Development Of Osteolytic Bone Lesions Research Articles

The checkpoint inhibitor PD-1H/VISTA controls osteoclast-mediated multiple myeloma bone disease

Multiple myeloma bone disease is characterized by the development of osteolytic bone lesions. Recent work identified matrix metalloproteinase 13 as a myeloma-derived fusogen that induces osteoclast activation independent of its proteolytic activity. We now identify programmed death-1 homolog, PD-1H, as the bona fide MMP-13 receptor on osteoclasts. Silencing PD-1H or using Pd-1h-/- bone marrow cells abrogates the MMP-13-enhanced osteoclast fusion and bone-resorptive activity. Further, PD-1H interacts with the actin cytoskeleton and plays a necessary role in supporting c-Src activation and sealing zone formation. The critical role of PD-1H in myeloma lytic bone lesions was confirmed using a Pd-1h-/- myeloma bone disease mouse model wherein myeloma cells injected into Pd-1h-/-Rag2-/- results in attenuated bone destruction. Our findings identify a role of PD-1H in bone biology independent of its known immunoregulatory functions and suggest that targeting the MMP-13/PD-1H axis may represent a potential approach for the treatment of myeloma associated osteolysis.

Biclonal myeloma in renal failure.

Multiple myeloma is a clonal proliferation of the plasma cell line that accounts for approximately 10% of all hematological malignancies. It is characterized by abnormal growth of plasma cells producing monoclonal immunoglobulin or light chain (paraprotein), with subsequent development of osteolytic bone lesions, anemia, hypercalcemia, and renal failure. In 3-6% of myeloma patients, more than one monoclonal protein (usually two) is discovered, with different heavy or light chain or both. These additional monoclonal proteins may be identified at the time of diagnosis or appear later during an observation or therapy. The authors describe two patients with biclonal myeloma, one diagnosed during evaluation for newly discovered renal failure, and one identified in the course of treatment of monoclonal gammopathy. The discussion of the diagnosis, natural history, and prognosis in patients with biclonal myeloma are also reported.

Checkpoint Inhibitor PD-1H/VISTA Functions As MMP-13 Receptor on Osteoclasts and Mediates MMP-13 Induced Osteoclast Activation in Multiple Myeloma

Introduction Multiple myeloma (MM) bone disease is characterized by the development of osteolytic bone lesions due to the over-activation of osteoclast and inhibition of osteoblast cells. MM cells secret pro-osteoclastogenic factors which lead to osteoclast (OCL) activation. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor which is highly secreted by MM cells. MMP-13 induces osteoclast fusion and bone-resorption by triggering the ERK1/2-dependent up-regulation of the cell fusogen, DC-STAMP. This process operates independently of the MMP-13 proteolytic activity. The fact that MMP-13 regulates OCL signaling via a proteolytic independent mechanism suggests that MMP-13 functions as a paracrine message protein mediating the crosstalk between MM cells and OCL. However, the mechanism is yet to be identified. Methods and Results To screen for MMP-13 cellular binding proteins/receptors, we performed a MMP-13 pull-down assay wherein recombinant MMP-13-His6 was incubated with mouse mononuclear bone marrow cells (BMCs) lysates and Ni-NTA magnetic beads were used to pull-down MMP-13- associated proteins. Following mass spectral analysis, programmed death-1 homolog (PD-1H) was identified as a major MMP-13-binding protein. PD-1H, also known as V-domain Ig suppressor of T cell activation (VISTA), is a critical negative checkpoint regulator expressed on myeloid cells and involved in immune responses. Binding of MMP-13 and PD-1H was confirmed by co-expressing both proteins in HEK293 cells and subsequent co-immunoprecipitation assay. Further, following PD-1H expression in HEK293 cells, an MMP-13-GFP fusion fluorescence protein docked to the cell surface where mutagenesis studies demonstrated that the PD-1H extracellular domain (ECD) mediates the specific binding interaction. Western blot and immunohistochemistry revealed that PD-1H was highly expressed in mononuclear BMCs, pre-OCL and mature OCL. ShRNA-mediated knockdown of PD-1H in mouse mononuclear BMCs blocked the ability of MMP-13 to induce osteoclast fusion and activation. These results were further confirmed by using BMCs from Pd-1h-/- mice and WT littermates for in vitro osteoclast differentiation. While MMP-13 induced WT OCL fusion and activation, these effects were completely blocked in Pd-1h-/-OCLs in tandem with a loss in MMP-13 induced ERK1/2 phosphorylation, NFATc1 and DC-STAMP upregulation. Conclusions Taken together, our study, for the first time, revealed that checkpoint inhibitor PD-1H is highly expressed in pre- and mature osteoclast. More importantly, we identified PD-1H as the critical receptor for MMP-13 in OCL, thereby mediating MMP-13-induced OCL fusion, activation and bone resorption. Hence, MMP-13 as a novel PD-1H ligand might not only induce bone disease, but also play a potential role in the regulation of T-cell activity in MM. As such, targeting MMP-13 and PD-1H interactions may represent a novel therapeutic strategy to treat MM bone disease and modulate the MM immune environment. Disclosures Lentzsch: Abbvie: Consultancy; Clinical Care Options: Speakers Bureau; Sanofi: Consultancy, Research Funding; Multiple Myeloma Research Foundation: Honoraria; International Myeloma Foundation: Honoraria; Karyopharm: Research Funding; Columbia University: Patents & Royalties: 11-1F4mAb as anti-amyloid strategy; Bayer: Consultancy; Janssen: Consultancy; Takeda: Consultancy; BMS: Consultancy; Proclara: Consultancy; Caelum Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Glutamine Depletion By Addicted Myeloma Cells Inhibits Osteoblastic Differentiation of Bone Marrow Mesenchymal Stromal Cells Limiting Asparagine Availability: A Possible New Mechanism for Myeloma Bone Disease

Metabolic alterations of cancer cells, aimed at sustaining their growth, may also influence the behavior of the tumor microenvironment. Our group has recently demonstrated that multiple myeloma (MM) is a highly glutamine(Gln)-addicted tumor that utilize huge amounts of Gln to fuel its metabolism through the enzyme glutaminase (GLS). For this reason, MM cells exhibits increased Gln uptake, mainly through the ASCT2 transporter. Interestingly, lower bone marrow (BM) plasma Gln concentration (down to a median value of 0.4 mM vs. a median value of 0.6 mM) was found in MM patients as compared with smoldering MM (SMM) and Monoclonal Gammopathy of Uncertain Significance (MGUS). The main feature of MM BM microenvironment is the suppression of osteoblastic (OB) differentiation leading to the development of osteolytic bone lesions, the hallmark of MM. Most recently, it has been demonstrated that Gln metabolism is needed to sustain bone mass formation in murine models and that GLS inhibition decreases OB differentiation of human mesenchymal stromal cells (hMSCs). However, no information is yet available on the role of Gln depletion imposed by MM cell metabolism on OB differentiation into the BM. This topic has been investigated in the present study. Firstly, human MM cells were co-cultured with BM hMSCs, and Gln medium concentration was evaluated with mass spectrometry (MS), demonstrating a MM-induced depletion of the amino acid. Upon Gln depletion, MSC exhibited a sustained induction of Glutamine Synthetase (GS). On the contrary, when differentiated in osteogenic medium (D-MEM + 5% Fetal Bovine Serum, supplemented with 2 mM Gln, ascorbic acid and dexamethasone), GS was suppressed. Conversely, GLS (both KGA and GAC isoforms) and SLC38A2, the gene for the concentrative Gln transporter SNAT2, were induced. These data suggest that hMSCs differentiation in OBs is associated with an increased dependence upon extracellular Gln. Consistent with this conclusion, the activity of SNAT2 was absent in undifferentiated hMSCs but well detectable after 14 days of OB differentiation, when total Gln uptake was also increased. Under the same conditions, OB differentiation markers (RUNX2, COL1A1, ALPL expression and ALPL activity or staining) were significantly induced but their expression was blunted by incubation in low-Gln (0.4 mM) medium or in the presence of the SNAT2 inhibitor MeAIB. The incubation in Gln-free D-MEM suppressed the induction of GLS and SLC38A2 along with OB differentiation, which was restored by the supplementation of Non-Essential Amino Acids (NEAA). Among NEAA, only asparagine (Asn) was able to rescue OB differentiation in the absence of Gln. The determination of intracellular amino acids with MS indicated that OB differentiation was associated with the increase of cell Asn, without significant changes of Gln, glutamate (Glu) or aspartate (Asp). Asparagine Synthetase (ASNS), the Gln-dependent enzyme that accounts for Asn synthesis, was also found induced during OB differentiation of hMSCs. Gene Expression Profiles of primary BM hMSCs and OBs from bone biopsies of both healthy donors (n=7) and MM patients (n=16) indicated that GLS, ASNS, and SLC38A2 are more expressed in OBs, while the expression of GLUL, the gene for GS, is higher in undifferentiated hMSCs from healthy donors. Overall, these results indicate that (1) OB differentiation of hMSCs is Gln-dependent; (2) the partial Gln depletion, imposed by Gln-addicted MM cells in the BM microenvironment, contributes to the impairment of osteoblastic differentiation of hMSCs; (3) hindrance of differentiation may depend on the limited availability of intracellular Asn derived from Gln-dependent ASNS. These results support the evidence that Gln addiction of MM cells affects bone microenvironment leading to the inhibition of OB differentiation and, consequently, to the development of MM bone disease. Disclosures Giuliani: Janssen: Research Funding.

Selective Targeting of Multiple Myeloma By Bcma-Specific Central Memory CD8+ cytotoxic T Lymphocytes: A Potential Immunotherapeutic Application in Multiple Myeloma and Other Plasma Cell Disorders

Background:Multiple myeloma (MM) is a B-cell malignancy characterized by the clonal proliferation and accumulation of malignant plasma cells in the bone marrow and development of osteolytic bone lesions. Despite recent advances in treatment using novel therapeutics, MM remains incurable with high mortality rates. We have demonstrated in preclinical studies that CD8+ cytotoxic T lymphocytes (CTL) generated with immunogenic HLA-A2 or HLA-A24 peptides targeting XBP1(X-box binding protein 1), CD138 (Syndecan-1) and CS1 (SLAMF7) antigens induces robust cytotoxic activities against MM. The Phase 1/2a trials were completed or are in progress in the patients with smoldering multiple myeloma or triple negative breast cancer, respectively, using the HLA-A2 XBP1/CD138/CS1 multipeptide vaccine. The clinical data demonstrated that the multipeptide vaccine is safe and induces the XBP1/CD138/CS1-specific immune responses, evidenced by expansion of peptides-specific Tetramer+/CD45RO+memory CTL and Th-1 specific immune responses. Moreover, clinical trials combining with Lenalidomide or checkpoint inhibitor enhanced the CD8+ CTL activities induced by multipeptide vaccine, indicating the benefit of combination therapy. To expand the breadth and extent of the antigens-specific immunotherapy beyond XBP1/CD138/CS1, we have recently identified additional tumor-associated antigens (TAA) on tumor cells obtained from newly diagnosed MM patients (N=616). Here, we introduce a novel heteroclitic peptides specific to BCMA, the receptor forbinding of B cell activating factor (BAFF) and a proliferation-inducing ligand (APRIL). Due to its restricted expression pattern on MM cells and plasma cells along with its critical role in promoting MM cell growth, survival and drug resistance, we are currently in development of novel immunotherapeutic to target BCMA on MM cells with different therapeutic approaches.Objective:The aims of current study were to target BCMA as a TAA by generating the antigen-specific memory CD8+ CTL to induce effective and long-lasting immune response against MM.Findings:We report on immunogenic HLA-A2-restricted peptides derived from BCMA, which are capable of evoking antigen-specific immune responses against MM. The heteroclitic BCMA peptides displayed improved binding affinity/stability to HLA-A2 molecules from their native BCMA peptides. To define immunogenicity of the selected peptides, we generated BCMA-specific CTL (BCMA-CTL) by repeated stimulationof CD3+ T cells with respective heteroclitic peptide. The BCMA-specific engineered peptides evoked the expansion of antigen-specific CD8+ CTL and generated BCMA-CTL displaying high T cell activation (CD38, CD69) and co-stimulatory (CD40L, OX40, GITR) molecules expression. Additionally, a gradual expansion was observed in BCMA-specific memory CD8+ T cells, with a corresponding decrease in naïve CD8+ T cells. The BCMA-CTL demonstrated robust poly-functional immune responses with Th1-specific anti-MM activities [high IFN-g/IL-2/TNF-aproduction, CD8+ T cells proliferation, cytotoxicity] in antigen-specific and HLA-A2-restricted manner. The functional activities were directly correlated with the expansion of central memory CD8+ CTL in the BCMA-CTL generated from different HLA-A2+ individuals (Donor 1 BCMA-CTL: 81.0%, Donor 2 BCMA-CTL: 82.6%, Donor 3 BCMA-CTL: 67.0%). Finally, the combination with checkpoint inhibitor (anti-LAG3) or immune agonist (anti-OX40) enhanced the anti-tumor activities of BCMA-CTL, along with the induction of cytotoxic activities by central memory CD8+ T cell subset. Therefore, these studies suggest that heteroclitic BCMA peptides offer a therapeutic potential to effectively generate BCMA-specific CD8+ CTL targeting MM.Significance:Here, we introduce novel immunogenic engineered heteroclitic BCMA peptides capable of inducing antigen-specific memory CD8+ CTL with robust poly-functional immune responses against MM. These results provide the framework for therapeutic application of heteroclitic BCMA peptides in MM patients. They further support combination treatment options incorporating BCMA peptides-specific vaccine or BCMA peptides-specific adoptive T cells immunotherapy with anti-LAG3 and/or anti-OX40for patients with myeloma or other diseases expressing BCMA. DisclosuresMunshi:OncoPep: Other: Board of director. Anderson:Celgene: Consultancy; Takeda Millennium: Consultancy; Bristol Myers Squibb: Consultancy; Gilead: Membership on an entity's Board of Directors or advisory committees; Oncopep: Equity Ownership; C4 Therapeutics: Equity Ownership.

Role of Osteocytes in Myeloma Bone Disease: Anti-sclerostin Antibody as New Therapeutic Strategy.

Osteocytes are terminally differentiated cells of the osteoblast lineage. They are involved in the regulation of bone remodeling by increasing osteoclast formation or decreasing bone formation by the secretion of the osteoblast inhibitor sclerostin. Monoclonal antibody anti-sclerostin, Romosozumab, has been developed and tested in clinical trials in patients with osteoporosis. In the last years, the role of osteocytes in the development of osteolytic bone lesions that occurs in multiple myeloma, have been underlined. Myeloma cells increase osteocyte death through the up-regulation of both apoptosis and autophagy that, in turn, triggers osteoclast formation, and activity. When compared to healthy controls, myeloma patients with bone disease have higher osteocyte cell death, but the treatment with proteasome inhibitor bortezomib has been shown to maintain osteocyte viability. In preclinical mouse models of multiple myeloma, treatment with blocking anti-sclerostin antibody increased osteoblast numbers and bone formation rate reducing osteolytic bone lesions. Moreover, the combination of anti-sclerostin antibody and the osteoclast inhibitor zoledronic acid increased bone mass and fracture resistance synergistically. However, anti-sclerostin antibody did not affect tumor burden in vivo or the efficacy of anti-myeloma drugs in vitro. Nevertheless, the combination therapy of anti-sclerostin antibody and the proteasome inhibitor carfilzomib, displayed potent anti-myeloma activity as well as positive effects on bone disease in vivo. In conclusion, all these data suggest that osteocytes are involved in myeloma bone disease and may be considered a novel target for the use of antibody-mediated anti-sclerostin therapy also in multiple myeloma patients.

Abstract 38: Mitophagy deficiency promotes osteolytic bone metastasis in breast cancer

Abstract Introduction: Metastasis of breast cancer to the bone is a common complication and occurs in 65-80% of late-stage patients. Specifically, the development of osteolytic bone lesions increases patient morbidity and mortality with debilitating skeletal complications. Mitophagy is a selective form of autophagy that removes damaged or excessive mitochondria in response to various metabolic stresses. However, the role of mitophagy in development of cancer remains largely unclear. The Janus-faced role of mitophagy during tumorigenesis leads to a novel challenge when targeting it for cancer therapies. This study focuses on the role of mitophagy deficiency in breast cancer osteolytic bone metastasis, and explores the potential of rescuing mitophagy deficiency as possible therapeutic targets for metastatic disease. Methods: Specific single-guide RNAs (sgRNAs) were used to deplete ULK1 expression in breast cancer cell lines. Mitophagy was monitored by detecting Tom20 and Hsp60. In addition, mitochondrial DNA (mtDNA) was used to further confirm the reduction of mitochondrial mass. For bone metastasis studies, luciferase-tagged tumor cells were injected into the left cardiac ventricle of anesthetized female nude mice. Metastatic progression was monitored by bioluminescence imaging (BLI). Micro-CT imaging was used to detect bone volume, cortical wall thickness, trabecular thickness and number in mice of tibia. TRAP assay was used to detect osteoclast differentiation assays. Results: We found that the core Atg protein ULK1 was not only expressed at low level in breast cancer, but also required for hypoxia-induced mitophagy. ULK1-related mitophagy deficiency promoted the formation of metastatic osteolytic lesions in experimental bone metastasis mouse model with MDA-MB-231 cells. TRAP staining revealed that mature osteoclasts significantly increased in RAW264.7 cells treated with CM from ULK1-knockout breast cancer cells. In addition, MEK inhibitor trametinib was sufficient to upregulate the expression of ULK1 and reduced the release of cytokines, which is key to osteoclast differentiation via triggering mitophagy, thereby effectively blocking breast cancer osteolytic bone metastasis. Conclusion: In conclusion, we demonstrate the functional importance of mitophagy defect in breast cancer for promoting osteolytic bone metastasis. We also provide strong preclinical evidence for using MEK-ERK pathway inhibitors to disrupt ERK kinase-involved ULK1 degradation and induce mitophagy as a breast cancer bone metastasis therapy. Citation Format: Rong Deng, Hai-Liang Zhang, Rui-Zhao Cai, Jun Tang, Xiao-Feng Zhu. Mitophagy deficiency promotes osteolytic bone metastasis in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 38.

Label-free Raman spectroscopy provides early determination and precise localization of breast cancer-colonized bone alterations.

Breast neoplasms frequently colonize bone and induce development of osteolytic bone lesions by disrupting the homeostasis of the bone microenvironment. This degenerative process can lead to bone pain and pathological bone fracture, a major cause of cancer morbidity and diminished quality of life, which is exacerbated by our limited ability to monitor early metastatic disease in bone and assess fracture risk. Spurred by its label-free, real-time nature and its exquisite molecular specificity, we employed spontaneous Raman spectroscopy to assess and quantify early metastasis driven biochemical alterations to bone composition. As early as two weeks after intracardiac inoculations of MDA-MB-435 breast cancer cells in NOD-SCID mice, Raman spectroscopic measurements in the femur and spine revealed consistent changes in carbonate substitution, overall mineralization as well as crystallinity increase in tumor-bearing bones when compared with their normal counterparts. Our observations reveal the possibility of early stage detection of biochemical changes in the tumor-bearing bones - significantly before morphological variations are captured through radiographic diagnosis. This study paves the way for a better molecular understanding of altered bone remodeling in such metastatic niches, and for further clinical studies with the goal of establishing a non-invasive tool for early metastasis detection and prediction of pathological fracture risk in breast cancer.

Biology and Treatment of Skeletal Manifestations in Multiple Myeloma

MM is frequently associated with the development of osteolytic bone lesions, osteoporosis and pathological fractures. Bone destruction in MM is caused by osteoclasts recruited in areas adjacent to myeloma plasma cells; their contact triggers both cell types to secrete soluble factors sustaining one each other’s activation and proliferation. Osteoclasts differentiate and maturate upon binding of the receptor activator of NF-kappaB ligand (RANKL), secreted by bone marrow microenvironmental cells, to its receptor (RANK) on osteoclast progenitors, while osteoprotegerin (OPG), a natural decoy receptor, can block the aforementioned ligation. At the same time osteoblasts are inactivated by the Wnt/β-catenin signaling pathway inhibitor, Dickkopf-1 protein (DKK-1), secreted by malignant plasma cells. Furthermore, DKK-1 deregulates the OPG/RANKL equilibrium, promoting osteoclastogenesis. Myeloma bone disease (MBD) can be treated with myeloma-directed chemotherapy and agents inhibiting bone resorption such as aminobisphosphonates, although new promising biology driven monoclonal antibodies targeting osteoclastogenesis mechanisms are emerging. Palliative MBD treatment includes analgesics, orthotics, radiation therapy, vertebroplasty and kyphoplasty. In case of spinal cord compression, radiation therapy or surgical decompression, should be instantly performed, along with steroid administration. Surgery may also be an option especially in case of weight-bearing bone fractures. MBD is a morbid complication and should be carefully managed because it deteriorates patients’ quality of life and worsens disease outcome.

Oleocanthal Inhibits Proliferation and MIP-1α Expression in Human Multiple Myeloma Cells

Multiple myeloma (MM) is a plasma cell malignancy that causes devastating bone destruction by activating osteoclasts in the bone marrow milieu. MM is the second of all hematological malignancies. Thus, the search for new pharmacological weapons is under intensive investigation being MM a critically important public health goal. Recently, it has been demonstrated that macrophage inflammatory protein 1- alpha (MIP-1 α) is crucially involved in the development of osteolytic bone lesions in MM. Phenolic components of extra virgin olive oil are reported to have anti tumor activity. However, the underlying mechanisms and specific targets of extra virgin olive oil remain to be elucidated. In the present study, we investigated the effects of a recently isolated novel extra virgin olive oil polyphenol, oleocanthal, on the human multiple myeloma cell line ARH-77. Here we report that this natural compound has a remarkable in vitro activity by inhibiting MIP-1 α expression and secretion in MM cells. In addition, we also demonstrated that oleocanthal inhibits MM cells proliferation by inducing the activation of apoptosis mechanisms and by down-regulating ERK1/2 and AKT signal transduction pathways. This in vitro study suggests a therapeutic potential of oleocanthal in treating multiple myeloma.

Inhibiting activin-A signaling stimulates bone formation and prevents cancer-induced bone destruction in vivo

Cancers that grow in bone, such as myeloma and breast cancer metastases, cause devastating osteolytic bone destruction. These cancers hijack bone remodeling by stimulating osteoclastic bone resorption and suppressing bone formation. Currently, treatment is targeted primarily at blocking bone resorption, but this approach has achieved only limited success. Stimulating osteoblastic bone formation to promote repair is a novel alternative approach. We show that a soluble activin receptor type IIA fusion protein (ActRIIA.muFc) stimulates osteoblastogenesis (p < .01), promotes bone formation (p < .01) and increases bone mass in vivo (p < .001). We show that the development of osteolytic bone lesions in mice bearing murine myeloma cells is caused by both increased resorption (p < .05) and suppression of bone formation (p < .01). ActRIIA.muFc treatment stimulates osteoblastogenesis (p < .01), prevents myeloma-induced suppression of bone formation (p < .05), blocks the development of osteolytic bone lesions (p < .05), and increases survival (p < .05). We also show, in a murine model of breast cancer bone metastasis, that ActRIIA.muFc again prevents bone destruction (p < .001) and inhibits bone metastases (p < .05). These findings show that stimulating osteoblastic bone formation with ActRIIA.muFc blocks the formation of osteolytic bone lesions and bone metastases in models of myeloma and breast cancer and paves the way for new approaches to treating this debilitating aspect of cancer.

Abstract 5394: Sequential treatment with zoledronic acid and doxorubicin has no additive effects in an established mouse model of bone metastasis

Abstract Bone metastases still present an unmet need for effective treatment. One preclinical study has suggested a beneficial effect with sequential treatment of doxorubicin (DOX) and zoledronic acid (ZOL). Furthermore, bisphosphonates are used as adjuvant therapy, but controversy of their anti-tumor effects remains. We tested clinically relevant doses of DOX, ZOL and their sequential combination in a treatment setting in a widely used nude mouse model of breast cancer bone metastasis. GFP-transfected MDA-MB-231(SA) human breast cancer cells were inoculated into five-week-old female nude mice via left cardiac ventricle. Subsequent tumor growth and development of osteolytic bone lesions were quantitated by fluorescence imaging and radiography, respectively, on days 14 and 25. The mice were randomized to four groups (n=8/group) on day 14 according to body weight and the presence of osteolytic lesions. Starting on day 15, groups received either weekly dose of vehicle (control group), DOX 2.5 mg/kg i.p. weekly, ZOL 0.1 mg/kg s.c. on day 15 and vehicle weekly, or weekly dose of DOX and a single injection of ZOL 24 hrs after the first dose of DOX (combination group). Bone resorption marker tartrate-resistant acid phosphatase 5b (TRACP 5b) was determined in serum samples collected on days 1, 9, 17 and 24. Bone samples were collected on day 25, and tumor area, trabecular bone area and apoptotic index (TUNEL and apoptotic morphology) were quantitated by histomorphometry. Statistical analysis was performed using One-way ANOVA followed by Dunnett's test or Kruskal-Wallis test followed by Mann-Whitney U-test. All animals in the control and DOX groups had lesions at sacrifice, whereas 12.5% of the mice in the ZOL group and 42.9% in the combination group had lesions. Osteolytic lesion area at sacrifice was significantly lower in both groups treated with ZOL compared to the control group, with no increase from day 14. Lesion area in the DOX-treated group was not different from control. Whole body tumor burden or the tumor area in hind limbs did not differ between the groups, indicating no inhibition on tumor growth. Apoptotic index was increased in groups treated with DOX. ZOL inhibited the increase in serum TRACP 5b, although less effectively in the combination group. Taken together, clinically relevant doses of DOX, ZOL or a sequential combination treatment of these two were not able to inhibit tumor growth in the established disease in this aggressive model of breast cancer bone metastasis. However, DOX increased the apoptosis of cancer cells and ZOL potently inhibited cancer-induced osteolysis, but the sequential treatment did not have additive affects on either tumor growth or osteolytic lesion area. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. 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 5394.

Inhibiting Dickkopf-1 (Dkk1) Removes Suppression of Bone Formation and Prevents the Development of Osteolytic Bone Disease in Multiple Myeloma

Multiple myeloma (MM) is associated with the development of osteolytic bone disease, mediated by increased osteoclastic bone resorption and impaired osteoblastic bone formation. Dickkopf-1 (Dkk1), a soluble inhibitor of wingless/int (Wnt) signaling and osteoblastogenesis, is elevated in patients with MM and correlates with osteolytic bone disease. In this study, we investigated the effect of inhibiting Dkk1 on the development of osteolytic lesions in the 5T2MM murine model of myeloma. We showed that Dkk1 is expressed by murine 5T2MM myeloma cells. Injection of 5T2MM cells into C57BL/KaLwRij mice resulted in the development of osteolytic bone lesions (p < 0.05), mediated by increased osteoclast numbers (p < 0.001) and a decrease in osteoblast numbers (p < 0.001) and mineralizing surface (p < 0.05). Mice bearing 5T2MM cells were treated with an anti-Dkk1 antibody (BHQ880, 10 mg/kg, IV, twice weekly for 4 wk) from time of paraprotein detection. Anti-Dkk1 treatment prevented 5T2MM-induced suppression of osteoblast numbers (p < 0.001) and surface (p < 0.001). Treatment increased mineralizing surface by 28% and bone formation rate by 25%; however, there was no change in mineral apposition rate. Inhibiting Dkk1 had no effect on osteoclast numbers. muCT analysis showed that anti-Dkk1 treatment significantly protected against 5T2MM-induced trabecular bone loss (p < 0.05) and reduced the development of osteolytic bone lesions (p < 0.05). Treatment had no significant effect on tumor burden. These data suggest that inhibiting Dkk1 prevents the suppression of bone formation and in doing so is effective in preventing the development of osteolytic bone disease in myeloma, offering an effective therapeutic approach to treating this clinically important aspect of myeloma.

Geranylgeranyl transferase type II inhibition prevents myeloma bone disease

Geranylgeranyl transferase II (GGTase II) is an enzyme that plays a key role in the isoprenylation of proteins. 3-PEHPC, a novel GGTase II inhibitor, blocks bone resorption and induces myeloma cell apoptosis in vitro. Its effect on bone resorption and tumor growth in vivo is unknown. We investigated the effect of 3-PEHPC on tumor burden and bone disease in the 5T2MM model of multiple myeloma in vivo. 3-PEHPC significantly reduced osteoclast numbers and osteoclast surface. 3-PEHPC prevented the bone loss and the development of osteolytic bone lesions induced by 5T2MM myeloma cells. Treatment with 3-PEHPC also significantly reduced myeloma burden in bone. The magnitude of response was similar to that seen with the bisphosphonate, risedronate. These data show that targeting GGTase II with 3-PEHPC can prevent osteolytic bone disease and reduce tumor burden in vivo, and represents a novel approach to treating tumors that grow in bone.

Intraosseous injection of RM1 murine prostate cancer cells promotes rapid osteolysis and periosteal bone deposition

The molecular mechanisms associated with prostate cancer (PCa) progression within bone remain a topic of intense investigation. With the availability of transgenic mouse strains, a model of PCa for use in immune competent/transgenic mice would be highly beneficial. This study was designed to explore the utility of RM1 mouse PCa cells in investigations of tumor:bone interactions. The efficacies of several implantation techniques were examined for reliably producing intra-bone RM1 tumor growth and bone lesion formation in immune competent mice. Longitudinal monitoring of bone remodeling and lesion phenotypes was conducted by microcomputed tomography (muCT) and histological analyses. Our results indicate that direct intrabone injections of RM1 cells are necessary for tumor growth within bone and direct implantation promotes the rapid development of osteolytic bone lesions with periosteal bone deposition post-cortical breach. In vitro, RM1 cells promote the proliferation of osteoblast (MC3T3-E1) and osteoclast (Raw264.7) progenitors in a dose dependent manner. Conditioned culture media from RM1 cells appears to promote earlier expression of genes/proteins associated with osteoblastic differentiation. While clearly stimulating osteoclast function in vivo, RM1 cells had little effect on differentiation and tartate resistant acid phosphatase (TRAP) expression by Raw264.7 cells. These data, coupled with in vivo muCT images, indicate the ability of RM1 cells to induce mixed, yet predominentally osteolytic, responses in bone and illustrate the potential of RM1 cells as a model of investigating prostate tumor:stroma interactions in immune competent/transgenic mice on a C57BL/6 background.

OC7. Inhibiting dickkopf-1 (Dkk-1) prevents the development of osteolytic bone disease in multiple myeloma

Multiple myeloma (MM) is associated with the development of osteolytic bone disease caused by increased osteoclastic bone resorption and impaired osteoblastic bone formation. Dickkopf-1 (Dkk-1), a soluble inhibitor of Wnt signaling and osteoblastogenesis, is elevated in the serum of patients with MM and correlates with the presence of osteolytic bone disease. In this study, we investigated the effect of inhibiting Dkk-1, on the development of osteolytic bone lesions in the 5T2MM murine model of MM.

P1. Breast cancer cells cause a reduction in osteoblast number – An early event in the development of osteolytic bone lesions

P1. Breast cancer cells cause a reduction in osteoblast number – An early event in the development of osteolytic bone lesions

Inhibition of p38α Mitogen-Activated Protein Kinase Prevents the Development of Osteolytic Bone Disease, Reduces Tumor Burden, and Increases Survival in Murine Models of Multiple Myeloma

The bone microenvironment plays a critical role in supporting the growth and survival of multiple myeloma as well as in the development of osteolytic bone disease. Signaling through p38alpha mitogen-activated protein kinase (MAPK) mediates synthesis of multiple myeloma cell growth factors, and its inhibition reduces proliferation in vitro. However, it is unclear whether targeting p38alpha MAPK prevents multiple myeloma growth and the development of bone disease in vivo. In this study, we determined whether SCIO-469, a selective p38alpha MAPK inhibitor, inhibits multiple myeloma growth and prevents bone disease in the 5T2MM and 5T33MM models. SCIO-469 decreased constitutive p38alpha MAPK phosphorylation of both 5T2MM and 5T33MM cells in vitro. This was associated with decreased DNA synthesis and an induction of apoptosis when the cells were cultured with bone marrow stromal cells. Treatment of C57Bl/KaLwRij mice bearing 5T33MM cells with SCIO-469 inhibited p38alpha MAPK phosphorylation and was associated with a significant decrease in serum paraprotein, an almost complete reduction in tumor cells in the bone marrow, a decrease in angiogenesis, and a significant increase in disease-free survival. Injection of 5T2MM murine myeloma cells into C57Bl/KaLwRij mice resulted in myeloma bone disease characterized by increased osteoclast occupation of the bone surface, reduced cancellous bone, and the development of osteolytic bone lesions. Treatment of 5T2MM-injected mice with SCIO-469 reduced this development of bone disease. Together, these data show that targeting p38alpha MAPK with SCIO-469 decreases myeloma burden in vivo, in addition to preventing the development of myeloma bone disease.

Myeloma Cells and Their Interactions With the Bone Marrow Endothelial Cells

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by a monoclonal proliferation of plasma cells in the bone marrow (BM), secretion of paraprotein in serum, development of osteolytic bone lesions and angiogenesis in the BM. In the BM, MM cells receive signals to survive and proliferate due to the existence of functional, mutual interactions between the MM cells and the BM stromal cells. This stroma not only offers protection against apoptosis and leads to growth stimulation, but also has a role in the clinical very relevant drug resistance. As such, it became clear that the BM stroma can also become a therapeutical target in addition to the MM cells. In the past, the BM fibroblasts were considered to be the main stromal cells that interact with the MM cells. However, since the observation of a myeloma associated angiogenesis in patients and in the 5TMM mouse model, it became clear that the endothelial cells (EC) are also an important participant of the BM stromal cells. BMEC are involved in the specific homing of MM cells and are a source of paracrine growth factors. In this review, the interaction between BMEC and MM cells will be discussed. Keywords: Multiple myeloma, bone marrow microenvironment, bone marrow stromal cells, endothelial cells

Hypercalcemia and Osteolytic Bone Lesions in B-Cell Chronic Lymphocytic Leukemia: A Case Report

Hypercalcemia and osteolytic bone lesions are rare associations of chronic lymphocytic leukemia. Different pathogenetic mechanisms have been postulated. We report a case of chronic lymphocytic leukemia with hypercalcemia, osteolytic bone lesions, and pathologic fractures without the evidence of Richter's transformation. Hypercalcemia was present at diagnosis and resolved with chemotherapy but recurred with disease progression and development of osteolytic bone lesions. A review of the literature is presented.