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.

Description: Abstract 2918 Natural killer cells (NK) have the unique ability to kill target cells without priming. While their therapeutic potential against various malignancies is becoming more apparent, it has been restricted to the allogeneic setting; NK cells are inhibited by autologous targets by engaging killer immunoglobulin-like receptors with their ligands. Another major challenge to the clinical utility of NK cells is obtaining a sufficient number of NK cells for infusion. Co-culture of blood mononuclear cells (PBMNC) with the leukemic cell line K562, genetically modified to express membrane-bound IL15 and the co-stimulatory molecule 41BBL (K562mbIL15-41BBL) in the presence of IL2 results in robust expansion and activation of NK cells. To determine if NK cells derived from myeloma (MM) patients can be used therapeutically in the autologous setting, we explored the expansion of NK cells from MM patients, their gene expression profiles (GEP), and their ability to kill autologous and allogeneic MM cells from high-risk patients in vitro and in vivo, and compared these to NK cells from healthy donors (HD). PBMNC from MM patients (N=30) co-cultured with irradiated K562mbIL15-41BBL cells expanded a median of 351 fold (range20–10, 430), comparable to the expansion of HD-derived NK cells (N=15, median 803, range 127–1, 727; p=0.5). GEP of MM non-exp-NK differed from HD non-exp-NK in the expression of only one gene (PRKCi), underexpessed in MM (false discovery rate (FDR)

Description: Abstract 3329 Development of inhibitory antibodies that block factor VIII (FVIII) pro-coagulant function requires T-cell help. In order for T-cell stimulation to occur, one or more FVIII-derived peptides must first bind effectively to MHC Class II (e.g. HLA-DRB1) receptors on the surface of antigen presenting cells. If this Class II-peptide complex is recognized by a receptor on a helper T cell, then high-avidity Class II-peptide-T-cell receptor interactions can (but may not) cause proliferation and cytokine secretion leading to anti-FVIII antibody production. Thus the initial binding of a FVIII peptide to a Class II receptor can initiate an HLA-restricted cascade of events that leads to inhibitor formation in hemophilia A (HA) patients. Non-hemophilic, non-synonymous single nucleotide polymorphisms (ns-SNPs) in the human F8 gene were identified previously (Viel et al., Blood 109:3713–24, 2007) that result in the FVIII sequence variations R484H, R776G, D1241E and M2238V. Recombinant FVIII proteins with R484, R776, either D1241 or E1241, and M2238 are currently used therapeutically. The potential immunogenicity of FVIII peptides corresponding to these sequence variations is now being analyzed directly using in vitro assays employing cellular fractions of HA blood, recombinant proteins and synthetic peptides. These studies are motivated by a concern that some HA patients who express even very low levels of their endogenous, hemophilic FVIII, which should tend to promote immune tolerance to exogenous FVIII, could in principle develop inhibitors provoked solely by amino acid sequence mismatch(es) at sites in FVIII encoded by ns-SNPs. This could happen in patients with specific HLA types if their corresponding Class II receptors presented a FVIII peptide that then stimulated a helper T-cell response. Synthetic FVIII peptides containing both alternate sequences encoded by these 4 ns-SNPs were tested for binding to the monomeric extracellular domains of 10 common DRB1 alleles using competitive binding assays. The results are as follows: (1) 484: R484 peptides had strong affinity (IC50 〈 1 mM) for DRB1*15:01 and DRB1*03:01; H484 peptides had moderate affinity (IC50 = 1–10 mM) for DRB1*03:01 and DRB1*15:01; both R484 and H484 peptides bound with moderate affinity to DRB1*11:04 and DRB1*04:04; (2) 776: R776 and G776 peptides had moderate affinity for DRB1*09:01; (3) 1241: E1241 peptides had strong affinity for DRB1*09:01 and moderate affinity for DRB1*01:01, D1241 peptides bound weakly (IC50 = 30–50 mM) to DRB1*09:01 and DRB1*01:01; (4) 2238: Both M2238 and V2238 peptides had strong affinity for DRB1*03:01 and moderate affinity binding to DRB1*01:01, DRB1*11:04, DRB1*11:01 and DRB1*04:04. Results of the binding assays compared reasonably well with computer-predicted affinities of the peptides for some of the better-characterized HLA-DRB1 alleles. T-cell stimulation and MHC Class II tetramer staining experiments (which can unambiguously identify antigen-specific T cells) were next carried out using CD4 T cells from (1) 4 HA subjects (1 with a current inhibitor, 1 with a previous inhibitor and 2 without inhibitors) whose F8 gene encoded the V2238 variant and therefore had been treated with a therapeutic FVIII produce that was mismatched at this site with respect to the endogenous sequence encoded by their hemophilic F8 gene; and (2) from 4 HA subjects with the M2238 variant. All 8 patients expressed either the DRB1*03:01 or DRB1*11:01 alleles (one expressed both). No peptides corresponding to regions encoded by either version of this ns-SNP were recognized as T-cell epitopes by CD4 cells from these subjects, despite moderate to strong binding affinity of the peptides for DR0301 and DR1101 proteins. This suggests that the immunogenicity of these FVIII regions in HA patients who express these F8 and HLA genotype combinations may be less than might have been anticipated based on the binding data. HA patients with HLA-DRB1 receptors that do not effectively bind peptides encoded by ns-SNPs in the F8 gene would be expected to have a low risk of developing helper T-cell responses to FVIII sequences encoded by these ns-SNPs. Thus, the negative as well as positive peptide-binding assay results have significant clinical and pharmaceutical relevance. Results of assays such as those described above could be useful in developing improved methods to assess the relative risk of individual HA patients developing an inhibitor. Disclosures: Pratt: Bayer, CSL Behring, Pfizer: Research Funding; Puget Sound Blood Center: Patents & Royalties.

Description: Abstract 3739 Primary lymphomas of the breast are very rare (0.2–1.5% of breast malignancies) and the vast majority (95%) is of B-cell origin. Recently, a series of anaplastic large-cell kinase (ALK)-negative, T-cell, anaplastic, non-Hodgkin lymphomas (T-ALCL) have been reported worldwide in patients with saline and silicone breast implants (Lechner et al. Cancer 2010, Lazzeri D et al. Clin Breast Cancer 2011, Carty et al. Plast Reconstr Surg 2011, Popplewell et al. Leuk Lymphoma 2011, Roden et al. Mod Pathol 2008, de Jong et al. JAMA 2008, and others). These cases are striking for their homogeneous clinical presentation and pathology. Histological and cytogenetic analyses of the primary tumor biopsy specimens consistently demonstrates CD30+ EMA(+) PAX5− lymphoma cells with significant chromosomal atypia that lack the NPM-ALK (2;5) translocation, express pan T-cell markers, and show monoclonal TCR gene rearrangements. Increasing evidence suggests that these seroma-associated ALK− ALCLs are a distinct clinicopathologic entity from either systemic ALK− ALCL or cutaneous ALCL, and while the majority of cases are indolent, several patients have died from this disease. In order to understand the nature of this newly emerging clinical entity, we have established three new cell lines, (TLBR-1, TLBR-2, TLBR-3,) from the primary tumor biopsy specimens of three patients diagnosed with breast implant-associated ALCL. Characterization of these pre-clinical models confirmed fidelity to the original tumor biopsy specimens and highlighted unique features that may aid the development of effective treatments for these cancers. Cytogenetics and FISH performed on TLBR-1, -2, and -3 revealed nuclear atypia with partial or complete trisomy (modal chromosome number 47, 76, and 81, respectively) and absence of the NPM-ALK (2;5) translocation. Phenotypic characterization of TLBR cell lines by flow cytometry and immunocytochemistry showed strong positivity for CD30, CD71, pan T cell CD2/5/7, natural killer (CD56), and antigen presentation (HLA-DR, CD80, CD86) markers, and IL-2 receptors (CD25, CD122). Multiplex or consensus sequence PCR confirmed monoclonal TCR gene rearrangements and showed no onco-virus incorporation (EBV, HTLV1/2), respectively, for TLBR-1, -2, and -3. To identify effective therapies for patients with breast-implant associated ALCL, in vitro studies were used to evaluate the mechanisms by which TLBR evades immune detection and promotes cell cycle dysregulation. Gene expression analysis for proto-oncogenes, tumor suppressor genes, and regulators of apoptosis demonstrated significant up-regulation of survivin and down-regulation of pro-apoptotic genes (BID, BAK, BBC3) by all TLBR cell lines relative to healthy donor T cells. Interestingly, TLBR cell lines shared many features with human regulatory T cells, including phenotype (CD4+/8+, CD25+), high FoxP3 expression, up-regulation of immunosuppressive cytokines IL-10 and TGFβ, and suppression of T cell responses to stimuli. Inhibitor studies were used to identify critical signaling pathways in survival and proliferation of breast implant-associated ALCL models TLBR-1, -2, and -3. Data from these studies revealed that inhibition of the JAK/STAT, PI3K/mTOR, and WNT/beta-catenin signaling pathways resulted in a substantial increase in cell death in the TLBR cell lines. Significantly, TLBR-2, which was derived from a fatal breast implant associated ALCL, showed increased resistance to all inhibitors compared with TLBR-1 and TLBR-3, which were derived from more indolent cases. Analysis of protein lysates showed elevated cleaved Notch1 in TLBR-2, compared with cell lines TLBR-1 and -3, which may drive increased cell proliferation and resistance to apoptotic regulators. Since the treatment of these lymphomas has not been studied, we evaluated the sensitivity of the TLBR cell lines to mainstay chemotherapy regimens (CHOP, methotrexate). These studies found that doxorubicin and vinblastine exert potent cytotoxic effects against all three TLBR cell lines, whereas cyclophosphamide was largely ineffective. In summary, TLBR-1, -2, and -3 closely resemble the primary breast implant-associated lymphomas from which they were derived, and as such provide valuable preclinical models to study the cell of origin and biology of this newly emerging clinical entity. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 884 Myeloma cells typically grow in bone, recruit osteoclast precursors and induce their differentiation and activity in areas adjacent to tumor foci. Clonal B-lymphocytes and plasma cells harboring lymphocytic phenotypes have a proposed role in sustaining myeloma. Bruton's tyrosine kinase (BTK) is expressed in hematopoietic cells and is particularly involved in B-lymphocyte function and osteoclastogenesis. The SDF-1/CXCR4 signaling pathway is reportedly involved in homing to bone of myeloma cells and osteoclast precursors. The aims of the study were to investigate the possible association between CXCR4 and BTK signaling in myeloma cells and osteoclast precursors, and the consequences of BTK inhibition on myeloma cell migration and clonogenicity, and osteoclastogenesis. By global gene expression profiling (GEP), BTK expression was moderately higher in clinical myeloma cells (n=559) than normal plasma cells (n=25, p

Description: 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 (p4 folds (p5 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