Deregulated Cellular Iron Metabolism Factors Mediate Iron Overload in Myeloma Cells and Osteoclasts, and Promote Myeloma Growth and Bone Disease,

Abstract 3941

Iron overload is a significant clinical feature in multiple myeloma (MM) and has been implicated in osteoporosis. MM patients also frequently suffer from anemia presumably due to elevated hepcidin secretion and dysfunctional erythropoiesis. The aims of the study were to shed light on molecular mechanisms associated with iron overload in MM cells and study the effect of the novel iron chelator, Dp44mT, on MM cell growth, osteoclastogenesis and MM bone disease in vitro and in vivo. In our clinical global gene expression profiling (GEP) data the main iron transporter gene TFRC (transferrin receptor) was >3 folds higher (p<0.0001) in newly diagnosed MM cells (n=556) than normal plasma cells (n=25) while the iron exporter ferroportin was downregulated in MM cells by >4 folds (p<0.0001). Deregulated TFRC and ferroportin expression were more profound in the molecularly classified proliferation (PR) subtype. Osteoclasts which are known to have abundant mitochondria due to high energy consumption express excessive TFRC (>5 folds higher than highly proliferating MM cells). In primary MM cell-osteoclast cocultures (n=8) TFRC expression was upregulated in cocultured MM cells than baseline MM cells (p<0.03) while ferroportin was lower in cocultured osteoclasts than control osteoclasts (p<0.04). Our GEP, qRT-PCR and immunohistochemistry analyses revealed expression of hepcidin by osteoclasts but not MM cells. Hepcidin was not detected in conditioned media from osteoclasts cultured alone or cocultured with MM cells using ELISA, suggesting an autocrine role of hepcidin in maintaining excess iron in osteoclasts. In vitro, Dp44mT dose dependently inhibited growth of MM cell lines (n=3) at low nanomolar levels (IC50 at 3±0.8 nM, p<0.03, 48 hrs). In contrast, known chelators such as Deferoxamine and Deferasirox inhibited myeloma cell growth at 10–50 micromoles range. At 1nM Dp44mT also suppressed formation of multinucleated osteoclasts by 87% (p<0.001) and bone resorbing activity of mature osteoclasts on dentine slices by 94% (p<0.03). Dp44mT induced upregulation of BMP2 expression in osteoblast precursors and promoted osteoblast differentiation. In vivo, SCID-rab mice engrafted with luciferase-expressing U266 MM line (6 mice/group) or the Hg MM line (maintained through in vivo passaging, 10 mice/group) were subcutaneously treated with vehicle or Dp44mT (1 mg/kg/day) for 2–3 weeks. Using live-animal imaging, Dp44mT reduced growth of U266 cells by 3 folds from pretreatment levels (p<0.01) while in control group tumor burden was increased by 52 folds from pretreatment levels (p<0.002). Dp44mT also inhibited growth of Hg MM cells determined by measurement of circulating human immunoglobulins in mice sera (p<0.01). Osteoclasts numbers were lower by 36% (p<0.003) while osteoblasts numbers were higher by 59% (p<0.017) in myelomatous bones from hosts treated with Dp44mT than control vehicle. Our data suggest that interaction of myeloma cells with osteoclasts alters expression of distinct iron metabolism associated factors which elicit iron overload in both cell types, resulting in increased myeloma cell proliferation and osteoclast activity. This study also suggests that Dp44mT is an effective iron chelator with marked anti-MM activity.