidium iodide. Mean and SD.

Western blots. It became evident that decreased cell We therefore performed Western blotting experiments proliferation and G1 cell cycle arrest was accompanied by and incubated NCI-H929 and OPM-2 myeloma cells with a decrease of cdc25a, cyclin D2, cyclin E, cyclin A, and pRb. 200 Amol/L A771726 for up to 48 h and blotted proteins p21 protein was up-regulated, whereas the cyclin-depen- of the Akt signaling pathway and proapoptotic and dent kinases as well as cyclin D1 seemed not to be affected antiapoptotic mitochondrial proteins. We revealed that (Fig. 3C). These findings were more obvious in the NCI- A771726 strongly inhibits phosphorylation of Akt, p70S6K, H929 than in the OPM-2 cell line. and 4E-binding protein-1 (Fig. 4A). Interestingly, phos- In summary, inhibition of cell cycle progression and pho-mammalian target of rapamycin slightly increased induction of a G1 cell cycle arrest represent further on incubation with A771726. Also, proapoptotic and anti- characteristics of A771726. apoptotic mitochondrial proteins were deregulated: the Inhibition of DHODH Leads to Decreased Phosphor- expression of the antiapoptotic bcl-xL protein was de- ylation of Akt, p70S6K, and 4E-Binding Protein-1,Mod- creased and the expression of the proapoptotic proteins ulation of bcl-2 Family Proteins, and Cellular Uridine bim and bax was induced (Fig. 4B). Depletion DHODH is the rate-limiting enzyme in the synthesis Because we were able to show that the incubation of chain of uridine, a nucleotide needed for many cellular multiple myeloma cells with A771726 induces apoptosis, functions. We asked whether A771726 exerts its effects on cell growth, and cell cycle arrest, we asked whether multiple myeloma cells through inhibition of DHODH. We A771726 leads to a modulation of signaling pathways in therefore incubated NCI-H929 and RPMI-8226 multiple multiple myeloma cells. myeloma cells with increasing concentrations of A771726

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together with 100 Amol/L uridine for 48 h and deter- Our findings suggests that (a) multiple myeloma cells mined apoptosis after staining with Annexin V-FITC and have a high need of uridine and (b) induction of apoptosis, propidium iodide. A771726 led to apoptosis at 50 and as well as cell growth and cell cycle arrest induced by 100 Amol/L, which was completely reversed by 100 Amol/L A771726, is not exclusively due to inhibition of DHODH. uridine in the NCI-H929 cell line and almost com- Additional mechanisms, such as inhibition of the Akt pletely reversed in the RPMI-8226 cell line. At 200 Amol/L pathway and modulation of proapoptotic and antiapoptotic A771726, uridine did not reverse induction of apoptosis proteins, contribute to the effect seen in multiple myeloma (Fig. 4C). cells treated with A771726. Because we observed a modulation of the Akt pathway as A771726 Inhibits Stromal Cell Medium-Induced Cell well as a modulation of mitochondrial and cell cycle Growth and Leads to a Down-regulation of Adhesion proteins, we asked whether these changes are dependent Molecules and Cytokine Receptors on DHODH. NCI-H929 myeloma cells were incubated with In multiple myeloma, the malignant plasma cell clone is or without 200 Amol/L A771726 and with or without localized in the bone marrow microenvironment, which has 100 Amol/L uridine for 48 h and blotted proteins, which been shown to support survival, drug resistance, and cell were shown to be modulated by A771726. In this setting, proliferation of multiple myeloma cells. This is mediated by uridine did not completely reverse any of the changes of both cellular adhesion to bone marrow stromal cells and protein expression/phosphorylation described above. Only growth factors such as insulin-like growth factor-I and a slight reversal of protein modulation was seen for pAkt cytokines such as interleukin (IL)-6 through activating the (Thr308) and cyclin D2 (Fig. 4D). Akt pathway.

Figure 3. A771726 inhibits multiple myeloma cell proliferation and induces a G1 cell cycle arrest. A, NCI-H929, OPM-2, RPMI-8226, and U266 cells were incubated with 0, 5, 50, 100, and 200 Amol/L A771726 for 72 h. After the incubation period, cell proliferation was determined usingthe BrdUrd proliferation assay. Results from two independent experiments. *, P < 0.05 versus control. B, OPM-2, NCI-H929, RPMI-8226, and U266 myeloma cells were incubated with increasingconcentrations of A771726 as indicated. DNA content was determined by flow cytometry after fixation with ethanol and stainingthe cells with propidium iodide. Results from two independent experiments done in triplicates. C, NCI-H929 and OPM-2 myeloma cells were incubated with 200 Amol/L for 4, 24, and 48 h. Cells were then lysed and directly subjected to SDS-PAGE, transferred to membranes, and blotted with the indicated antibodies.

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Figure 4. Inhibition of DHODH leads to cellular uridine depletion, decreased phosphorylation of Akt, p70S6K, and 4E-bindingprotein-1, and modulation of bcl-2 family proteins in myeloma cells. A and B, NCI-H929 and OPM-2 myeloma cells were incubated with 200 Amol/L A771726 for 4, 24, and 48 h. Cells were then lysed and directly subjected to SDS-PAGE, transferred to membranes, and blotted with the indicated antibodies. C, NCI-H929 and RPMI- 8226 myeloma cells were incubated with or without 100 Amol/L uridine and with increasingconcentrations of A771726 for 48 h as indicated. Cells were then analyzed by flow cytometry after stainingwith Annexin V-FITC and propidium iodide. Results from two independent experiments. *, P < 0.05 versus control. In all figures, the used cell line is indicated in the control experiment. D, NCI-H929 cells were incubated for 48 h with or without 100 Amol/L uridine and with or without 200 Amol/L A771726. After that, cells were lysed and directly subjected to SDS-PAGE, transferred to membranes, and blotted with the indicated antibodies.

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We therefore asked whether A771726 inhibits condi- wasseenforthecombinationswithtreosulfanand tioned medium-induced increase of cell growth and bortezomib. Analysis using the Calcusyn software showed incubated NCI-H929, U266, and RPMI-8226 multiple CI values < 0.9 for combinations with bortezomib and myeloma cells with 25% conditioned medium obtained treosulfan, indicating that the combination with A771726 from a 48 h HS-5 stromal cell culture together with 50 or leads to synergistic effects. Combinations with melphalan 100 Amol/L A771726. HS-5 conditioned medium is known and doxorubicin lead to CI values of 0.9 to 1.1, indicating to contain several stimulating cytokines, including vast additive activity in multiple myeloma cells. Addition of amounts of IL-6 (22, 23). After 48 h, cell growth was dexamethasone did not lead to additive or synergistic determined using the WST-1 growth assay. Figure 5A activity (Table 1). shows that, in all three cell lines, the stimulation with HS-5 stromal cell conditioned medium leads to an increase in myeloma cell growth (NCI-H929: 54%, RPMI-8226: 198%, Discussion and U266: 28%), which was nearly completely overcome Leflunomide and its active metabolite A771726 show both by A771726. Because A771726 seemed to effectively inhibit immunosuppressive and virostatic characteristics. Leflu- the stimulation of HS-5 medium, we further analyzed the nomide is rapidly taken up in the gastrointestinal tract effect of A771726 on myeloma cell surface molecules and and is rapidly and completely converted to its active incubated NCI-H929, OPM-2, and U266 myeloma cells with metabolite A771726. In the therapy of rheumatoid arthritis, increasing concentrations of A771726 and determined the the drug is established in common treatment regimens (24). surface expression of the integrins CD11a, CD49d, and Usually, after an initial loading dose of 100 mg/d for CD54 and the cytokine/growth factor receptors CD126 3 days, treatment is then continued with 20 mg/d, which (IL-6 receptor) and CD221 (insulin-like growth factor-I can lead to concentrations of 63 Ag/mL, corresponding to receptor) by flow cytometry. For primary multiple myelo- 233 Amol/L (25). In the treatment of the BK virus or cyto- ma cells, only the surface expression of CD49d and CD126 megalovirus, higher doses are needed and a plasma level of was analyzed. We found that A771726 inhibits expression 50 to 100 Ag/mL, corresponding to 150 to 300 Amol/L, of CD126 and CD49d (Fig. 5B) in multiple myeloma cell should be achieved. Even this dosage (usually 40 mg/d) is lines as well as in primary multiple myeloma cells. The well tolerated, as described in a previously published expression of CD11a, CD54, and CD221 was not changed report (16). (data not shown). Our study shows that the clinically available DHODH Because proliferating lymphocytes need increased inhibitor A771726 also has strong anti-myeloma effects. amounts of pyrimidines and proliferation of multiple Up to 95% inhibition of multiple myeloma cell growth myeloma cells is induced by conditioned HS-5 medium was observed at clinically achievable drug concentrations. as well as cytokines such as IL-6 (23), we asked whether Apoptosis was induced in multiple myeloma cell lines as HS-5 medium or IL-6 increases the expression of DHODH well as in primary multiple myeloma cells obtained from in myeloma cells. We therefore incubated NCI-H929 three patients. A771726 also strongly inhibited multiple myeloma cells with medium obtained from a 48-h-old myeloma cell proliferation at lower concentrations. We culture of HS-5 stromal cell or 15 ng/mL recombinant observed a G1 cell cycle arrest with corresponding changes human IL-6 for 24 or 48 h and analyzed the expression of in the expression of cell cycle regulatory proteins. These DHODH using Western blotting. Figure 5C clearly shows results stand in line with results from others, who have that stimulated multiple myeloma cells express increased seen an induction of a G1 arrest in mitogen-activated T amounts of DHODH. lymphocytes (26) and B lymphocytes from patients with A771726 Synergizes with Common and New Anti- chronic lymphocytic leukemia (27). Cyclin D proteins are Myeloma Agents expressed at low levels in quiescent cells. In response to Drug combinations often lead to synergistic effects and growth factors, they are up-regulated in proliferating cells reduce side effects during cytostatic treatment. Therefore, (28). Cyclin D is dysregulated in virtually all multiple we next asked whether the incubation with A771726 might myeloma tumors. Whereas IgH translocations directly led synergize with commonly used anti-myeloma agents and to a modulation of cyclin D1 and cyclin D3 expression, preincubated OPM-2, NCI-H929, and U266 myeloma cells most other multiple myeloma tumors, which show differ- with 50 or 100 Amol/L A771726 for 1 h. After the ent chromosomal aberrations, show overexpression of preincubation period, either 300 or 600 nmol/L doxorubi- cyclin D2. Fifty-four percent of all multiple myeloma cin, 5 or 10 Amol/L melphalan, 0.5 or 5 Amol/L dexa- tumors overexpress cyclin D1, 48% overexpress cyclin D2, methasone, 2 or 4 nmol/L bortezomib, or 30 or 60 Amol/L 3% overexpress cyclin D3, and 8% overexpress both cyclin treosulfan was added to the culture. A sample of pri- D1 and cyclin D2 (29). Therefore, the finding that A771726 mary multiple myeloma cells was incubated with 50 and decreases cyclin D2 expression is important and suggests 100 Amol/L A771726 and with or without 5 or 10 Amol/L an additional mechanism of action for A771726. melphalan. After the incubation period of 48 h, cell growth We suspected inhibition of changes in cellular signal- was determined by the WST-1 assay. Figure 6 shows that ing on incubation with higher concentrations of A771726 A771726 reduces cell growth by f50% at the used and found that 200 Amol/L A771726 strongly inhibited the concentrations. Maximum increase of cell growth inhibition Akt pathway. This pathway confers to cell survival and

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Figure 5. A771726 inhibits cytokine-induced increase of multiple myeloma cell growth. A, NCI-H929, RPMI-8226, and U266 myeloma cells were incubated with or without 25% of conditioned medium obtained from a 48-h-old HS-5 stromal cell culture for 72 h. Additionally, cell growth was inhibited by adding50 or 100 Amol/L A771726 to the culture. Cell growth was determined by using the WST-1 assay. Results from two independent experiments. B, NCI-H929, OPM-2, U266, and primary myeloma cells were incubated with 200 Amol/L A771726 for 48 h. Cells were then harvested and washed. After that, cells were stained with monoclonal FITC- or PE-conjugated antibodies raised against CD49d or CD126, which was followed by flow cytometry analysis. C, NCI-H929 myeloma cells were incubated with or without 25% conditioned medium obtained from a 48-hour-old culture of HS-5 stromal cells or with or without 15 ng/mL recombinant human IL-6 for 24 and 48 h. After the incubation period, cells were lysed and directly subjected to SDS-PAGE, transferred to membranes, and blotted with antibodies raised against actin or DHODH. In all figures, the used cell line is indicated in the control experiment. proliferation and therefore is crucial in multiple myeloma. ments revealed that changes in cellular signaling induced These results are consistent with the findings from others by A771726 are only partly reversed by the coincubation (30), who have shown A771726-induced Akt inhibition in with uridine. This shows that effects mediated by A771726 mast cells. In addition, we saw changes in the regulation of are not exclusively due to inhibition of DHODH. Addi- bcl-2 family proteins. Proapoptotic bax and bim-EL were tional mechanisms such as inhibition of the Akt path- up-regulated, whereas antiapoptotic bcl-xL was down- way and modulation of proapoptotic and antiapoptotic regulated. These findings suggest that apoptosis induced proteins contribute to the effect seen in multiple myeloma by A771726 is induced through inhibition of the Akt cells treated with A771726 and seem to be independent pathway and deregulation of bcl-2 family members. of DHODH. The apoptotic effect of lower concentration of A771726 on Primary multiple myeloma cells are mostly localized in multiple myeloma cells was partly reversed by 100 Amol/L the bone marrow and are embedded in the bone marrow uridine, the final product of the pyrimidine synthesis chain. microenvironment. Integrins such as VLA-4 and LFA-1 Apoptosis induced by higher concentrations of A771726 provide cellular adhesion of multiple myeloma cells to was not overcome by uridine. Western blotting experi- bone marrow stromal cells. This has been shown to lead to

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Figure 6. A771726 exerts synergistic activity on multiple myeloma cells when it is combined with common and new anti- myeloma agents. NCI-H929, OPM-2, and U266 myeloma cells were preincubated with/without 50 or 100 Amol/L A771726 for 1 h. After the incubation period, 300 nmol/L doxorubicin, 5 Amol/L melphalan, 2 nmol/L bortezomib, 30 Amol/L treosulfan, or 5 Amol/L dexamethasone was added to the culture and cells were given to 96-well plates. Additionally, primary multiple myeloma cells were preincubated with 50 or 100 Amol/L A771726 and 5 or 10 Amol/L melphalan was added after 1 h. After the incubation period for 48 h, cell growth was determined by the WST-1 assay. Results from two independent experiments. *, P < 0.05 versus control.

increased survival in multiple myeloma cells (31–33). signal-regulated kinase, and Akt/mammalian target of Furthermore, paracrine and autocrine secretion of cyto- rapamycin/p70S6K. kines and growth factors is provided by the bone marrow We therefore asked whether A771726 may diminish the milieu, which results again in the stimulation of cell survival signals of soluble factors and incubated multiple growth, proliferation, and survival. IL-6 and insulin-like myeloma cells with conditioned medium obtained from HS-5 growth factor-I are the major soluble factors in multiple stromal cells with and without A771726. We found that, myeloma that lead to phosphorylation and activation of the besides inhibition of basal cell growth, incubation with signaling pathways JAK/STAT, Raf/MEK/extracellular A771726 inhibited the stimulatory effect of HS-5 medium. To clarify this, we asked whether A771726 modulates the expression of integrins and growth factor/cytokine receptor and Table 1. A771726 exerts synergistic activity against myeloma cells when combined with common and new anti-myeloma found that incubation with A771726 decreased expression of agents VLA-4 and IL-6 receptor on the multiple myeloma cell surface. There are several reports suggesting that a high pyrim- A771726 Doxorubicin FA CI idine pool is necessary for proliferating lymphocytes (11). 50 0.3 0.240923 0.963 To increase the pool of pyrimidines, lymphocytes require 100 0.6 0.152991 1.297 both the nucleoside uptake transporters and the de novo A771726 Melphalan FA CI synthesis of pyrimidines (34). We therefore asked whether 50 5 0.267689 0.996 the expression of DHODH is modulated after stimulation 100 10 0.118814 0.795 A771726 Dexamethasone FA CI with conditioned stromal cell medium or recombinant 50 0.5 0.419247 1.068 human IL-6 and found that DHODH is increasingly 100 5 0.30704 1.585 expressed on cytokine stimulation. A771726 Bortezomib FA CI Additionally we asked whether A771726 acts synergisti- 50 0.002 0.341808 0.593 cally with melphalan, doxorubicin, dexamethasone, or 100 0.004 0.173886 0.284 bortezomib. Because treosulfan, which is becoming increas- A771726 Treosulfan FA CI ingly in use in multiple myeloma therapy, has shown to be 50 30 0.280906 0.737 active in myeloma (35, 36), we also tested this promising 100 60 0.156365 0.586 compound in combination. In our experiments, additive (melphalan and doxorubicin) and synergistic (bortezomib NOTE: Data obtained from the experiments shown in Fig. 6 were analyzed using the Calcusyn software to show additive/synergistic effects. In and treosulfan) effects were seen when A771726 was this experiment, NCI-H929 cells were preincubated with/without 50 or combined with established myeloma drugs. These results A 100 mol/L A771726 for 1 h. After the incubation period, 300 nmol/L suggest potential clinical activity of combination therapies. doxorubicin, 5 Amol/L melphalan, 2 nmol/L bortezomib, 30 Amol/L treosulfan, or 5 Amol/L dexamethasone was added to the culture and cells In summary, we show that the DHODH inhibitor A771726/ were given to 96-well plates. After the incubation period for 48 h, cell leflunomide is strongly active in myeloma cells. Therefore, growth was determined by the WST-1 assay. Values obtained from the NCI- H929 cell line were analyzed. CI = 1.1 to 0.9 indicates additive effects, our results provide the framework for clinical studies in whereas CI < 0.9 indicates synergism. myeloma patients to improve myeloma patients’ prognosis.

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Mol Cancer Ther 2009;8(2). February 2009

Dihydroorotate dehydrogenase inhibitor A771726 (leflunomide) induces apoptosis and diminishes proliferation of multiple myeloma cells

Philipp Baumann, Sonja Mandl-Weber, Andreas Völkl, et al.

Mol Cancer Ther 2009;8:366-375. Published OnlineFirst January 27, 2009.

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