ALBERT

Description: Gene expression profiling (GEP) of purified plasma cells 48 hours after thalidomide and dexamethasone test doses showed these agents' mechanisms of action and provided prognostic information for untreated myeloma patients on Total Therapy 2 (TT2). Bortezomib was added in Total Therapy 3 (TT3), and 48 hours after bortezomib GEP analysis identified 80 highly survival-discriminatory genes in a training set of 142 TT3A patients that were validated in 128 patients receiving TT3B. The 80-gene GEP model (GEP80) also distinguished outcomes when applied at baseline in both TT3 and TT2 protocols. In context of our validated 70-gene model (GEP70), the GEP80 model identified 9% of patients with a grave prognosis among those with GEP70-defined low-risk disease and 41% of patients with favorable prognosis among those with GEP70-defined high-risk disease. PMSD4 was 1 of 3 genes common to both models. Residing on chromosome 1q21, PSMD4 expression is highly sensitive to copy number. Both higher PSMD4 expression levels and higher 1q21 copy numbers affected clinical outcome adversely. GEP80 baseline-defined high risk, high lactate dehydrogenase, and low albumin were the only independent adverse variables surviving multivariate survival model. We are investigating whether second-generation proteasome inhibitors (eg, carfilzomib) can overcome resistance associated with high PSMD4 levels.

Description: Abstract 808 Background: Bone disease is one of the most debilitating complications in patients with multiple myeloma (MM). The molecular mechanisms by which MM triggers bone disease are not fully understood. We have previously demonstrated that Dkk1 is highly expressed in primary MM plasma cells, and associated with bone disease in MM patients by inhibiting Wnt signaling-promoted mesenchymal stem cell differentiation and osteoprotegerin production in osteoblast cells. We have also reported that increase in Wnt signaling in the bone marrow microenvironment by overexpression of Wnt3a in myeloma cells or administration of rWnt3a, or indirectly increasing Wnt signaling by administration of anti-Dkk1 neutralizing antibody also decreased in osteoclast numbers. However, Dkk1 is less frequently expressed in MM cell lines that are derived mostly from late stage of MM; and injection of these MM cell lines into human fetal bone also is able to induce bone lesion in MM animal model. These results indicate that additional factors may be involved in induction of the bone disease at the stage of the disease. The members of the sFRPs family of secreted proteins (including sFRP-1, -2, -3 and -4) directly bind to Wnts, thereby preventing Wnts from binding to the cellular Wnt receptor complex. It has also been reported that sFRP-1 and -2 augment canonical Wnt3a activated signaling in fibroblast. MM cells from pateints with advanced bone lesions express sFRP2 mRNA. Like sFRP2, sFRP3 mRNA is highly expressed in MM plasma cells, but it's function in MM bone disease remains unknown. We sought to investigate the role of sFRP3 in MM-triggered bone lesions using the osteoblast (OB) cell lines CH3T1/2 and C2C12, and serum from MM pateints those MM cells expressed high level of sFRP3. Methods/Results: RT-PCR analysis showed that sFRP3 is expressed in primary MM plasma cells and certain MM cell lines. Recombinant sFRP3 protein did not inhibit, but synergized with Wnt3a to increase beta-catenin protein, while Dkk1 significantly inhibited this process. Similarly, sFRP3 treatment of OB cells increase Wnt-3a-induced TCF transcript activity in OB cells transfected with TOPflash luciferase report constructs. sFRP3 also increased MSC differentiation, as evidenced by increase in alkaline phosphatase activity (ALP) and increased in mineralization by Alizarin red staining. sFRP3 treatment also increases OPG mRNA and protein production in these cells. Similar to sFRP3, sFRP1 and sFRP2 synergistically acted with Wnt3a to induce MSC differentiation and OPG expression in osteoblasts, while Dkk1 significantly inhibited these processes. To confirm the synergistic effects of sFRPs with canonical Wnt signaling on MSC differentiation, we employed R-podin1, a well-known agonist of canonical Wnt signaling. Treatments of MSC cells with R-podin1 led to increase in beta-catenin protein and TCF transcriptional activity and in ALP activity, and increase in OPG mRNA and protein. Pretreatment of the cells with sFRP2 and sFPP3 proteins further enhanced the function of R-podin1. In contrast, Dkk1 protein showed negative effect on R-Spodin1 functions, indicating that sFRP2 and sFRP3 synergized with R-Spodin1 to induce activation of canonical Wnt signaling and subsequent MSC differentiation and OPG production. Conclusion: Taken together, these data suggest that sFRP2 and sFRP3 augment canonical Wnt signaling to induce MSC differentiation and indirectly inhibit osteoclastogenesis by regulating OPG in MSC cells. These results also indicate that Dkk1 may be most important in MM-induced bone disease. Disclosures: Barlogie: Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties. Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.

Description: Abstract 688 Hepcidin, a liver-expressed peptide hormone, plays a central role in iron homeostasis by regulating the level of ferroportin. Ferroportin, the only known mammalian cellular iron exporter, is found on enterocytes, hepatocytes, and macrophages and allows the flux of iron from these cells into plasma. Upon binding, hepcidin induces the internalization and degradation of ferroportin, thereby modulating the absorption of iron from the diet and release of iron from cellular stores. Inappropriate hepcidin levels can therefore lead to dysregulation of iron homeostasis. In particular, inappropriately elevated hepcidin levels can limit iron needed for heme production, resulting in anemia. Elevated hepcidin is thought to play an important role in anemia of chronic disease and anemia of chronic kidney disease. RNA interference (RNAi) is a fundamental cellular mechanism for silencing genes and other transcribed elements, including those currently “undruggable” by small molecule and antibody therapeutics. RNAi has already transformed biological research by serving as a powerful tool for studying gene function and is now poised to form the basis of a new class of therapeutics. Small interfering RNAs (siRNAs) were developed against HAMP, the hepcidin gene, as well as other targets involved in iron metabolism (including HJV, TFR2, HFE, Neogenin, BMP6, BMPRI, BMPRII, SMAD4, and IL6R), both as a means to identify a therapeutic for the treatment of anemia and as a means to further characterize the hepcidin signaling pathway. Hepcidin was effectively silenced in mice, rats, and nonhuman primates, resulting in concomitant dose-dependent increases in serum iron, with approximately 100% transferrin saturation achievable after a single administration. Treatment with HAMP-targeting siRNA was able to blunt hepcidin induction, hypoferremia, and the onset of anemia in a turpentine-induced mouse model of inflammatory anemia. Silencing other members of the hepcidin pathway resulted in decreases in HAMP expression and increases in transferrin saturation to varying degrees. In particular, TFR2 was found to be an especially attractive target, leading to potent downregulation of HAMP, and rapid and durable increases in transferrin saturation. A single 0.1 mg/kg dose of a TFR2-targeting siRNA, resulted in ∼80% silencing of both TFR2 and HAMP and 100% transferrin saturation within 24 hours post-administration in mice. HAMP silencing and elevated transferrin saturation persisted for over two weeks, with levels returning to baseline after 4 weeks. In addition, TFR2 targeting resulted in the resolution of anemia in rodent models of anemia of inflammation. These data indicate that siRNAs directed at HAMP, TFR2, and other members of the hepcidin pathway represent an attractive novel therapeutic approach for the treatment of anemia of chronic disease and anemia of chronic renal disease. Disclosures: Akinc: Alnylam Pharmaceuticals: Employment. Chan-Daniels:Alnylam Pharmaceuticals: Employment. Sehgal:Alnylam Pharmaceuticals: Employment. Foster:Alnylam Pharmaceuticals: Employment. Bettencourt:Alnylam Pharmaceuticals, Inc.: Employment. Hettinger:Alnylam Pharmaceuticals, Inc.: Employment. Racie:Alnylam Pharmaceuticals, Inc.: Employment. Aubin:Alnylam Pharmaceuticals: Employment. Kuchimanchi:Alnylam Pharmaceuticals: Employment. Epstein-Barash:Alnylam Pharmaceuticals: Employment. Nakayama:Alnylam Pharmaceuticals: Employment.