Abstract
Multiple myeloma (MM) is characterized by a clonal proliferation of immunoglobulin-secreting plasma cells in the bone-marrow (BM) and remains an incurable disease, despite the use of high-dose chemotherapies. Since a marked (BM)-angiogenesis is the hallmark of MM, but not of monoclonal gammopathies of undetermined significance (MGUS), validation of novel agents targeting MM tumor cells and their permissive BM-stroma is crucial to improve patient outcome. Patients fulfilling the International Myeloma Working Group diagnostic criteria for MM (n = 21) and MGUS (n = 14) were studied. Healthy donors or patients with benign anemia (due to vitamin B12 deficiency) were also incuded as controls. In plasma cells and endothelial cells (ECs) isolated from BM-aspirates by anti-CD138 and Ulex Europaeus agglutinin-1 (UEA-1) coated-beads, we dissected the contribution of activity against individual targets such as platelet-derived growth factor (PDGF)-receptor beta (PDGF-Rb) and c-Src tyrosine kinases (TKs), to the anti-tumor/vessel efficacy of dasatinib (BMS-354825), a novel orally bioavailable TK inhibitor. The PDGF-BB/PDGF-Rb kinase-axis was found constitutively activated in plasma cells from patients with MM but not with MGUS or benign anemias, thus supporting its pathophysiological role in MM. PDGF-Rb activated, independently of vascular endothelial growth factor (VEGF)-receptors (VEGF-R1 and VEGF-R2), the mitogen-activated protein kinases (ERK1/2) and the phosphatidylinositol 3-kinase (PI3-K)/AKT-dependent cascade, thereby increasing MM plasma cell growth. Expression of PDGF-Rb, at both mRNA and protein levels, was also increased in MMECs compared to MGECs, correlating with AKT phosphorylation. Exposure to recombinant PDGF-BB or conditioned media from MM plasma cells triggered PDGF-Rb phosphorylation and MMEC migration and spontaneous sprouting in vitro (both being mandatory for angiogenesis). Dasatinib abrogated PDGF-elicited tumor/vessel growth and impaired VEGF-signaling via c-Src TK-inhibition (IC50=25–100nM) in both MM-patient tumor and ECs. The use of small-interfering (si)-RNAs validated c-Src as a key VEGF-downstream effector of MMEC proliferation, migration and capillarogenesis in vitro. Nevertheless, the inhibitory effect elicited by siSrc was partially rescued by recombinant PDGF-BB which sustained the expression of pro-angiogenic factors such as VEGF, interleukin (IL)-8, basic fibroblast growth factor (bFGF), and hepatocyte growth factor (HGF) in MMECs. Dasatinib reversed all these transcriptional effects, thereby abrogating MMEC angiogenesis in the CAM assay as well as the neovascularization and tumor growth of MM-xenografts in vivo. More importantly, low-dose dasatinib showed synergistic cytotoxicity in vitro when tested in combination with conventional MM drugs (i.e. bortezomib and thalidomide), thereby increasing therapeutic efficacy and overcoming drug resistance. These findings indicate that:
the PDGF-BB/PDGF-Rb kinase-axis elicits direct effects on MM plasma cells and could promote the MM “angiogenic switch”, hence disease progression; the inhibition of this pathway could provide the rationale for clinical trials with dasatinib which interferes with shared growth-signaling cascades in MM-patient isolated plasma cells and ECs, involving PDGF-Rb and cytosolic c-Src TKs.
Identification of a liver growth factor as an albumin-bilirubin complex
