B029 Dihydroorotate Dehydrogenase (DHODH) is a Promising Novel Target in the Treatment of Multiple Myeloma

STAT3, suggesting that CS1 induces central growth and survival signaling pathways in MM cells. Serum deprivation markedly blocked survival at earlier time points in CS1 knockdown compared with control MM cells, associated with earlier activation of caspases, PARP, and proapoptotic proteins BNIP3 and BIK. CS1 knockdown further delayed development of OPM2 tumor and prolonged survival in mice. Concomitantly, we overexpressed CS1 in CS1-low expressing U266 cells by transfecting an expressing plasmid pFLAG-CS1 or control vector. Enforced CS1 expression enhanced U266 cell growth and survival. In contrast to the majority of U266 cells that grow in suspension in standard tissue culture flasks, all U266 CS1 cells exhibited adherent growth and homotypic adhesion. Importantly, overexpressed CS1 increased adhesion of U266 and MM1S cells to BMSCs. Furthermore, U266 CS1 cells formed more and larger colonies in methylcellulose than U266 cells. Tumors that developed in mice injected with U266 cells expressed significantly higher levels of CS1 than injected U266 cells. Exercised tumors grew in an adherent manner in vitro. We next performed gene expression profiling to define overlapping differentially expressed genes in U266CS1 versus U266 and CS1null OPM2 versus cntOPM2. Significantly, c-Maf pathway was upregulated in U266CS1 versus U266 cells and downregulated in CS1null OPM2 versus cntOPM2 cells, as evidenced by differentially expressed c-Maf and its target genes, ie, cyclin D2, integrin b7 at both mRNA and protein levels. Myeloma cell adhesion–induced VEGF secretion by BMSCs was greater with U266 CS1 than U266 cells (P < .03). Moreover, c-Maf, integrin b7, and cyclin D2 was augmented in CS1-overexpressing tumors developed in mice receiving U266 cells. These studies provide direct evidence of the role of CS1 in myeloma pathogenesis, define molecular mechanisms regulating its effects, and further support novel therapies targeting CS1 in MM.

.05). The Annexin-V assay revealed statistically significant induction of apoptosis of MM cell lines and primary MM cells (NCI-H929: 74%; OPM-2: 70%; RPMI-8226: 49%; IM-9: 63%; and U266: 26%; primary MM: 68%; P < .05). The BrdU assay showed that inhibition of cell growth was partly due to inhibition of MM cell proliferation (NCI-H929: -92%; OPM-2: -94%; and U266: -86%; P < .05). A771726 induced G1 cell-cycle arrest via modulation of cyclin D2 and pRB expression. A771726 decreased phosphorylation of Akt, P70S6k, and 4E-BP-1 and led to an inhibition of Bcl-XL and activation of Bax and Bim-EL. Furthermore, we show that the stimulatory effect of conditioned medium of HS-5 bone marrow stromal cells on MM cell growth is completely abrogated by A771726. In addition, synergism studies (CalcuSyn) revealed synergistic and additive activity of A771726 together with the established anti-MM agents melphalan, doxorubicin, treosulfan, dexamethasone, and bortezomib. Conclusion: With these data taken together, we show that inhibition of DHODH by A771726/leflunomide is effective in MM. Considering the favorable toxicity profile and the great clinical experience with leflunomide in rheumatoid arthritis, this drug represents a potential new candidate for targeted therapy in MM.

B033 Aurora-Kinase Inhibition for Tailored RiskAdapted Treatment of Multiple Myeloma D Hose,1 T Rème,2 T Meißner,3 J Moreaux,2 A Seckinger,3 J Lewis,4 V Benes,4 A Benner,5 M Hundemer,3 T Hielscher,5 JD Shaughnessy Jr.,6 B Barlogie,6 K Neben,3 A Krämer,3 J Hillengass,3 U Bertsch,3 A Jauch,7 J De Vos,2 JF Rossi,2 T Möhler,3 J Blake,4 J Zimmermann,4 B Klein,2 H Goldschmidt3 1

Universitätsklinikum Heidelberg; 2CHU Montpellier and INSERM

B029

U847; 3Medizinische Klinik V, Universitätsklinikum Heidelberg;

Dihydroorotate Dehydrogenase (DHODH) is a Promising Novel Target in the Treatment of Multiple Myeloma

4

European Molecular Biology Laboratory, Heidelberg; 5Abteilung

für Biostatistik, Deutsches Krebsforschungszentrum; 6Myeloma Institute for Research and Therapy, University of Arkansas for

P Baumann, S Mandl-Weber, A Völkl, C Adam, I Bumeder, F Oduncu, R Schmidmaier

Heidelberg

Purpose: Multiple myeloma (MM) is an incurable disease; therefore, new therapeutic agents are needed. A771726 is the active metabolite of the immunosuppressive drug leflunomide, which is applied in the treatment of rheumatoid arthritis. Nothing is known about the role of dihydroorotate dehydrogenase (DHODH) in MM. Because proliferating lymphocytes expand their pyrimidine pools and therefore require DHODH, this enzyme represents an attractive target in MM. Methods: A771726 was characterized by several assays. Cell growth rates of MM cells were measured by the WST-1 proliferation assay, apoptosis by flow cytometric Annexin-V-FITC surface staining, and cell proliferation was determined using the BrdU assay. Expression and activity of signal transduction molecules was revealed by Western blotting. Results: We show that DHODH is expressed in both MM cell lines and primary cells. A771726 inhibited cell growth in different MM cell lines at clinically achievable concentrations in a time- and dose-dependent manner (NCI-H929: -95%; OPM-2: -97%; RPMI-8226: -96%; and U266: -91%; P <

Background: Genetic instability and cellular proliferation have been associated with Aurora-kinase expression in several cancer entities, including multiple myeloma. Patients and Methods: Expression of Aurora-A, -B and -C was assessed using Affymetrix DNA-microarrays in 784 samples including 2 independent sets of 233 and 345 CD138-purified myeloma-cells from previously untreated patients. Chromosomal aberrations were assessed by comprehensive iFISH and proliferation of primary myeloma-cells was determined by propidium iodine staining. The effect of the clinical Aurorakinase inhibitor VX680 on proliferation of 20 human myeloma cell lines and survival of 5 primary myeloma cell-samples was tested. Results: We found Aurora-A and -B to be expressed at varying frequencies in primary myeloma-cells of different patient cohorts. Given that Aurora-C is restricted to germ cells, expression was found in testis-samples only. Myeloma-cell-samples with detectable AuroraA expression show a significantly higher proliferation rate, whereas the number of chromosomal aberrations (aneuploidy) is not higher

Medical Sciences; 7Institut für Humangenetik, Universitätsklinikum

Clinical Lymphoma & Myeloma Supplement February 2009

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