Myeloma Bone Research Articles

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

Abstract 3941Iron 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. Disclosures:Barlogie:Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties. Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.

The Activation of Wnt5a-Mediated Non Canonical Wnt Signaling in Human Bone Marrow Osteoprogenitor Cells Increases Osteoblastogenesis and Counterbalances the Inhibitory Effect of Myeloma Cells on Ror2/FZD5 Expression,

Abstract 3928Multiple Myeloma (MM) is characterized by the impairment of osteogenic differentiation of bone marrow (BM) mesenchymal stromal cells (BMSC) and osteoblast suppression. Canonical Wnt signal pathway is critical in the regulation of bone formation. However recent evidences suggest that non-canonical Wnt activation by Wnt5a, rather than canonical one by Wnt3a stimulates the osteogenic properties of BMSC. Non-canonical Wnt signaling, mainly activated by Wnt5a, is transduced through FZD receptor (FDZ5) and Ror2 co-receptor to several cascades such as Disheveled pathways involving the Rho family small GTPases or the Ca++ dependent pathways/PKC involving the nuclear factor of activated T cells (NFATc).Actually, the effect of MM cells on non-canonical Wnt signaling and the role of the activation of this pathway on MM-induced osteoblastic exhaustion are still unknown and have been investigated in this study.First we checked the expression of non-canonical Wnt related molecules by BMSC and osteoprogenitor cells (PreOB) and found that both cells expressed Wnt5a but lack of express Wnt1, Wnt3a and Wnt8. The presence of the Wnt5a receptors FZD2 and FZD5 was also detected in both cell types as well as of Ror2. Interestingly we found that osteogenic differentiation of BMSC towards preOB significantly increased Ror2 and FZD5 expression. Secondly, we performed a series of co-culture between PreOB and MM cells using either IL-6 dependent (XG-1), and independent (JJN3) human myeloma cell lines or purified primary CD138+ MM cells. We found that XG-1 and CD138+ MM cells inhibit Ror2 and FZD5 expression in PreOB and consistently the activity of NFATc1 at nuclear level.Thereafter the activation of non-canonical WNT pathway in PreOB, checked by the intracytoplasmatic increase of Ca++influx, phospho-PKC expression and NFATc1 activity, was induced either by Wnt5a treatment or by Wnt5a overexpression through a lentivirus vector. Ror2 overexpression was also performed by lentivirus vector in PreOB. The transcriptional profiles of both PreOB overexpressing Wnt5a and Ror2 have been evaluated by GeneChip® HG-U133Plus 2.0 arrays. The raw intensity signals were extracted from CEL files and normalized using the RMA package for Bioconductor and custom GeneAnnot-based Chip Definition Files in R software. We found that Wnt5a treatment as well as Wnt5a overexpression significantly increased osteogenic differentiation and the expression of the osteogenic markers alkaline phosphatase and collagen I in PreOB. Consistently with these observations, we also demonstrated that siRNA-mediated Wnt5a silencing inhibited these osteogenic markers in the same cell type. Moreover we found that the activation of non-canonical WNT signal pathway in PreOB, blunted the inhibitory effect of MM cells on the osteogenic differentiation process in co-culture. Finally, we show that Ror2 overespression in PreOB activated non-canonical Wnt signaling, increased osteogenic differentiation and restored the osteogenic properties of PreOB in co-culture with MM cells.In conclusion, our data indicate that activation of non-canonical Wnt5a/Ror2 pathway in BM osteoprogenitor cells increases osteogenic differentiation and counterbalances the inhibitory effect of MM cells suggesting that modulation of Wnt5/Ror2 pathway in the microenvironment could be a target for MM bone disease. Disclosures:Bolzoni:Celgene Italy: Research Funding. Giuliani:Celgene: Research Funding; Novartis: Research Funding.

The TGF- β Family Member Growth Differentiation Factor 15 (GDF15) Regulates the Self-Renewal of Multiple Myeloma Cancer Stem Cells

Abstract 2954Multiple myeloma (MM) cancer stem cells (CSCs) possess both enhanced tumorigenic potential and relative drug resistance suggesting they play a major role in disease relapse and progression. Therefore, a better understanding of the processes regulating MM CSCs may lead to the development of novel therapies that prevent tumor regrowth and improve long-term outcomes. Normal stem cells are tightly regulated by factors within the local microenvironment that include both soluble factors and direct contact with accessory cells. However, external factors regulating MM CSCs have not been identified. Recent studies have demonstrated that stromal cells in the MM bone marrow microenvironment secrete growth differentiation factor 15 (GDF15), a member of the TGF-b family. We initially studied the role of this cytokine in the pathogenesis of MM by examining circulating GDF15 levels in MM patients. Compared to healthy volunteers, we found that median GDF15 levels were significantly increased in MM patients (821 vs. 390 pg/ml; n=16; p<0.05) and increased with disease stage (Stage II=585 pg/ml, Stage III=1, 004 pg/ml). To examine the functional effects of GDF15 on MM cells, we cultured human MM cell lines (NCI-H929, RPMI 8226) with recombinant GDF15 and found that it induced the expansion of isolated CD138neg MM CSCs in a dose-dependent manner but had little impact on the growth of CD138+ plasma cells (Fig). Furthermore, GDF15 enhanced clonogenic myeloma growth as evidenced by increased colony formation that was maintained during serial replating, a surrogate for self-renewal. This effect appeared to be GDF15 specific since it could be blocked using anti-GDF15 antibody. Similarly, GDF15 treatment increased the in vitro clonogenic growth of MM CSCs from primary clinical bone marrow specimens. We also investigated the down-stream cellular pathways potentially mediating the effects of GDF15 and found that it activates the AKT signaling pathway known to improve the self-renewal of embryonic (ES) and normal hematopoietic stem cells. GDF15 also induced expression of the SOX2 transcription factor known to be upregulated in CD138neg MM CSCs. Since SOX2 is required for the self-renewal of ES cells and the generation of induced pluripotent stem (iPS) cells, its induction by GDF15 may also increase the self-renewal of MM CSCs. GDF15 is the first soluble factor identified that regulates MM CSCs, and its effects are mediated by the activation of highly conserved self-renewal programs. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

The Effect of ICAM-1 Antibody Therapy in the SCID-Hu Mouse Model Using Primary Myeloma Cells

Abstract 2914 Background:Myeloma (MM) - stromal cell-cell interactions play a crucial role in MM cell growth, survival, and drug resistance. Intercellular adhesion molecule-1 (ICAM-1) is up-regulated in different cancers, including MM, and represents one of the major adhesion molecules mediating tumor-stromal cell-cell contacts. Previous investigations have shown that ICAM antibodies induce antitumor activity in SCID mice xenografted with MM cell lines, likely involving Fc :Fc gamma receptor dependent anti-tumor mechanisms. We have examined the anti-MM activity in the SCID-hu mouse model of a specific humanized ICAM1 antibody (BI-505, Bio-Invent, Sweden) using primary MM cells. Materials and Methods:The expression of ICAM1 was examined in plasma cells from 22 donors, 351 newly diagnosed MMs, and 9 MM cell lines using Affymetrix U133 Plus2 chips. Human fetal femurs and tibias, obtained at 17 to 22 gestational weeks, were cut into fragments and implanted subcutaneously in 16 SCID mice (SCID-hu) at age 6 to 8 weeks. Four weeks after bone implantation, approximately 5 ×106 CD138+ plasma cells from MM patients were injected directly into the human bone of the SCID-hu mice in final volume of 30 to 40μl of phosphate-buffered saline(PBS). Human immunoglobin (hIg) levels by ELISA were used as an indicator of myeloma cell growth. When hIg level reached 10μg/mL or higher in 2 consecutive samples after 4 weeks of injection of the tumor cells, the mice were divided into four groups (n=4), Control group (not injected MM cells and no drug treatment); the other 3 groups were injected with MM cells: the isotype control group (isotype IgG 2mg/kg, i.p., twice weekly), BI-505 group (2mg/kg, i.p., twice weekly) and bortezomib group (1mg/kg, i.p., twice weekly). Bone remolding was detected by X-radiography and by measuring bone mineral density (BMD). Tumor cells were detected by immunohistochemistry using the CD138 antibody. Results:High expression of ICAM1 was observed in normal plasma cells, primary MM cells and MM cell lines. With a follow- up of 10 weeks, the tumor burden in the mice treated with BI-505 or bortezomib was significantly lower compared with the isotype control group (p: BI505 < 0.01; p: bortezomib < 0.01). Also, the number of MM tumor cells stained with the CD138 antibody was significantly less in samples treated with either BI-505 or bortezomib than in the isotype control group (p < 0.01). In addition, 6 weeks after injection of tumor cells, X-rays showed severe bone resorption in the isotype-control group, while there was no obvious bone resorption in the fetal bones after treatment with BI-505 or bortezomib. The BMD (0.0715±0.006 g/cm2) of isotype control was significantly lower than that in other 3 groups including control: 0.1278±g/cm2, BI-505 group: 0.102±0.0064 g/cm2, and bortezomib group: 0.106±0.0059g/cm2. Importantly, the number of TRAP-positive cells was significantly higher in the isotype control group than in the other 3 groups (p < 0.01). Conclusion:ICAM1 expression presents in all subtypes of myeloma. The ICAM1 antibody BI-505 significantly inhibits primary MM cell growth and bone destruction in the SCID-hu mice to the same degree as bortezomib, indicating its clinical applicability in therapy of MM. Disclosures:No relevant conflicts of interest to declare.

NAMPT/PBEF Enzymatic Activity Is Indispensable for Myeloma Cell Growth and Osteoclast Activity

Abstract 886Clinical MRI and PET-CT analyses implicated hypermetabolic activity of multiple myeloma (MM) cells in focal lesions with tumor burden, metastasis and survival. Osteoclasts induced by MM are also naturally hypermetabolic suggesting that targeting cellular metabolism may contribute to controlling MM and associated bone disease. The coenzyme nicotinamide adenine dinucleotide (NAD) not only is essential for proper cellular metabolism but also affects activity of NAD-dependent enzymes, such as PARP1 and sirtuins. Nicotinamide phosphoribosyl transferase (NAMPT/PBEF/Visfatin) is a rate-limiting enzyme in NAD biosynthesis from nicotinamide and also functions extracellularly. APO866 (TopoTarget, Switzerland), an inhibitor of NAMPT, depletes cellular NAD particularly in cancer cells. The aims of the study were to evaluate expression of NAMPT (PBEF1) in clinical and experimental MM samples using global gene expression profiling (GEP) and investigate the effect of APO866 on MM cell growth and osteoclastogenesis. Our molecular classification data revealed elevated expression of PBEF1 in the PR, MAF and LB subtypes of newly diagnosed MM patients (n=796) and in MM cell lines (n=41). Based on our GEP70 risk model, PBEF1 expression was higher in high risk of the CD-1, HY, and MS subtypes relative to their low risk counterparts, and in focal lesions at relapse than at diagnosis. PBEF1 expression was 2.5 fold higher in myelomatous versus nonmyelomatous human bones from SCID-hu mice (p<0.0001). In primary MM cell-osteoclast cocultures (n=8) PBEF1 expression was upregulated by >3 folds in cocultured MM cells or osteoclasts compared to baseline MM cells (p<0.02) or control osteoclasts (p<0.04). APO866 dose dependently inhibited growth of 6 MM cell lines at low nanomolar concentrations (IC50 at 0.1–5 nM, 72 hrs) and similarly inhibited growth of MM cells in coculture with stromal cells or osteoclasts. NAD depletion by APO866 (5 nM) in 3 MM cell lines was consistently associated with reduced enzymatic activities of PARP1 by 73 ± 5% (p<0.001) and of SIRT1 by 49 ± 3% (p<0.005). In vivo, SCID-rab mice engrafted with our DAS MM cell line (MF subtype) were intraperitoneally treated with saline or APO866 (10 mice/group, 20 mg/kg, twice weekly) for 3 weeks. At experiment’s end circulating human Ig levels in control and APO866 groups were 43 ± 15 μg/ml and 1 ± 0.6 μg/ml, respectively (p<0.01). APO866 prevented bone loss as assessed by X-rays and measurement of bone mineral density (p<0.05). Histological examination confirmed high infiltration of MM cells in myelomatous bones of control hosts while in APO866-treated hosts the implanted bones reduced numbers of osteoclasts by 53% (p<0.0007). In vitro, at concentration of ≥0.1 nM, APO866 significantly suppressed osteoclast differentiation and bone resorption activity of mature osteoclasts on dentine slices (p<0.007). We conclude that NAMPT/PBEF/Visfatin is a potential target in certain molecularly classified and high risk MM patients, and that MM cells and osteoclasts are highly sensitive to NAD depletion. Disclosures:Barlogie:Celgene, Genzyme, Novartis, Millennium: Consultancy, Honoraria, Patents & Royalties. Shaughnessy:Myeloma Health, Celgene, Genzyme, Novartis: Consultancy, Employment, Equity Ownership, Honoraria, Patents & Royalties.

Clinical Features of Patients with Multiple Myeloma and C-MYC Rearrangements

Abstract 5090 Background:The proto-oncogene c-MYC encodes a transcription factor that regulates cell proliferation, growth, and apoptosis. c-MYC is located on the long arm of chromosome 8 (8q24.1) and its rearrangement has been reported in 15% of myeloma patients independent of the disease stage (Avet-Loiseau et al. Blood 2001). However, the clinical significance of c-MYC rearrangement is not well described. Here we report the characteristics and outcome of 7 myeloma patients with c-MYC rearrangements that were treated at our institution. Methods:Between July 2007 and May 2011, we identified 7 patients (4 males, 3 females) who had c-MYC rearrangements on fluorescence in situ hybridization (FISH) analyses at the time of diagnosis. The primary objective of this study was to describe the patient characteristics, response to therapy, time to progression (TTP), and overall survival (OS). Results:Median age at diagnosis was 58 years (49–72). There were 4 Caucasians, 2 African-Americans and 1 Asian. International Staging System stage was I, II, and III in 3, 2, and 2 patients, respectively. Two patients had serum creatinine of ≥2 mg/dL as well as Hgb ≤10 g/dL, two other patients had hypercalcemia at diagnosis. Six (85%) patients had myelomatous bone lesions at diagnosis. Plasma cell counts in bone marrows ranged from 20%-78%, with a mean of 51%. Abnormalities involving chromosome 8q24.1, the c-MYC locus, were detected on conventional cytogenetics in all 7 patients, including t(8;14)(q24.1) in 3 cases, a t(2;8)(p12;q24.1), a variant of the t(8;14), in 2 cases, and an abnormal chromosome 8 with unknown material attached to the 8q24.1 region confirmed by FISH in two cases. This is in contrast to an earlier report where only 25% of patients with c-MYC rearrangement on FISH had corresponding cytogenetic abnormalities. Three patients also had a del(13)(q14.1)/RB1, one of whom also had a del(17)(p13)/TP53, while two other patients had t(11;14)(q13;q32) involving CCND1-XT/IGH rearrangements. Six patients (85%) received induction with a bortezomib+ dexamethasone regimen, and one patient received thalidomide+ dexamethasone. Five patients achieved a partial (PR) or very good partial remission (VGPR) to induction, while 2 patients had <PR, with an overall response rate of 71%. Four patients went on to receive high-dose chemotherapy and autologous hematopoietic stem cell transplantation (auto-HCT). Three of these patients were in first remission, while one patient progressed prior to auto-HCT. Median follow up in all patients was 17.7 months (range 2.6–22.4). Two patients progressed at 4.4 and 4.5 months after the start of induction therapy, and 2 more patients progressed after auto-HCT, 14.4 and 16.1 months after the start of induction. Median TTP was 14.4 months, and median OS was 20.2 months. The TTP and OS appear to be shorter than what has been reported for standard-risk myeloma patients (24 and 72 months, respectively). Conclusion:This is the first report describing clinical characteristics of myeloma patients with c-MYC rearrangements. All 7 patients had concurrent translocations involving chromosome 8q24 on conventional cytogenetics, and had a shorter TTP and OS than our historical data. The role of c-MYC rearrangement in myeloma needs to be explored in prospective studies. Disclosures:No relevant conflicts of interest to declare.

The impact of osteoporosis (as measured by lumbar spine quantitative computed tomography) on disease activity and survival in myeloma patients: A 13‐year prospective study

Myeloma is a progressive neoplastic disease characterized by plasma cell dyscrasia, progressive bone loss, and pathological fractures. We conducted a prospective, single center study on myeloma patients to determine the impact of osteoporosis on disease activity and survival. Data collected on 108 patients followed for 13 years included clinical demographics, markers of myeloma activity, skeletal radiography, and bone densitometry. There were 56 men and 52 women with mean age of 69 years. Of these, 78% presented with stage I/II disease, 11% underwent stem cell or bone marrow transplants, 78% received adjuvant intravenous bisphosphonates, and 80% died during the course of the study (median survival of 47.3 months). There were 66% with osteoporosis, 54% with fractures, and 56% with lytic bone lesions. Femoral neck dual energy X-ray absorptiometry (DXA) and lumbar spine quantitative computed tomography (QCT) were major independent predictors of patient survival. Kaplan-Meier survival estimates demonstrated that patients presenting with a lumbar spine QCT T-score ≤ -3.5, died on average 18 months earlier than those with a lumbar spine QCT T-score > -3.5. These data suggest that the severity of osteoporosis, as defined by initial BMD T-score values, significantly impact on patient survival.

Abstract 3532: Antitumor activity of SR16388 on multiple myeloma targets factors needed for adaptation to bone marrow microenvironment

Abstract Multiple myeloma (MM) is a B-cell malignancy that is characterized by accumulation of clonal plasma cells in the bone marrow. The bone marrow microenvironment plays a critical role in MM cell pathogenesis and progression. In the bone marrow milieu, MM cells adhere to the bone marrow stromal cells and trigger the secretion of growth factors such as vascular endothelial growth factor (VEGF). VEGF in turn upregulates the secretion of interleukin-6 (IL-6), the major cytokine that mediates MM cell growth and survival in part through the activation of Janus kinase (Jak)/signal transducers and activators of transcription 3 (STAT3). VEGF also stimulates the proliferation of endothelial and stromal cells and induces angiogenesis via its receptors. Thus, targeting both myeloma cells and bone marrow microenvironment is a valid approach for the development of therapeutic agents for MM. At SRI International, we have developed an antiestrogen, SR16388, that binds to ER-α, ER-β and ERR-α with potent antitumor and antiangiogenic properties. We have previously reported that SR16388 inhibited MM cell proliferation in vitro and MM tumors in vivo. In the current study, we are presenting data to show that the inhibitory effect of SR16388 on MM cell proliferation was achieved by inducing apoptosis, and arresting cells at the G2 phase. Furthermore, at nanomolar concentrations SR16388 inhibited IL-6-induced cell proliferation and blocked the activation of STAT3 induced by IL-6 in RPMI-8226 human MM cells. In human endothelial cells, SR16388 inhibited VEGF-induced cell proliferation and VEGF-induced phosphorylation of STAT3. In a RPIM-8226 tumor xenograft model, the microvessel density in the tumor tissue was markedly reduced by treatment with SR16388. We are currently investigating the effect of SR16388 in combination with known therapeutic agents on the growth of MM tumors in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3532. doi:10.1158/1538-7445.AM2011-3532

Abstract 789: Cytokine interleukin-16 is an important growth-promoting factor for multiple myeloma

Abstract BACKGROUND: Multiple myeloma is a malignancy characterized by the expansion of a plasma cell clone that localizes to the human bone marrow. Myeloma cells and bone marrow stroma cells both produce soluble factors promoting the survival and progression of multiple myeloma. Interleukin-(IL)-16 is involved in regulating migration and proliferation of normal leukocytes, however, it has been unclear whether IL-16 also plays a role in the pathophysiology of human cancers. METHODS: Using an antibody array we screened BM plasma of MM patients for the presence of a large number of cytokines/chemokines. We confirmed cytokine expression by myeloma cell lines and malignant plasma cells from MM patients applying real-time PCR, western blots, ELISA, and flow cytometry. We used transfection with inhibitory RNA to analyze cytokine function and we applied anti-cytokine antibodies to evaluate possible therapeutic options for MM. RESULTS: We found IL-16 to be strongly overexpressed in the bone marrow of myeloma patients. Myeloma cell lines as well as primary tumor cells from myeloma patients constitutively expressed IL-16 and its receptors CD4 and/or CD9 and spontaneously secreted soluble IL-16. Silencing of IL-16 had an anti-proliferative effect on the tumor cells which was reversed by the addition of a recombinant C-terminal IL-16 peptide. Most importantly, the application of a monoclonal antibody directed against IL-16 had a strong growth-inhibiting influence on myeloma cells. CONCLUSIONS: These findings suggest that cytokine IL-16 is an important growth-promoting factor in MM and might represent an important diagnostic and therapeutic target for this incurable human malignancy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 789. doi:10.1158/1538-7445.AM2011-789

Human Placenta-Derived Adherent Cells Prevent Bone loss, Stimulate Bone formation, and Suppress Growth of Multiple Myeloma in Bone

Human placenta has emerged as a valuable source of transplantable cells of mesenchymal and hematopoietic origin for multiple cytotherapeutic purposes, including enhanced engraftment of hematopoietic stem cells, modulation of inflammation, bone repair, and cancer. Placenta-derived adherent cells (PDACs) are mesenchymal-like stem cells isolated from postpartum human placenta. Multiple myeloma is closely associated with induction of bone disease and large lytic lesions, which are often not repaired and are usually the sites of relapses. We evaluated the antimyeloma therapeutic potential, in vivo survival, and trafficking of PDACs in the severe combined immunodeficiency (SCID)-rab model of medullary myeloma-associated bone loss. Intrabone injection of PDACs into nonmyelomatous and myelomatous implanted bone in SCID-rab mice promoted bone formation by stimulating endogenous osteoblastogenesis, and most PDACs disappeared from bone within 4 weeks. PDACs inhibitory effects on myeloma bone disease and tumor growth were dose-dependent and comparable with those of fetal human mesenchymal stem cells (MSCs). Intrabone, but not subcutaneous, engraftment of PDACs inhibited bone disease and tumor growth in SCID-rab mice. Intratumor injection of PDACs had no effect on subcutaneous growth of myeloma cells. A small number of intravenously injected PDACs trafficked into myelomatous bone. Myeloma cell growth rate in vitro was lower in coculture with PDACs than with MSCs from human fetal bone or myeloma patients. PDACs also promoted apoptosis in osteoclast precursors and inhibited their differentiation. This study suggests that altering the bone marrow microenvironment with PDAC cytotherapy attenuates growth of myeloma and that PDAC cytotherapy is a promising therapeutic approach for myeloma osteolysis.

Consequences of Daily Administered Parathyroid Hormone on Myeloma Growth, Bone Disease, and Molecular Profiling of Whole Myelomatous Bone

BackgroundInduction of osteolytic bone lesions in multiple myeloma is caused by an uncoupling of osteoclastic bone resorption and osteoblastic bone formation. Current management of myeloma bone disease is limited to the use of antiresorptive agents such as bisphosphonates.Methodology/Principal FindingsWe tested the effects of daily administered parathyroid hormone (PTH) on bone disease and myeloma growth, and we investigated molecular mechanisms by analyzing gene expression profiles of unique myeloma cell lines and primary myeloma cells engrafted in SCID-rab and SCID-hu mouse models. PTH resulted in increased bone mineral density of myelomatous bones and reduced tumor burden, which reflected the dependence of primary myeloma cells on the bone marrow microenvironment. Treatment with PTH also increased bone mineral density of uninvolved murine bones in myelomatous hosts and bone mineral density of implanted human bones in nonmyelomatous hosts. In myelomatous bone, PTH markedly increased the number of osteoblasts and bone-formation parameters, and the number of osteoclasts was unaffected or moderately reduced. Pretreatment with PTH before injecting myeloma cells increased bone mineral density of the implanted bone and delayed tumor progression. Human global gene expression profiling of myelomatous bones from SCID-hu mice treated with PTH or saline revealed activation of multiple distinct pathways involved in bone formation and coupling; involvement of Wnt signaling was prominent. Treatment with PTH also downregulated markers typically expressed by osteoclasts and myeloma cells, and altered expression of genes that control oxidative stress and inflammation. PTH receptors were not expressed by myeloma cells, and PTH had no effect on myeloma cell growth in vitro.Conclusions/SignificanceWe conclude that PTH-induced bone formation in myelomatous bones is mediated by activation of multiple signaling pathways involved in osteoblastogenesis and attenuated bone resorption and myeloma growth; mechanisms involve increased osteoblast production of anti-myeloma factors and minimized myeloma induction of inflammatory conditions.

Systemically Injected Mesenchymal Stem Cells Traffic to Myelomatous Bone and Inhibit Bone Disease, and Intralesionally Promotes Bone Formation and Delays Myeloma Progression During the Disease Active Stage or Remission

AbstractAbstract 980Mesenchymal stem cells (MSCs) cytotherapy has been clinically tested in various applications including bone regeneration, autoimmune diseases, and cancer. The aims of the study were to test the effect of MSCs cytotherapy on myeloma (MM) bone disease and tumor growth, and determine their ability to traffic into myelomatous bone during active disease stage and induction of remission by melphalan. We exploited the SCID-rab model for MM and a novel human myeloma cell line, Hg, established through our previously reported procedure (Xin et al., BJH 2007). Hg myeloma cells do not grow in culture but are capable of sequential passaging in SCID-rab mice. Microarray analysis revealed similar gene expression profiling between the Hg cell line and the original patient's myeloma plasma cells, and that these cells are classified in the MMSET subgroup and express DKK1, indicating their authenticity and clinical relevancy. The human fetal MSCs were transduced with a luciferase/GFP reporter in a lentiviral vector for in vivo tracking. In Hg-bearing hosts (5 mice/group), intra-bone injection of MSCs (1×106 cells/mouse) increased bone mineral density (BMD) by 21±4% while in control, diluent injected bones it was reduced by 14±5% (p<0.001). Increased bone formation by MSCs was associated with reduced tumor growth by 50% (p<0.01). Bioluminescence analysis revealed disappearance of the majority of the intralesionally injected MSCs within 4 weeks, suggesting that MSCs exert their effects on bone remodeling as a bystander cells (trophic effect). To test effect on relapse, remission was induced by treating Hg-bearing hosts with a total of 4 subcutaneous injections of melphalan (10 mg/kg/4 days), followed by intra-bone injection of diluent or MSCs (10 mice/group). Three weeks post-cytotherapy BMD was increased by 23±5% in bones injected with MSCs and reduced by 23±3% in bones injected with diluent (p<0.001). Eleven weeks post-cytotherapy, BMD was reduced by 33±6% and 9±7% in bones injected with diluent and MSCs, respectively (p<0.03). Following melphalan treatment circulating immunoglobulin (Ig) level (MM burden) was undetected while at 2 weeks after cytotherapy it was detected in 80% and 30% of hosts injected with diluent and MSCs, respectively. At experiment's end, Ig levels were significantly lower by 6 folds in hosts treated with MSCs than diluent (p<0.01). To further validate clinical relevancy, MSCs or diluent were intravenously injected into Hg-bearing hosts. In contrast to a single injection, 4 weekly, intravenous injections of MSCs (10 mice/group) prevented reduction of the BMD of the myelomatous bone while in control hosts the BMD was reduced by 14±3% (p<0.006 vs. pretreatment). MM growth was not affected by single or multiple intravenous injections of MSCs. Ex vivo imaging of tissues from hosts with active MM or treated with melphalan detected MSCs in implanted bones and murine lungs indicating that myeloma cells or conditions induced by MM or melphalan attract MSCs to myelomatous bones. We conclude that intra-bone injection of MSCs effectively promotes bone formation and delays MM progression during the disease active stage or remission. We also conclude that exogenous MSCs are capable of trafficking to myelomatous bone and that systemic, weekly injections of MSCs inhibit MM bone disease. Disclosures:No relevant conflicts of interest to declare.

Consequences of Intermittent PTH Treatment on Myeloma Growth, Bone Disease and Molecular Profiling of Whole Myelomatous Bone

AbstractAbstract 4063Intermittent PTH (iPTH) treatment is bone anabolic and has been successfully used for treatment of osteoporosis. The aims of the study were to investigate the effects of iPTH-induced bone formation on myeloma progression and unravel molecular mechanisms associated with these effects, using the SCID-rab and SCID-hu models. Using our reported procedure (Xin et al., BJH 2007), we established a novel myeloma cell line, Hg, capable of sequential passaging in experimental models. Hg cells have similar gene expression profiling (GEP) as the original patient's plasma cells, are classified in the MMSET subtype and express DKK1. SCID-hu or SCID-rab (8-10 hosts/group in each model) mice engrafted with Hg myeloma cells were subcutaneously treated with saline or iPTH (80 ug/kg/day) for 4 weeks. Overall, whereas BMD of the myelomatous bones in saline-treated hosts was similarly reduced in SCID-rab and SCID-hu mice by 14±5%, it was increased by 10±2% in iPTH-treated hosts (p<0.002 saline vs. iPTH-treated hosts). Histologic and histomorphometric analyses revealed increased bone formation parameters and no effect on number of osteoclasts. The bone anabolic effect of iPTH was associated with reduced myeloma growth by >50% (p<0.03) assessed by measurement of human immunoglobulin level in mice sera and histologically. Treatment with iPTH also increased BMD and attenuated myeloma growth in SCID-rab mice engrafted with myeloma cells from 10 patients. We found by qRT-PCR that type-1 PTH receptor was not expressed by myeloma cells and that PTH had no direct effect on in vitro growth of myeloma cells indicating that PTH anti-myeloma effect is indirectly mediated through modulation of the BM microenvironment. To shed light on molecular mechanisms associated with iPTH effects, human GEP and qRT-PCR validation of selected genes were preformed on whole myelomatous human bones from Hg-bearing SCID-hu mice treated with saline or iPTH for 4 weeks (5 hosts/group). Mice were sacrificed 2 hours after the last injection. Treatment with iPTH upregulated 343 genes and downregulated 410 genes ('2 folds, p<0.05) in myelomatous bones. There was a remarkable alteration in expression of genes associated with cAMP (e.g. RGS1/2 upregulation) and Wnt (e.g. LRP4 upregulation; DKK1 downregulation) signaling, upregulation of growth factors and receptors involved in bone remodeling (e.g. FGFR1/2, FDGFA, FDGFRA, TGFB, TGFBR1), and increased expression of osteoblast markers (e.g. osteocalcin, RUNX2). Interestingly, although iPTH induced upregulation of RANKL there was a reduction in expression of osteoclastic genes (e.g. ACP5/TRAP, NFATC1), probably due to increased osteoblast numbers, downregulation of inflammatory genes (e.g. AIF1) and upregulation of anti-inflammatory genes (e.g. TNFAIP6, CXCL14). Moreover, iPTH reduced expression of typical myeloma associated genes (e.g. CD38, WHSC1, IRF4) and had no effect on expression of myeloma growth factors such as IL6 and IGF1. Expression of certain documented anti-myeloma factors was upregulated by iPTH (e.g. decorin). We conclude that iPTH-induced bone formation in myelomatous bones is mediated by activation of multiple signaling pathways involved in osteoblastogenesis, and attenuated bone resorption and myeloma growth through mechanisms involving increased production of anti-myeloma factors by osteoblasts and minimizing inflammatory conditions induced by myeloma. Treatment with iPTH may be a promising approach for myeloma bone disease and tumor progression. Disclosures:No relevant conflicts of interest to declare.

Human Placenta-Derived Adherent Cells Delivered Intralesionally Inhibit Myeloma Bone Disease and Tumor Growth, While Intravenously Are Capable of Trafficking to Myelomatous Bone.

AbstractAbstract 3717Human placenta has emerged as a valuable, uncontroversial source of transplantable cells for many cytotherapeutic purposes, including modulation of inflammation, bone repair, and cancer. Placenta-derived adherent cells (PDAC) are mesenchymal like adherent cells isolated from postpartum human placenta and capable of supporting bone formation in vivo. Multiple myeloma (MM) is closely associated with induction of bone disease and large lytic lesions, which are often not repaired and are usually the sites of relapses. The aim of the study was to evaluate the antimyeloma therapeutic potential, in vivo survival, and trafficking of PDAC in the SCID-rab model of MM-associated bone disease. SCID-rab system constructed by implanting a 4-weeks old rabbit bone into which primary human myeloma cells were directly injected (Yatta et al., Leukemia 2004; Yaccoby et al., Blood 2007). Bone disease was evaluated by measurements of bone mineral density (BMD) and X-rays. MM growth was determined by human immunoglobulin (hIg) ELISA and histologically. For in vivo tracking PDAC were transduced with a luciferase/GFP reporter in a lentiviral vector. SCID-rab mice engrafted with primary myeloma cells from 2 patients. Upon establishment of MM growth, PDAC (1×106 cells/bone) or vehicle were injected into the implanted myelomatous bone (Patient's 1, 5 mice/group; Patient's 2, 7 mice/group). While BMD of the implanted bones was significantly reduced in control hosts, intralesional PDAC cytotherapy significantly increased BMD of the implanted bones from pretreatment levels by >37% (p<0.01 versus control) and inhibited MM growth in the 2 sets of experiments (p<0.04). The bone anabolic effect of PDAC was associated with increased number of osteoblasts (p<0.003) and reduced number of osteoclasts (p<0.004). Intralesional PDAC cytotherapy also promoted bone formation in nonmyelomatous SCID-rab mice. Intralesional but not subcutaneous engraftment of PDAC inhibited bone disease and tumor growth in SCID-rab mice. In contrast to intra-bone injection in SCID-rab mice, intra-tumor injection of PDAC had no effect on subcutaneous growth of the H929 myeloma cell line in SCID mice (8 mice/group). Live-animal imaging revealed that the majority of PDAC disappeared from the injected bones within 4 weeks. To test their systemic behavior, PDAC were intravenously injected into 18 SCID-rab mice engrafted with H929 myeloma cells. The presences of PDAC in various organs were evaluated 1, 2 and 7 days after injection. Ex vivo bioluminescence analysis of the implanted myelomatous bones detected PDAC in two of five bones on day 1, in four of four bones on day 2, and in four of nine bones on day 7. Intravenously injected PDAC were also detected in lungs not in any other murine tissues. Immunohistochemical staining for GFP in myelomatous bone sections detected GFP-expressing PDAC in rabbit marrow areas infiltrated with myeloma cells, supporting bioluminescence analysis. Our study suggest that PDAC stimulate bone formation by acting as bystander cells that increase endogenous osteoblastogenesis and inhibit osteoclastogenesis, and that alteration of the bone marrow microenvironment by PDAC attenuates growth of MM. PDAC cytotherapy is a promising therapeutic approach for myeloma bone disease. Disclosures:Khan:Celgene Cellular Therapeutics: Research Funding. Heidaran:Celgene Cellular Therapeutics: Employment. Pal:Celgene Cellular Therapeutics: Employment. Zhang:Celgene Cellular Therapeutics: Employment. He:Celgene Cellular Therapeutics: Employment. Zeitlin:Celgene Cellular Therapeutics: Employment. Abbot:Celgene Cellular Therapeutics: Employment. Faleck:Celgene Cellular Therapeutics: Employment. Hariri:Celgene Cellular Therapeutics: Employment.

Bruton's Tyrosine Kinase (BTK) Is Indispensable for Myeloma Cell Migration towards SDF-1 and Induction of Osteoclastogenesis and Osteolytic Bone Disease

AbstractAbstract 447Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase of the TEC family of protein tyrosine kinases, is preferentially expressed in the hematopoietic cells and involved in B-lymphocytes survival and differentiation, and their migration towards SDF-1 gradient. BTK is also expressed in osteoclast precursors and plays indispensable role in osteoclastogenesis (Shinohara et al., Cell 2008). LFM-A13 is small molecule inhibitor of BTK and TEC, and can be tolerated even when animals are given daily dose of 100 mg/kg. The aims of the study were to investigate the effect of LFM-A13 on myeloma cell migration and growth in vitro, and on myeloma bone disease and tumor growth in the SCID-rab model. Our clinical gene expression profiling data indicate that in contrast to most myeloma cell lines, primary myeloma cells express BTK and that relative to normal plasma cells, BTK expression is upregulated in myeloma cells molecularly classified in the CD-1, CD-2, HY, LB and MF subtypes. Expression of BTK at the RNA and proteins levels in myeloma cells and osteoclast precursors was validated using qRT-PCR and Western Blot. To test effect on migration, myeloma cells expressing BTK and cell surface CXCR4 (n=5) were placed in the top of transwell inserts in the absence and presence of LFM-A13 (50 μM) and their migration towards SDF-1 (30 nM) was evaluated after 18 hours. SDF-1 induced migration of myeloma cells by >2.5 folds, an effect that was markedly inhibited by LFM-A13 (p<0.01). At similar concentration, this agent modestly reduced growth of BTK-expressing myeloma cells by 20%, assessed by MTT assay. LFM-A13 dose dependently inhibited osteoclast formation by 25% at 10 μM (p<0.0005) and by 80% at 40 μM (p<0.0001). In vivo, SCID-rab mice engrafted with a novel myeloma cell line, DAS, established through sequential passaging in this animal model (Xin et al., BJH 2007). DAS cells do not grow in culture, are molecularly classified as MF subtype and express high level of BTK. Upon establishment of myeloma growth, 2 weeks after tumor cell injection, hosts were intraperitoneally treated with 40 mg/kg LFM-A13 or vehicle (10 mice/group) twice a day for 3 weeks. Myeloma bone disease was evaluated by x-rays and analysis of bone mineral density (BMD). Tumor growth was analyzed by measurement of circulating human Ig and histologically. Whereas in the control group BMD of the implanted bone was reduced by 15±4% from pretreatment levels, it remained unchanged (0.8±4% change from pretreatment level) in the LFM-A13 treated hosts (p<0.01 versus control). X-ray radiographs revealed induction of osteolytic lesions in implanted bones of control hosts and that LFM-A13 effectively prevented these effects. LFM-A13 reduced the number of TRAP-expressing osteoclasts in myelomatous bones by >43% (p<0.004). At the end of the experiment, tumor burden was insignificantly lower in hosts treated with the LFM-A13 (Ig levels 42±15 and 19±5 μg/ml in control and LFM-A13 groups, respectively). We conclude that BTK is indispensable for SDF-1-indcued myeloma cell migration and stimulation of osteoclastogenesis, and that pharmacologic inhibition of BTK effectively inhibits myeloma-induced bone resorption and may interrupt with myeloma cell homing and metastasis to bone. Disclosures:No relevant conflicts of interest to declare.

An Acidic Milieu Created In Myeloma-Osteoclast Interaction Enhances Tumor Growth, but Triggers Anti-Myeloma Activity of Reveromycin A, a Novel Anti-Resorptive Agent

AbstractAbstract 454Multiple myeloma (MM) develops and expands in the bone marrow, and causes devastating bone destruction by enhancing osteoclastic bone resorption in their close vicinity. In MM bone lesions, thus induced osteoclasts (OCs) in turn enhance MM cell growth and survival, thereby forming a vicious cycle between the progression of bone destruction and MM tumor expansion. Such cellular interactions create an acidic milieu not only through acids produced by OCs but also through a large amount of lactate by proliferating tumor cells (Warburg effect). Reveromycin A (RM-A), a small microbial metabolite, preferentially induces cellular apoptosis in an acidic milieu, and draws considerable attention as a novel anti-resorptive agent. In the present study, we explored whether an acidic condition induced by MM-OC interaction affects MM expansion and whether RM-A targets not only OCs but also such an acidic microenvironment to regress tumor expansion in MM. INA6 and RPMI8226 MM cells potently enhanced osteoclastogenesis and osteoclastic pit formation when cocultured with rabbit bone marrow cells on bone slices. Notably, large multinucleated OCs were almost completely disappeared and pit formation on bone slices was abolished upon the treatment with RM-A at concentrations as low as 100nM. The cocultures with rabbit bone marrow cells stimulated INA6 MM cell growth; RM-A at 1microM was however able to substantially decrease the MM cell viability in the cocultures after 12 hours, although RM-A at this concentration did not affect MM cell growth when MM cells were cultured alone at pH7.4. The suppression of INA6 MM cell viability by RM-A was obviously more potent than that under bisphosphonate treatment in which mature OCs and pits on bone slices similarly decreased in number, suggesting that the anti-MM effects of RM-A is not merely due to depletion of mature OCs. Blockade of acid release by the proton pump inhibitor concanamycin A abolished such RM-A effects. Because an acidic microenvironment increases cell permeability of RM-A to cause apoptosis, it is plausible that a highly acidic milieu created by OC-MM interaction allows RM-A to act on nearby MM cells as well as OCs. In order to clarify a role of tumor acidity in RM-A-triggered cell death, we examined the effects of RM-A on MM cell growth upon acidification with lactic acid. When lactic acid was added to media to adjust their pH to be 7.0 and 6.75, the growth of INA6 and RPMI8226 MM cells was enhanced up to 150 and 120%, respectively, after 24 hours compared to that at pH7.4. However, RM-A at 1microM induced cell death in these MM cells at pH7.0 (60-70% reduction of alive MM cells compared to those at pH7.4) and at pH6.75 (>90%), suggesting cytocidal effects of RM-A on lactate-producing MM cells densely proliferated in an acidic milieu. Because metoformin, anti-diabetic agent, up-regulates lactate production through stimulation of glycolysis, we next examined the effects of RM-A on MM cells in combination with metoformin. Metoformin dose-dependently enhanced lactate production by MM cells to decrease pH in their culture media over time; RM-A at 1microM showed potent cytotoxic effects on MM cells upon 24-hour preceded treatment with metoformin at 5 mM even when MM cells were started to be cultured at pH7.4, suggesting induction of anti-MM activity of RM-A with metoformin. Finally, in vivo RM-A effects were studied using INA6 MM cell-bearing SCID-rab mice. We injected RM-A sc at 4mg/kg twice daily for 18 days to the mice after confirming MM cell growth at 4 weeks after the MM cell inoculation. The RM-A treatment substantially decreased osteolytic lesions in X-ray and microCT images and MM tumor area in bone sections along with a reduction of INA6 cell-derived human soluble IL-6 receptor levels in mouse sera as a marker of MM tumor burden. These results collectively suggest that acidic microenvironment produced by MM-OC interaction enhances MM tumor progression but can trigger cytotoxic effects of RM-A on MM cells besides acid-producing OCs. RM-A may become a candidate for a novel therapeutic agent against MM with extensive bone resorption. Disclosures:No relevant conflicts of interest to declare.

Systemically Transplanted Human Bone Marrow Mesenchymal Stem Cells Primarily Traffic to Mesenteric Lymph Nodes

AbstractAbstract 2580Intravenously administered mesenchymal stem cells (MSCs) are trapped in pulmonary vascular bed and only few MSCs home to bone or other tissues in physiological or pathological conditions. Following intracardiac injection MSCs pass the lung barrier but their homing to bone and tissue localization is uncertain. The aim of the study was to investigate trafficking and exact localization of human MSCs following intracardiac injection into unchallenged mice and a xenograft bone tumor model. MSCs were isolated from human fetal bones (ABR Inc, Alameda CA) and expanded in DMEM-LG medium supplemented with 10% FBS. Global gene expression profiling revealed that the cultured MSCs were devoid of hematopoietic cells and expressed typical mesenchymal markers such as CD166, CD146 and CD90. We have previously shown that these MSCs are capable of differentiation into osteoblasts and adipocytes and retain their differentiation potential after multiple passages (Haematologica 2006). The MSCs were transduced with a luciferase/GFP reporter in a lentiviral vector and were maximally passaged 8 times before used in vivo. Detection of MSCs in mice was determined by live-animal imaging and ex vivo bioluminescence activity using the IVIS system, by microscopic examination of GFP-expressing cells and by immunohistochemistry for GFP. MSCs (1×106 cells/mouse) were intracardiacly injected into unconditioned SCID mice (n=8) using Dovetail Slide Micromanipulator that ensures accurate injection. Following 2 or 7 days after MSC injection to SCID mice, live-animal imaging revealed bioluminescence activity mainly in the mice abdomen but not bone, while ex vivo examination detected MSCs in various abdominal organs, primarily in reproductive organs, intestine and pancreas. Careful microscopic examination revealed localization of MSCs in draining lymph nodes attached to these organs by connective tissue. Immunohistochemistry showed GFP-expressing MSCs in the adjacent mesenteric lymph nodes but not within the organs. To confirm our findings, MSCs were intracardially injected into C57BL6 mice (n=6) that harbor functional lymph nodes. Evans blue dye which is known to accumulate in and identify lymph nodes, was injected into the rear footpad or lateral tail base of the mice, 3 hours after MSC injection and 30 minutes prior to bioluminescence and florescence analyses. The Evans blue dye and GFP positivity were co-localized, indicating specific trafficking of MSCs to lymph nodes. Culturing of the dissected lymph nodes resulted in release of GFP-expressing MSCs which regained their in vitro morphology. For testing MSCs trafficking in a xenograft model, we used our SCID-rab system constructed by implanting a 4-weeks old rabbit bone into which human myeloma cells were directly injected (Leukemia 2004; Blood 2007). In this model myeloma cells grow restrictively in the implanted bone. MSCs injected intracardiacly into SCID-rab mice were mostly found in mesenteric lymph nodes but were also detected in the myelomatous bone 72 hours after MSCs injection, validating the ability of tumor cells to attract MSCs and that these MSCs are capable of transmigration. We conclude that MSCs primarily traffic to draining lymph nodes, partially explaining their in vivo immunomodulatory activity, and that understanding the mechanism by which MSCs traffic to lymph nodes may help develop approaches to shift their homing to desired organs. Disclosures:No relevant conflicts of interest to declare.

Epidemiology and risk factors for osteonecrosis of the jaw in cancer patients

Osteonecrosis of the jaw (ONJ), previously an entity associated with radiation therapy to the head and neck, has been observed in patients treated with bisphosphonates. Patients with metastatic breast cancer and myelomatous bone disease, commonly treated with high-potency nitrogen-containing bisphosphonates for a prolonged period of time, have the greatest risk of ONJ development. The reported frequency of ONJ ranges from 0.6% to 6.2% in breast cancer and from 1.7% to 15% in patients with multiple myeloma. Osteonecrosis of the jaw has also been observed in patients with other cancers such as prostate cancer and in benign bone disorders such as osteoporosis and Paget's disease in which the incidence is low. Risk factors associated with the development of ONJ include dental extractions, length of bisphosphonate treatment, and the type of bisphosphonate used. In this review, we summarize the reported incidence and risk factors associated with ONJ.

Assessment of Bishosphonate Activity In Vitro

Bisphosphonates are a class of drugs developed over the past three decades for the treatment of metabolic bone diseases with high bone turnover, such as Paget's disease, tumor associated osteolysis and osteoporosis. The exceptional pharmacokinetic profile of bisphosphonates makes them very suitable and safe drugs for the treatment of bone diseases, because, by conventional administration, osseous tissue and bone resorbing osteoclasts are the targets for these drugs as a result of the very high affinity of bisphosphonates for bone mineral. Several recent studies have demonstrated, however, that bisphosphonates decrease tumor burden in bone in rodent models of myeloma and metastatic bone disease, with suggestions of antitumor effects also in patients. Although decreased tumor burden could be a consequence of inhibition of bone resorption, there is increasing evidence that bisphosphonates might also have direct effects on tumor cell in vivo, since effects on tumors outside of skeleton or at doses not inhibiting bone resorption have been reported. Recent studies also suggest that bisphosphonates have inhibitory effect also on endothelial cell function and angiogenesis in tumor tissue. These findings suggest that the target cells for bisphosphonates as well as their molecular mechanism of action may be more diverse and complex than realized so far. This review highlights the main methodologies used to monitor the action of BPs in vitro cell models, with a special emphasis on the detection of BP-induced ATP-analoques by mass spectrometry. In addition, cell death monitoring, immunomodulatory effects and inhibition of growth/proliferation are described.

Abstract 558: Constitutive activation of p38 MAPK signaling in tumor cells induces osteolytic bone lesions via regulating bone marrow microenvironment

Abstract Bone residing tumor cells cause osteolysis, characterized with severe bone pain, multiple fractures and hypercalcemia in patients. While it has been proposed that tumor cells activate osteoclasts and/or inhibit osteoblasts, leading to bone resorption, the mechanism underlying tumor-induced osteoclastogenesis activation in bone marrow microenvironment, which causes osteolytic bone lesions, is yet to be elucidated. P38 mitogen-activated protein kinase (MAPK) is commonly constitutively activated in malignant osteolytic tumors. We hypothesize that constitutive activity of p38 MAPK in tumor cells might contribute to osteolytic bone lesions via regulating bone marrow microenvironment. For this purpose, we applied of small hairpin RNAs (shRNAs) to disrupt p38 MAPK activity in myeloma ARP-1 and bone metastatic breast cancer MDA-MB-231 cells. To evaluate the role of tumor p38 MAPK in bone lesions, tumor cells with p38 MAPK-shRNAs or control vectors were intravenously injected to SCID mice. Lytic lesions in tibial bones were measured by radiograph and μ-CT, and further confirmed by histological examination. The results showed that lytic bone lesions were found in mice bearing of tumor cells with control vectors. In contrast, no or less bone lesions were found in mice with shRNA-expressing tumor cells. Moreover, decrease of osteoclast amounts covered bone surface, as well as circulating levels of collagen type I (bone resorption marker) and TRAP5b (active osteoclast marker), but increase of osteoblast amounts, and osteocalcin (bone formation marker) and ALP (active osteoblast marker), were found in those mice injected with shRNA-tumor cells. Noticeably, tumor p38 MAPK enhanced osteoclast differentiation and activity, as well as inhibited osteoblast maturation and function in vitro. However, disruption of p38 MAPK activity in tumor cells did not affect tumor growth and tumor cell homing to bone. We further identified that tumor p38 MAPK-derived monocyte chemotactic protein-1 (MCP-1) and dickkopf-1 (DKK-1) induced bone lesions via activation of RANK/RANKL-mediated NFκB, p38 MAPK and ERK1/2 signaling pathways. In conclusion, our results elucidate a novel mechanism that tumor p38 MAPK activity regulates bone marrow microenvironment and contributes to tumor-induced osteolytic bone lesions. Thus, inhibition of p38 MAPK signaling in tumor cells may have an important implication to treat bone lesions in patients with osteolytic tumors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 558.