Shiraishi, Norihiko

Abstract BAFF is a member of the tumor necrosis factor (TNF) family and is critical for the maintenance and homeostasis of normal B-cell development. Importantly, BAFF promotes the generation of rapidly dividing immunoglobulin secreting plasmablasts from activated memory B cells by enhancing their survival. Given that MM is a cancer of plasma cells and that the signaling cascades implicated in receptor ligand interactions of BAFF are crucial in MM cell biology, we hypothesized that this cytokine may play a critical role in MM cell development, survival, and proliferation. We performed gene expression profiling (GEP) on CD 138+ plasma cells isolated from 90 MM patients (45 newly diagnosed and 45 relapsed) and 11 healthy controls using the Affymetrix U133A arrays. Our data demonstrates increased expression of transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI) and B-cell maturation antigen (BCMA), 2 receptors used by BAFF to exert its effects. Our data also shows an increased expression of a proliferation-inducing ligand (APRIL), another member of the TNF family with homology to BAFF. Expression levels of BAFF and BAFF-R could not be determined because of lack of these probe sets on the Affymetrix U133A arrays. GEP analysis shows increased BCMA expression (p<0.0001, student T test) on newly diagnosed and relapsed MM versus normal plasma cells. Flow cytometry on MM cell lines demonstrated a differential expression of the three receptors of BAFF, with BCMA present on most cell lines but BAFF-R expressed at low levels only on LR5 cells and DOX40 MM cells. In contrast, flow cytometry performed on MM patient cells demonstrated the presence of all 3 receptors on CD 138+ cells. ELISA assays performed on 30 MM sera demonstrated a mean BAFF level of 618 pg/ml (range: 128–2126pg/ml) versus 235pg/ml (range: 158–326pg/ml) in 7 normal donor sera. Fifty six% (17/30) of MM patients had BAFF levels in excess of the highest value noted in normals. To understand the role BAFF might play in the biology of MM, we studied the effects of recombinant BAFF (rh-BAFF) on MM cells directly and in the context of its bone marrow microenvironment. (abstract # 554746) rh-BAFF conferred a survival advantage to MM cells and protected them against dexamethasone-induced cytotoxicity. Importantly, anti-apoptotic proteins Bcl2 and Mcl-1 were upregulated, as were growth and survival signals belonging to the JAK/STAT and MAPKinase pathways. Conversely, neutralizing antibody to BAFF blocked, at least in part, blocked the upregulation of anti-apoptotic proteins with associated growth and survival, confirming that these effects were due to BAFF. Importantly, all of these signals were downregulated even in the presence of bone marrow stromal cells (BMSCs). These data therefore show a role for BAFF mediating MM cell survival and provide the framework for inhibiting BAFF, either alone or in combination with dexamethasone, to improve patient outcome in MM.

Abstract Ribonucleotide reductase is the rate-limiting enzyme of deoxynucleoside triphosphate synthesis and is therefore an excellent target for cancer chemotherapy. Inhibition of ribonucleotide reductase results in inhibition of DNA synthesis and has anti-neoplastic effects. Several ribonucleotide reductase inhibitors (RRIs) are already in clinical practice, including Hydroxyurea and Gemcitabine. Here we examine the anti-MM activity of a novel RRI, Didox. Our data shows that Didox has potent cytotoxicity against MM cells in 48-hour cultures. Apoptosis induced in MM cell lines by Didox (200mmol/l), evidenced by increased sub-G1 cell fraction, is caspase dependent, confirmed by blocking experiments with Z-VAD-FMK. Unlike other RRIs which mainly target the pyrimidine metabolism pathway (hydroxyurea and gemcitabine), didox targets both the purine and pyrimidine metabolism pathways in MM, as demonstrated by transcriptional profiling using the Affymetrix U133A 2.0 gene chip. Specifically, our data shows a ≥ 2 fold downregulation of genes in these pathways as early as 12 hours after exposure to Didox. Importantly, ribonucleotide reductase (RR) M1 component transcript was downregulated, associated with downregulation of this enzyme by Western Blot analysis, and inhibition of DNA synthesis. Furthermore, genes involved in DNA repair mechanisms were also downregulated after exposure to Didox, demonstrated by heirachial clustering of gene chip data and further validation by western blotting. Specifically RAD 51 homologue, which is involved in nucleotide excision repair and recombination repair, was downregulated in MM cells both transcriptionally as well as at the protein level. This was accompanied by downregulation of the BCL family protein including Bcl-2, Bclxl, and XIAP both at the transcriptional and post transcriptional levels. Since Didox acts on MM cells by inhibiting DNA synthesis and repair, combination studies with melphalan, an alkylating agent commonly used in MM, were next performed. Our data shows a strong in vitro synergism, with combination indices of <0.7 by the Chou Talahay method. When combined with DNA damaging agents like doxorubicin, additive cytotoxicity was noted. These studies therefore provide the preclinical rationale for evaluation of Didox, alone and in combination with alkylating agents and anthracyclines, to improve patient outcome in MM.

Abstract The importance of bone marrow angiogenesis in multiple myeloma (MM) provided the initial rationale for using thalidomide in this disease, given its anti-angiogenic properties. Thalidomide (α-N-[phthalimido] glutarimide, C 13 H 10 N 2 O 4), a glutamic acid derivative, has proved useful in the treatment of both newly diagnosed and relapsed/refractory MM, however, its potential for teratogenicity lays a huge burden on its use as a therapeutic agent. Ongoing efforts have been directed towards development of analogues, which retain anti-MM activity without teratogenic effects. A product of cytochrome P450 2C19 isozyme biotransformation of thalidomide, 5′-OH-thalidomide, has been shown to be partly responsible for the anti-angiogenic effect of thalidomide. Based on the structure of this metabolite several N-substituted and tetraflourinated thalidomide analogues have been synthesized as potential anti-angiogenic agents and have shown pre-clinical activity in prostate cancer. In this study, we demonstrate potent anti-MM activity and delineate potential mechanisms of two such compounds, CPS11 (an N-substituted thalidomide) and CPS49 (a tetraflourinated thalidomide). Both CPS11 and CPS49 showed inhibition of proliferation of several MM cell lines as well as primary tumor cells from relapsed/refractory MM patients. These drugs had significant activity in myeloma cell lines resistant to conventional chemotherapy agents (dexamethasone, adriamycin, mitoxantrone and melphalan). Both the drugs were able to overcome the protective effects of growth factors like IL-6, IGF-1, and VEGF, and co-culture with bone marrow stromal cells (BMSCs), demonstrating their activity against myeloma cells in conditions simulating the natural microenvironment. In most of the cytotoxicity experiments, CPS49 was more potent compared to CPS11. An additive cytotoxic effect was noted when these agents were combined with dexamethasone and melphalan. Apoptosis was noted in MM1.S cells as evidenced by annexin and PI staining and a time- and dose-dependent increase in cleavage of poly ADP-ribosepolymerase (PARP) and caspase-8. Additionally, Z-VAD-FMK partially blocked these effects. Importantly, apoptosis triggered by these drugs was associated with down-regulation of anti-apoptotic proteins like Mcl1 and XIAP. In matrigel based angiogenesis assays both drugs demonstrated anti-angiogenic activity, CPS49 being more potent than CPS11. To further delineate their molecular mechanism of actions, we evaluated the effect of these drugs on various intracellular signaling pathways known to be important in myeloma. Both the drugs were able inhibit the PI3K/Akt and JAK/STAT pathways in MM1.S myeloma cells line. These encouraging in vitro data will provide the platform for testing these analogues initially in animal models and subsequently in the clinic.

AbstractBone marrow angiogenesis plays an important role in the pathogenesis and progression in multiple myeloma. Recent studies have shown that proteasome inhibitor bortezomib (Velcade, formerly PS-341) can overcome conventional drug resistance in vitro and in vivo; however, its antiangiogenic activity in the bone marrow milieu has not yet been defined.In the present study, we examined the effects of bortezomib on the angiogenic phenotype of multiple myeloma patient–derived endothelial cells (MMEC). At clinically achievable concentrations, bortezomib inhibited the proliferation of MMECs and human umbilical vein endothelial cells in a dose-dependent and time-dependent manner. In functional assays of angiogenesis, including chemotaxis, adhesion to fibronectin, capillary formation on Matrigel, and chick embryo chorioallantoic membrane assay, bortezomib induced a dose-dependent inhibition of angiogenesis. Importantly, binding of MM.1S cells to MMECs triggered multiple myeloma cell proliferation, which was also abrogated by bortezomib in a dose-dependent fashion. Bortezomib triggered a dose-dependent inhibition of vascular endothelial growth factor (VEGF) and interleukin-6 (IL-6) secretion by the MMECs, and reverse transcriptase-PCR confirmed drug-related down-regulation of VEGF, IL-6, insulin-like growth factor-I, Angiopoietin 1 (Ang1), and Ang2 transcription. These data, therefore, delineate the mechanisms of the antiangiogenic effects of bortezomib on multiple myeloma cells in the bone marrow milieu. (Cancer Res 2006; 66(1): 184-91)

AbstractCyclin-dependent kinase (CDK) inhibitors have the potential to induce cell-cycle arrest and apoptosis in cancer cells. Seliciclib (CYC202 or R-roscovitine) is a potent CDK inhibitor currently undergoing phase-2 clinical testing in lung and B-cell malignancies. Here we studied the in vitro cytotoxic activity of seliciclib against multiple myeloma (MM) cells. Our data demonstrate that seliciclib has potent cytotoxicity against MM cells that are both sensitive and resistant to conventional therapy as well as primary MM cells from patients. Cell-cycle and Western blot analysis confirmed apoptosis. Importantly, seliciclib triggered a rapid down-regulation of Mcl-1 transcription and protein expression independent of caspase cleavage. Adherence of MM cells to bone marrow stromal cells (BMSCs) induced increased Mcl-1 expression associated with signal transducer and activator of transcription 3 (STAT3) phosphorylation, which was inhibited in a time- and dose-dependent manner by seliciclib. Furthermore, seliciclib inhibited interleukin 6 (IL-6) transcription and secretion triggered by tumor cell binding to BMSCs. Up-regulation of Mcl-1 expression in cocultures was only partially blocked by neutralizing antibody to IL-6, suggesting alternative mechanisms of Mcl-1 modulation by seliciclib. Finally, combination studies of seliciclib with doxorubicin and bortezomib show in vitro synergism, providing the rationale for testing these drug combinations to improve patient outcome in MM.