ALBERT

Beschreibung: Multiple myeloma (MM) is a plasma cell malignancy that is the most frequent cancer to involve the skeleton. MM bone disease is characterized by the formation of lytic bone lesions adjacent to MM cells that rarely heal even when patients are in long-term remission. This is due to the persistent suppression of bone marrow stromal cell (BMSC) differentiation into osteoblasts. We previously reported that MM cells induce long-lasting suppression of osteoblast differentiation by repression of the Runx2 gene through elevated expression of the transcriptional repressor Gfi1. However, how Gfi1 activity in BMSC is regulated by MM cells remains unclear. Using bioinformatics analysis, we found that there are three putative phosphorylation sites in the Gfi1 protein for Aurora A kinase (AurA) at S216, S326, and T418. We confirmed that Gfi1 was phosphorylated by AurA at multiple sites using an in vitro kinase assay. Co-immunoprecipitation assays revealed that AurA physically interacted with Gfi1 and phosphorylated Gfi1 protein. The interaction with AurA stabilized Gfi1 protein by blocking Gfi1 protein turnover, thereby extending the Gfi1 half-life from 2 hrs to 6 hrs. Further, co-transfection studies using wildtype and mutant AurA and Gfi1 showed that AurA inhibition of Gfi1 protein turnover was dependent on AurA kinase activity and phosphorylation of the S326 and T418 amino acid residues of Gfi1. Studies with co-transfected Myc-ubiquitin, FLAG-Gfi1, and HA-AurA revealed that AurA decreased Gfi1 ubiquitination, thereby leading to increased Gfi1 protein stability. Amino acids S326 and T418 are in Gfi1 zinc fingers (ZF) 3 and 6, respectively. It is known that Gfi1 ZF3, 4, and 5 are required for DNA binding, and that the K403R mutation in ZF6 interferes with DNA binding. Therefore we investigated if AurA phosphorylation of Gfi1 interferes with DNA binding. Chromatin immunoprecipitation and mRunx2 promoter oligo-pull down assays demonstrated that phosphorylated Gfi1 can still bind the Runx2 promoter. However, co-transfection studies with AurA and Gfi1 expression vectors with mRunx2-promoter luciferase reporters demonstrated that AurA phosphorylation of Gfi1 blocked repression of the Runx2 promoter. These data indicate that although AurA increased the amount of Gfi1 protein present on Runx2, AurA phosphorylation of Gfi1 appeared to lock Gfi1 in an “Off” (inactive) status and abrogated Gfi1 repression of Runx2 expression in osteoblast precursor cells. Since AurA phosphorylation of Gfi1 is not blocking DNA binding, the difference between Gfi1 “OFF” and “ON” status probably involves altered protein-protein interactions between Gfi1 and other factors that regulate Runx2 transcription. TNFa treatment, which we showed also represses Runx2 via Gfi1 activity, decreased the AurA protein level in MC-4 osteoblast precursor cells. Importantly, we found that AurA mRNA was decreased in both MC-4 cells treated with MM cells in vitro, and in bone marrow stromal cells isolated from MM patients. In conclusion, these data indicate that MM cells lower the levels of AurA in bone marrow stromal cells, thereby decreasing AurA phosphorylation of Gfi1. This helps to maintain Gfi1 in the “ON” status and allows Gfi1 repression of the Runx2 gene, thereby preventing osteoblast differentiation. These data suggest that AurA is an important regulator of Gfi1 function in MM bone disease. Disclosures: Roodman: Amgen: Membership on an entity’s Board of Directors or advisory committees; Eli Lilly: Research Funding.

Beschreibung: CC-4047, an immunomodulatory analog of thalidomide, inhibits multiple myeloma with unknown effects on the human osteoclast lineage. Early osteoclast progenitors are of hematopoietic origin and differentiate into mature bone resorbing multinucleated osteoclasts. We investigated the effects of CC-4047 and thalidomide on human osteoclastogenesis, using in vitro receptor activator of NFκ-B ligand/macrophage colony-stimulating factor–stimulated bone marrow cell cultures. Treating bone marrow cultures with CC-4047 for 3 weeks decreased osteoclast formation accompanied by complete inhibition of bone resorption. The inhibitory effect was similar when cultures were treated for 3 weeks or for only the first week (90% inhibition), indicating that CC-4047 inhibits early stages of osteoclast formation. Inhibition of osteoclastogenesis by CC-4047 was mediated by a shift of lineage commitment to granulocyte colony-forming units at the expense of granulocyte-macrophage colony-forming units. Further studies revealed that this shift in lineage commitment was mediated through down-regulation of PU.1. Treatment with thalidomide resulted in significantly less potent inhibition of osteoclast formation and bone resorption. These results provide evidence that CC-4047 blocks osteoclast differentiation during early phases of osteoclastogenesis. Therefore, CC-4047 might be a valuable drug for targeting both tumors and osteoclastic activity in patients with multiple myeloma and other diseases associated with osteolytic lesions.

Beschreibung: Multiple myeloma (MM) is a plasma cell malignancy that is the most frequent cancer to involve the skeleton. MM bone disease is characterized by the formation of lytic bone lesions adjacent to MM cells that rarely heal even when patients are in long-term remission. This is due to the persistent suppression of bone marrow stromal cell (BMSC) differentiation into osteoblasts. We previously reported that MM cells induce long-lasting suppression of osteoblast differentiation by repression of the Runx2 gene through elevated expression of the transcriptional repressor Gfi1. However, how Gfi1 activity in BMSC is regulated by MM cells remains unclear. Using bioinformatics analysis, we found that there are three putative phosphorylation sites in the Gfi1 protein for Aurora A kinase (AurA) at S216, S326, and T418. We confirmed that Gfi1 was phosphorylated by AurA at multiple sites using an in vitro kinase assay. Co-immunoprecipitation assays revealed that AurA physically interacted with Gfi1 and phosphorylated Gfi1 protein. The interaction with AurA stabilized Gfi1 protein by blocking Gfi1 protein turnover, thereby extending the Gfi1 half-life from 2 hrs to 6 hrs. Further, co-transfection studies using wildtype and mutant AurA and Gfi1 showed that AurA inhibition of Gfi1 protein turnover was dependent on AurA kinase activity and phosphorylation of the S326 and T418 amino acid residues of Gfi1. Studies with co-transfected Myc-ubiquitin, FLAG-Gfi1, and HA-AurA revealed that AurA decreased Gfi1 ubiquitination, thereby leading to increased Gfi1 protein stability. Amino acids S326 and T418 are in Gfi1 zinc fingers (ZF) 3 and 6, respectively. It is known that Gfi1 ZF3, 4, and 5 are required for DNA binding, and that the K403R mutation in ZF6 interferes with DNA binding. Therefore we investigated if AurA phosphorylation of Gfi1 interferes with DNA binding. Chromatin immunoprecipitation and mRunx2 promoter oligo-pull down assays demonstrated that phosphorylated Gfi1 can still bind the Runx2 promoter. However, co-transfection studies with AurA and Gfi1 expression vectors with mRunx2-promoter luciferase reporters demonstrated that AurA phosphorylation of Gfi1 blocked repression of the Runx2 promoter. These data indicate that although AurA increased the amount of Gfi1 protein present on Runx2, AurA phosphorylation of Gfi1 appeared to lock Gfi1 in an “Off” (inactive) status and abrogated Gfi1 repression of Runx2 expression in osteoblast precursor cells. Since AurA phosphorylation of Gfi1 is not blocking DNA binding, the difference between Gfi1 “OFF” and “ON” status probably involves altered protein-protein interactions between Gfi1 and other factors that regulate Runx2 transcription. TNFa treatment, which we showed also represses Runx2 via Gfi1 activity, decreased the AurA protein level in MC-4 osteoblast precursor cells. Importantly, we found that AurA mRNA was decreased in both MC-4 cells treated with MM cells in vitro, and in bone marrow stromal cells isolated from MM patients. In conclusion, these data indicate that MM cells lower the levels of AurA in bone marrow stromal cells, thereby decreasing AurA phosphorylation of Gfi1. This helps to maintain Gfi1 in the “ON” status and allows Gfi1 repression of the Runx2 gene, thereby preventing osteoblast differentiation. These data suggest that AurA is an important regulator of Gfi1 function in MM bone disease. Disclosures: Roodman: Amgen: Membership on an entity’s Board of Directors or advisory committees; Eli Lilly: Research Funding.

Beschreibung: BM stromal cells (BMSCs) are key players in the microenvironmental support of multiple myeloma (MM) cell growth and bone destruction. A spliced form of the X-box–binding protein-1 (XBP1s), a major proximal effector of unfolded protein response signaling, is highly expressed in MM cells and plays an indispensable role in MM pathogenesis. In the present study, we found that XBP1s is induced in the BMSCs of the MM microenvironment. XBP1s overexpression in healthy human BMSCs enhanced gene and/or protein expression of VCAM-1, IL-6, and receptor activator of NF-κB ligand (RANKL), enhancing BMSC support of MM cell growth and osteoclast formation in vitro and in vivo. Conversely, deficiency of XBP1 in healthy donor BMSCs displayed a range of effects on BMSCs that were opposite to those cells with overexpression of XBP1s. Knock-down of XBP1 in MM patient BMSCs greatly compromised their increased VCAM-1 protein expression and IL-6 and RANKL secretion in response to TNFα and reversed their enhanced support of MM-cell growth and osteoclast formation. Our results demonstrate that XBP1s is a pathogenic factor underlying BMSC support of MM cell growth and osteoclast formation and therefore represents a therapeutic target for MM bone disease.

Beschreibung: The bone marrow of myeloma patients is a reservoir of marrow infiltrating lymphocytes (MILs) that upon activation with anti-CD3 and anti-CD28 antibodies show significant tumor specificity against both mature myeloma plasma cells and their clonotypic precursors, whereas activated peripheral blood lymphocytes (PBLs) fail to demonstrate any tumor specificity. Here we examine the major differences between MILs and PBLs of both myeloma patients and normal donors. In myeloma, CD4+/CD25+ MILs lack a regulatory T-cell (Treg) phenotype as shown by the absence of FoxP3 expression and the inability to suppress tumor-specific proliferation of effector T cells. In contrast, the CD4+/CD25+ PBLs express FoxP3 and suppress tumor specific proliferation. Analysis of MILs and PBLs from normal individuals reveals the exact opposite (more Tregs in the marrow than blood). Considering the abundance of plasma cell-derived IL-6 in myeloma marrow, we hypothesize that IL-6 skewing of MILs towards the effector Th17 phenotype could explain the reciprocal paucity of Tregs in myeloma and relative abundance in normal bone marrows. Intracellular staining shows increased expression of IL-17 in myeloma CD4 MILs (2.7%) as compared to either myeloma PBLs (0.02%) or normal MILs (0.7%). The Th17 cytokine secretion pattern also appears more pronounced in myeloma-derived MILs as compared to either myeloma PBLs or normal MILs respectively (see table). Th17 T cells have recently been implicated in osteoclastogenesis of rheumatoid arthritis. In fact, bone marrow serum IL-17 levels correlate with the degree of lytic bone disease. In experiments utilizing M-CSF and RANK-L as the osteoclast differentiation medium, we show that the addition of IL-17 to the culture medium increases the number of mature osteoclasts by 2.3 fold whereas γIFN reduces mature osteoclast numbers by 80%. These findings explain the profound differences seen in MILs of normal and myeloma patients and implicate Th17 MILs in the generation of lytic bone disease. Activation of myeloma MILs with anti-CD3/CD28 skews the population from Th17 to a γIFN-producing Th1 phenotype. As such, targeting Th17 T cells or utilizing adoptive T cell therapy with activated, γIFN-producing MILs may represent a novel therapeutic mechanism to target osteoclastogenesis and reduce bone disease in myeloma. Th-17 Cytokine Profile N=56 MM MILs MM PBLs NL MILs IL-6 (pg/ml) 30 3.7 6.5 IL-23 (pg/ml) 246 19.6 35.9 IL-17 (pg/ml) 12.2 0.4 5.1

Beschreibung: Abstract 3933 Multiple myeloma (MM) is characterized by bone destruction with suppressed new bone formation that is mediated by multiple factors including MIP-1α (CCL3), RANKL, IL-3, DKK1 and recently, Activin A (ActA). IL-3 is a bifunctional cytokine that indirectly increases osteoclastogenesis and suppresses osteoblastogenesis via CD14+ bone marrow monocytes (BMM). Additionally, IL-3 levels are elevated in the BM plasma of MM patients compared with normals, however the mediators of IL-3's effects on myeloma bone disease are unknown. To investigate this we performed gene expression profiling using Affymetrix GeneChip® analysis of IL-3 treated BMM from MM patients and found ActA gene expression was increased 180-fold and confirmed this finding at the protein level by ELISA. ActA is a negative regulator of bone mass that promotes osteoclastogenesis and is overproduced in MM patients. Interestingly, treatment of MM cells or bone marrow stromal cells (BMSC) with IL-3 did not induce secretion of ActA. We found that ActA was produced by MM and MGUS patient CD14+ cells treated with IL-3 to a significantly higher degree compared to healthy subjects. (Median ActA levels for MM and MGUS ActA levels were increased 66.57 and 51.6 fold respectively over untreated cells, while IL-3 treatment of normal cells increased ActA 8.5 fold.) ActA levels were also increased in freshly isolated marrow plasma of a cohort of patients with active MM as compared to patients with smoldering MM (SMM), MGUS, or healthy subjects (median ActA levels: active MM 453 pg/ml, SMM 328 pg/ml, MGUS 332 pg/ml, normal 286 pg/ml). ActA levels in MM patients with and without bone disease were not significantly different. (Median value with bone disease 463pg/ml vs. 407 pg/ml without bone disease.) ActA has also been reported to have a role in the differentiation and polarization of CD14+ tumor associated macrophages (TAMs), which are osteoclast precursors and can block MM cell apoptosis. Therefore, we examined the potential role of ActA in IL-3 mediated osteoclast (OCL) formation. Culture of normal BMM with ActA or IL-3 significantly enhanced osteoclastogenesis compared with control (mean number of OCL / 1×105 normal marrow non-adherent cells plated in IL-3 (100pg/ml) treated cultures 73; ActA (1ng/ml) treated cultures 123; cultures with vehicle alone, 8), and ActA enhanced RANKL-induced osteoclastogenesis. Osteoprotegerin treatment of normal donor BMM stimulated with ActA failed to block the osteoclastogenic effects of ActA, demonstrating that ActA's osteoclastogenic effects were RANKL independent. Importantly, the osteoclastogenic effect of IL-3 was dose-dependently inhibited by anti-ActA, and IL-3 induced ActA expression by BMM decreased during OCL differentiation. In support of early OCL precursors as the source of IL-3 induced ActA, we did not identify IL-3 receptors on mature OCL by flow cytometry. These results demonstrate that IL-3 induction of osteoclastogenesis is mediated by ActA produced by CD14+ BMM and is RANKL independent. Thus, we hypothesize that therapies targeting the ActA receptor, such as the recently developed ActA receptor antagonist, should block both IL-3 and ActA, and thereby significantly impact MM bone disease via their effects on TAMs. Disclosures: Bolzoni: Celgene Italy: Research Funding. Roodman:Millennium: Consultancy; Amgen: Consultancy. Giuliani:Celgene: Research Funding; Novartis: Research Funding.

Beschreibung: Multiple myeloma (MM) is an incurable neoplastic disease characterized by an accumulation of plasma cells in bone marrow. Osteolytic bone lesions are the major source of morbidity in MM patients and are associated with bone pain and fractures and hypercalcemia. The bone lesions result from increased osteoclastic bone destruction in areas adjacent to the myeloma cells. New bone formation that normally happens at sites of previous bone resorption still occurs in early stages of the disease but is absent in advanced MM. Although the molecular basis for the increased osteoclastic activity has been intensely investigated, the basis for the decreased osteoblast activity is just beginning to be understood. Recently, inhibitors of WNT signaling pathway, Dickkorpf1 (DKK1) and secreted Frizzle-Related Protein-2 (sFRP2) have been identified as factors involved in osteoblast suppression in MM. In addition, IL-3 and IL-7 are increased in plasma of MM patients and suppress osteoblastogenesis in cell culture models. However, the role of those factors in the osteoblastic activity in MM patients is unclear. Studies in patients are confounded by cytotoxic therapy as well as bisphosphonates, which are standard therapy for MM patients. Therefore, preclinical in vivo models are required to delineate the mechanisms responsible for the profound osteoblast suppression in MM. We have developed a mouse model of myeloma bone disease in which genetically modified myeloma cells can be selectively ablated without the confounding effects of cytotoxic therapies and allows us to tract the growth of MM cells. The 5TGM1 cell line which is the most common version of murine MM, was stably transfected with the thymidine kinase (TK) gene from herpes simplex virus, which permits eradication of myeloma cells with ganciclovir, as well as GFP and luciferase genes to detect the presence of MM cells. One ug/ml ganciclovir treatment in culture results in 100% death of the transfected 5TGM1 cells in 4 days. Importantly, ganciclovir treatment of primary marrow cell cultures had no effect on growth and differentiation of osteoblast and hematopoietic progentitors. Co-culturing of primary marrow cells with 5TGM1 expressing TK has no bystander effect on osteoblast differentiation with ganciclovir treatment. Subcutaneously implanted 5TGM1 cells into SCID mice were eradicated by intraperitoneal injection of 20mg/kg ganciclovir/d for 2 weeks. The dose of ganciclovir did not affect osteoblast differentiation of primary marrow culture from the mice treated with ganciclovir. Then we injected the 5TGM1 cells into tibia of SCID nude mice (n=4 per group). After measuring the increase of serum IgG2b level, half of the mice were treated with ganciclovir for 2 weeks and the other with saline. Our preliminary data show that osteogenic cultures of bone marrow from the ganciclovir treated mice had significantly higher alkaline phosphatase activity than cultures derived from the saline treated group (p=0.03). In addition, the ganciclovir treated mice had tendency of higher trabecular bone volume than the saline-treated group (p=0.08). These results demonstrate that this model should be useful for studying mechanisms of osteoblast suppression in MM.