Development Of Myeloma Bone Disease Research Articles

Significance of PET/CT Imaging in Myeloma Assessment: Exploring Novel Applications beyond Osteolytic Lesion Detection and Treatment Response

In multiple myeloma (MM), specific cytokines produced by plasma cells disrupt the equilibrium between osteoblasts and osteoclasts. As a result, MM patients experience an increase in osteoclast activity and a decrease in osteoblast activity. This disparity is fundamental to the development of myeloma bone disease. Lytic lesions, which are a feature of MM, can result in pathologic fractures and excruciating pain. For many years, whole-body X-ray radiography has been the standard imaging method for identifying lytic lesions. However, its sensitivity is limited because it can only detect lesions once the bone mass has been reduced by 30% to 50%. Hence, utilizing advanced and sensitive imaging modalities, such as positron emission tomography (PET) fused with computed tomography (CT), is crucial for the early detection of osteolytic lesions. Among radiotracers used in PET imaging, 1⁸F-fluorodeoxyglucose ([18F]FDG) is the most commonly employed in the field of oncology. Currently, most guidelines include [18F]FDG PET/CT in the assessment of myeloma patients, particularly for detecting osteolytic lesions, evaluating treatment response, and assessing extramedullary and residual disease. Nonetheless, in recent years, new applications of PET/CT for evaluating myeloma have been investigated. These include assessing aspects such as bone turnover, dual-time-point imaging (early and delayed scans), the impact of chemotherapy on the brain (commonly known as ‘chemo brain’), innovative PET radiotracers, and the use of artificial intelligence technology. This article aims to provide a comprehensive review of both conventional and innovative uses of PET/CT in evaluating multiple myeloma.

Pathophysiology and therapeutic advances in myeloma bone disease.

Bone disease is the most common complication in patients with multiple myeloma (MM), and it may lead to skeletal-related events (SREs) such as bone pain, pathological fractures, and spinal cord compression, which impair a patients' quality of life and survival. The pathogenesis of myeloma bone disease (MBD) involves disruption of bone reconstitution balance including excessive activation of osteoclasts, inhibition of osteoblasts, and participation of osteocytes and bone marrow stromal cells. Various factors, such as the receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin (OPG), dickkopf-1 (DKK-1), sclerostin, and activin-A, are involved in the development of MBD. Bisphosphonates and the anti-RANKL antibody denosumab are currently the main treatment options for MBD, delaying the onset of SREs. Denosumab is preferred in patients with MM and renal dysfunction. Although effective drugs have been approved, antimyeloma therapy is the most important method for controlling bone disease.

Significance of Detecting Serum Complement C3 and C4 in Patients with Multiple Myeloma

To investigate the significance of detecting serum complement C3 and C4 in patients with multiple myeloma (MM) and to explore its correlation with myeloma bone disease (MBD). The levels of serum complement C3 and C4 in 69 MM patients and 30 healthy people were examined by scatter nephelometry. The bone density of L1-4 vertebral body, bilateral femoral neck and bilateral hip joints were measured by dual energy bone density meter (DXA). The levels of serum complement C3 and C4 in MM patients significantly increased in comparison with that in healthy people (P＜0.01). The patients in advanced clinical stage exhibited a higher levels of C3 and C4 than those in stable stage (P＜0.01). In addition, the patients with grade C of MBD had a higher levels of serum complement C3 and C4 than those in patients with grade A and B of MBD (P＜0.01). The levels of serum complement C3 and C4 in MM patients negatively correlated with bone density in L1-4 vertebral body, bilateral femoral necks and hip joints. The correlation coefficients were r=-0.938, r=-0.659, r=-0.745, r=-0.748, r=-0.596 in complement C3 and r=-0.908, r=-0.623, r=-0.710, r=-0.714, r=-0.595 in complement C4, respectively. The levels of complement C3 and C4 positively correlate with the severity of bone disease and bone density in MM patients, which suggests that complement C3 and C4 plays important roles in the development of MBD. The levels of serum C3 and C4 may be the sensitive biomarkers of MBD.

Significance of Detecting the Plasma Levels of MMP-13 in Patients with Multiple Myeloma

To study the significance of detecting the plasma level of matrix metalloproteinase-13(MMP-13) in patients with multiple myeloma(MM) and to investigate the correlation of MMP-13 levels in MM patients with myeloma bone disease(MBD). The plasma level of MMP-13 was quantitatively analyzed in 53 newly diagnosed MM patients and 30 healthy controls by enzyme-linked immunosorbent assay(ELISA). Imaging examination was used to determine bone damage in patients with MM. At the same time, using a dual-energy X-ray absortionmetry(DXA), the bone mineral density was examined on vertebra L2 to L4 in the anteroposterior position and the proximal left femur in 17 MM patients and 15 healthy controls. The plasma level of MMP-13 in MM patients was significantly higher than that in the controls(P<0.01), and the MMP-13 level in MM patients with stage III of International Staging System(ISS) was significantly higher than that in patients with stage I-II(P<0.01). The MMP-13 level in MM patients without MBD was significantly higher than that in the controls(P<0.05). According to bone disease grading, 53 patients were divided into group A(bone grade 0-2, n=18)and group B(bone grade 3-4, n=35). Compared with group A, MMP-13 level group B was enhanced significantly (P<0.01). Further analyses revealed that the level of MMP-13 negatively correlated with the bone mineral density on L2 to L4, greater trochanter and Ward's triangle(r values were -0.693, -0.575 and -0.575, respectively, P<0.05), but not correlated with left femoral neck(r= -0.339)(P>0.05). The level of MMP-13 in MM patients is significantly high, and closely relates with ISS clinical stage, degree of MBD and the bone mineral density of MM patients. MMP-13 plays an important role in the development of MBD.

Knockdown of macrophage inhibitory cytokine-1 in RPMI-8226 human multiple myeloma cells inhibits osteoclastic differentiation through inhibiting the RANKL-Erk1/2 signaling pathway.

Patients with multiple myeloma (MM) often develop myeloma bone disease (MBD). The development of MBD from MM is considered to be caused by an abnormal bone marrow microenvironment. Macrophage inhibitory cytokine-1 (MIC-1) is a member of the transforming growth factor‑β superfamily. In patients with MM, MIC‑1 is expressed at high levels, however, whether this increased expression of MIC‑1 is associated with the development of MBD from MM remains to be elucidated. The present study investigated whether MIC‑1 is essential for the osteoclastic differentiation of peripheral blood mononuclear cells (PBMNCs) by using a co‑culture system, in which the PBMNCs were co‑cultured with RPMI‑8226 cells. The expression of MIC‑1 in the RPMI‑8226 cells was knocked down using RNA interference. Osteoclastic differentiation was evaluated using tartrate‑resistant acid phosphatase staining and lacunar resorption on dentine slices. The expression of receptor activator of nuclear factor‑κB ligand (RANKL) and phosphorylation of extracellular signal‑regulated kinase (Erk)1/2 were measured using Western blotting. It was found that the reduced expression of MIC‑1 in the RPMI‑8226 cells inhibited the osteoclastic differentiation of PBMNCs and decreased the expression levels of RANKL and phosphorylated Erk1/2. It was concluded that MIC‑1 promoted the osteoclastic differentiation of PBMNCs via the RANKL‑Erk1/2 signaling pathway and, therefore, MIC‑1 may offer potential as a target in the design of strategies to treat MBD.

Anti-Sclerostin Treatment Prevents Multiple Myeloma Induced Bone Loss and Reduces Tumor Burden

Multiple myeloma (MM) is a malignancy of plasma cells and is characterized by unrestricted tumor cell growth in bone marrow (BM). MM causes destructive osteolytic lesions causing bone fracture, bone pain, hypercalcaemia, and nerve-compression, resulting from increased bone resorption and suppressed bone formation. Despite the introduction of agents to inhibit bone resorption, such as bisphosphonates, which prevent further bone loss, approaches to preventing osteoblast suppression and repair bone lesions are limited and there are no agents available clinically.The wnt/β-catenin pathway plays a critical role in the regulation of bone formation. Production of the soluble wnt antagonist dickkopf1 (Dkk1) by MM cells has been implicated in MM inhibition of bone formation. As such, Anti-Dkk1 treatment prevents bone disease in pre-clinical models of MM and is in early clinical development. However, Dkk1 is not expressed by all myeloma cells; hence only a proportion of patients may respond to anti-Dkk1 therapy. Sclerostin (Scl) is a soluble wnt antagonist whose expression, unlike Dkk1, is restricted to osteocytes; therefore Scl targeted agents may have less off target effects. Anti-Sclerostin (Anti-Scl) treatment increased bone formation and bone volume in experimental models of osteoporosis, and increased bone mineral density in phase II osteoporosis clinical trials. However, Anti-Scl treatment effects on myeloma bone disease are unknown. Further, cells of the BM such as osteoblasts have been implicated in the regulation of MM cell survival and growth. Thus, in the present study we explored the potential for Anti-Scl therapy to prevent MM induced bone loss and inhibit MM growth in both murine and human xenograft MM models.Female C57BLKalwRij mice (n=8) were injected i.v, with 5TGM1/eGFP murine MM cells (1×106) and female SCID/beige mice (n=10) were injected i.v. with MM1S/Luc/eGFP human MM cells (4 × 106). 24 hours later, naïve mice (without tumor cells) or mice bearing MM cells were treated with anti-sclerostin antibody (Anti-Scl) (100mg/kg i.v) or control antibody. Mice were sacrificed at day 21 (MM1S) or day 28 (5TGM1) and the effect of Anti-Scl on bone structure in the femora and vertebrae were determined by microCT analysis. The effect of Anti-Scl on MM burden was determined by bioluminescent imaging (BLI) performed twice weekly from week 1 using a Xenogen IVIS system, whereas MM burden in 5TGM1/eGFP bearing mice was examined by FACS analysis.Anti-Scl treatment in naïve C57BLKalwRij mice increased trabecular bone volume fraction (BV/TV, 39%, p<0.01, Fig. 1B) in the femur, which was mediated by increases in trabecular thickness (Tb.Th, 42%, p<0.01). Treatment also increased cortical bone volume (22%, p<0.01) in the femur and increased trabecular BV/TV in the vertebra (32%, p<0.01). This demonstrated the potent bone anabolic effect of Anti-Scl independent of myeloma cells. Injection of 5TGM1 cells resulted in a decrease in femoral trabecular BV/TV (30%, p<0.01) through a 30% reduction in trabecular number (TbN) (p<0.01), but no effect on Tb.Th (Figs. 1A and B), whilst also reducing cortical bone volume (BV) by 6% (p<0.05). Vertebrae were also impacted by 5TGM1 tumor growth with a 29% reduction in Tb BV/TV through a 23% reduction in Tb.Th (p<0.01) and also a 15% reduction in cortical BV (p<0.01). Treatment of 5TGM1-bearing mice with Anti-Scl increased trabecular BV/TV (46%, p<0.01) and Tb.Th (30%, p<0.01) to values equivalent to femora of naïve, non-tumor bearing, control mice. Treatment with Anti-Scl also increased cortical BV by 16% (p<0.01), vertebral Tb BV/TV by 29% and cortical BV by 36% in 5TGM1 burdened mice (p<0.01).Treatment of 5TGM1-bearing mice with Anti-Scl had no effect on the proportion 5TGM1/eGFP cells in the BM or spleen. However Anti-Scl treatment significantly suppressed tumor progression in the MM1S model at 3, 3.5 and 4 weeks post cell injection, as determined by BLI imaging (p=0.02, wk 3; p=0.0019, wk 3.5, and p=0.0068, wk 4, 2-tailed t-tests, Fig. 1C).These data demonstrate that Anti-Scl antibody treatment can prevent development of myeloma bone disease. Furthermore, Anti-Scl treatment also suppressed tumor growth, supporting the possibility that targeting the BM microenvironment with this agent may slow disease progression. Our findings highlight the potential clinical application of Anti-Scl antibody treatment in patients with MM and other bone destructive cancers. [Display omitted] DisclosuresKneissel:Novartis Institutes for Biomedical Research, Novartis Pharma AG: Employment. Kramer:Novartis Pharma AG: Employment. Brooks:Spouse works for Boston Biomedical Inc: Employment.

Multiple Myeloma and Bone Marrow Microenvironment Immunohistochemical Study of the Expression of 15 Proteins Related to Myeloma Bone Disease

Objective: Neoplastic milieu is an integral part of all malignant diseases including multiple myeloma and plays variable role in their development, retention/adhesivity, resistency or sensitivity to therapeutic approach, homing and also paraneoplastic manifestations. Relatively genetically stable milieu may play an important role in new specific molecular therapeutic approaches and therefore should be contextually studied with neoplastic cells as complex neoplastic tissues. The expressions of 15 proteins with close relation to the development of myeloma bone disease (MBD) were analysed in consecutive multiple myeloma specimens.Methods: Bone marrow trephine biopsy specimens (n=57) with multiple myeloma were included in our prospective study. FFPE tissues were processed in app. 5microm sections and placed on charged slides. The indirect immunohistochemical staining was applicated after antigen retrieval and commercial primary antibodies were used for the detection of observed proteins. Standard secondary antibody and ABC method were included in visualisation. We analysed the expressions of MIP1alfa, Annexin A2, TRAP, DKK-1, RANK, RANKL, OPG, Sclerostin, Activin A, NFkappaB proteins (p50, p52, p65), p62 (sequestosome 1), MMP9 and RUNX2.Results: Bone marrow multiple myeloma specimens showed variable positivity of MIP1alfa in 60% (cut-off point 20%), Annexin A2 in 42% (myeloma cells, cut-off point 30%) and in 74% (stromal cells, cut-off point 5%), TRAP in 28% (cut-off point 5%), DKK-1 in 23% (cut-off point 30%), RANK in 53% (cut-off point 30%), RANKL in 70%, OPG in 39% (cut-off point 5%), Sclerostin in 95% (cut-off point 90%), Activin A in 35% (cut-off point 30%), cytoplasmic positivity of p50 in 5% (cut-off point 10%), p52 in 86% (cut-off point 10%), p62 in 91% (cut-off point 10%), p65 in 89% (cut-off point 10%), positivity of MMP9 in 22% (cut-off point 30%) and positivity of RUNX2 in 56% (cut-off point 30%).Conclusion: Our study showed variable expression of proteins related to MBD in multiple myeloma and its bone marrow microenvironment that imply biological heterogeneity, different development and stromal plasticity in this complex hemato-oncological disease. The exact and contextual knowledge of the engaged signaling pathways may suggest more specific or tailored therapeutic approaches (e.g. anti-RANKL, anti-DKK-1, anti-Sclerostin, anti-Activin A).Supported by the grant NT 14393. DisclosuresNo relevant conflicts of interest to declare.

Assessment of the Parameters of Myeloma Bone Disease Signaling to the Extent of Skeletal Involvement, Bone Mineral Turnover and Activity of Multiple Myeloma

Background: Myeloma bone disease (MBD) is present in up to 80% patients with newly diagnosed multiple myeloma (MM). It is known to be activated by three separate signaling pathways - receptor activator of nuclear factor κB/osteoprotegerin pathway (RANK/OPG), macrophage inflammatory factor pathway (MIP) and Wnt pathway. Except of these, several other factors have been described in relationship to MBD development. The aim of our study was to assess the activity of selected parameters of MBD signaling with respect to macroscopic skeletal changes detectable using imaging techniques, and in comparison with markers of bone turnover and the activity of MM.Patients and methods: We assessed 92 patients with monoclonal gammopathies: 56 patients with active multiple myeloma, 12 with smoldering myeloma and 24 individuals with monoclonal gammopathy of undetermined significance (MGUS). Following parameters of MBD signaling were measured in the patients´serum: hepatocyte growth factor (HGF, Human HGF Quantine ELISA), macrophage inflammatory factor alpha (MIP-1α, Human CCL3/MIP-1α Quantikine ELISA), syndecan-1 (Human Syndecan-1 (CD138) ELISA), osteoprotegerin (OPG, Human Osteoprotegerin ELISA), Activin A (Human Activin A Quantikine ELISA), Dickkopf-related protein-1 (DKK1, Human Dkk-1 Quantikine ELISA), Annexin A2 (Human Annexin A2 ELISA kit), nuclear factor kappa B (NF-κB, Human Nuclear factor-kappa B). The extent of skeletal involvement was evaluated using whole body magnetic resonance and low-dose computed tomography. The levels of the selected parameters of MBD signaling were compared to serum levels of Ca, P, PINP, ICTP, CTx, parathormone (PTH), calcitonine (CLC), 25-hydroxyvitamin D2 (D2), 1,25dihydroxyvitamin D3 (D3), bone isoenzyme of alkaline phosphatase (bALP) and sclerostin (SCL). The parameters were measured at the time of diagnosis and then after 4 months. In patients with MM we measured selected parameters of MBD in comparison with the stage of the disease according to Durie-Salmon (DS) and according to International Staging System (ISS). In MGUS we assessed the relationship of the parameters to the risk of transformation to MM based on the Mayo clinic stratification system. For statistical estimation we used Kruskal-Wallis test and Mann-Whitney U post hoc test with Bonferroni correction at p < 0,05.Results: Statistically significant difference between patients with low and high skeletal involvement was found in serum levels of MIP-1α, syndecan-1, DKK-1 and Annexin A2. None of the parameters correlated with the presence of extramedullary disease. With respect to markers of bone turnover we found following significant relationships: HGF to PINP, ICTP, CTx and inverse relationship to D3; MIP-1α to PINP, ICTP, CTx and inverse relationship to D3; Syndecan-1 to ICTP, CTx, and inversely to D2 and D3; OPG to ICTP and SCL; Activin A to PINP, ICTP, CTx and SCL; NF-κB to ICTP and CTx. We found significant differences between MGUS and MM in serum levels of HGF, MIP-1α, syndecan-1, DKK-1 and NF-κB. There was no significant relationship of any of the parameters to DS, however, we found significant relationship of HGF, syndecan-1, OPG and Activin A to ISS. MIP-1α only had a significant relationship to the risk stratification of MGUS. There was no significant change in the parameters in MM patients after 4 months of treatment with respect to treatment response.Conclusions: We confirmed the relationship of MBD signaling to the extent of skeletal changes using imaging techniques. All three signalling pathways were involved in the development of MBD in MM in comparison with MGUS. Signaling involved in osteoclast activation was more activated than the parameters preventing osteoblast differentiation. There was, however, no significant change in any of the parameters in MM patients with respect to treatment response after 4 months, suggesting that bone reparation processes are delayed after hematological response. Significant relationship to the markers of bone turnover and to the activity of MM suggests possible use of the parameters of MBD signaling as tools for early detection of bone marrow changes and timely indication for anti-resorption therapy.Supported by the grant NT 14393. DisclosuresNo relevant conflicts of interest to declare.

Effect of CCL3 on osteoblast in myeloma bone disease

To culture osteoblast in vitro and evaluate CCL3 receptor CCR1 expression in patients with multiple myeloma (MM). Bone marrow osteoblasts from MM patients were cultured in vitro with dexamethasone, β-sodium glycerophosphate and vitamin C, which were identified by alkaline phosphatase staining, Von Kossa's staining. The CCL3 receptor expression was evaluated by flow cytometry. The morphology and quantity of osteoblast were observed after exposure to CCL3. Bone marrow osteoblasts from MM patients could be cultured in vitro and be identified by positive staining of alkaline phosphatase and Von Kossa's. MM-derived osteoblasts expressed higher levels of CCR1 (74.48 ± 7.31)%, compared with normal controls (48.35 ± 8.81)%. Calcium deposition of osteoblasts after exposure to CCL3 was less than that of controls. Bone marrow osteoblasts could be cultured in vitro from MM Patients. CCL3 may contribute to the development of myeloma bone disease.

Immunomodulatory drugs thalidomide and lenalidomide affect osteoblast differentiation of human bone marrow stromal cells in vitro

Osteoblastic activity is severely impaired in active myeloma, contributing to the development of myeloma bone disease. Although several drugs reducing osteoclast-mediated bone degradation are in clinical use, approaches to specifically augment bone formation are at an early stage of development. Novel antimyeloma drugs not only directly act on myeloma cells, but impact on the microenvironment as well. Proteasome inhibitors were previously shown to have bone anabolic properties. Here we investigated the impact of immunomodulatory drugs (IMiDs) on bone formation. Treatment with thalidomide and lenalidomide significantly inhibited osteoblast development in vitro, as reflected by a reduction of alkaline phosphatase activity and matrix mineralization. The effects were upheld in combination with bortezomib. The IMiDs upregulated Dickkopf-1 (DKK1) and inhibin beta A, but blocking these molecules was not able to restore regular osteoblast development. We therefore performed gene expression profiling to reveal other osteoblast regulatory factors that might be involved in the IMiD-mediated effect on osteoblast development. Our data indicate that osteoblast inhibition is possibly an IMiD-class effect mediated by downregulation of major osteoblast regulators (e.g., runt-related transcription factor 2, distal-less homeobox 5, pleiotrophin) and concurrent induction of secreted inhibitors of osteoblast formation (e.g. DKK1, activin A, gremlin 1). Our results highlight the need for bone anabolic therapeutics in myeloma, counteracting the negative impact of prolonged IMiD exposure on bone metabolism.

Bone disease in multiple myeloma: pathophysiology and management.

Myeloma bone disease (MBD) is a devastating complication of multiple myeloma (MM). More than 80% of MM patients suffer from destructive bony lesions, leading to pain, fractures, mobility issues, and neurological deficits. MBD is not only a main cause of disability and morbidity in MM patients but also increases the cost of management. Bone destruction and lack of bone formation are main factors in the development of MBD. Some novel factors are found to be involved in the pathogenesis of MBD, eg, receptor activator of nuclear factor kappa-B ligand (RANKL), osteoprotegerin (OPG) system (RANKL/OPG), Wingless (Wnt), dickkopf-1 (Wnt/DKK1) pathway. The addition of novel agents in the treatment of MM, use of bisphosphonates and other supportive modalities such as radiotherapy, vertebroplasty/kyphoplasty, and surgical interventions, all have significant roles in the treatment of MBD. This review provides an overview on the pathophysiology and management of MBD.

Bone Turnover Biomarkers Are Useful In Monitoring Myeloma Bone Disease and As Early Predictor Biomarkers For Relapse Disease In Multiple Myeloma

BackgroundMultiple myeloma is a plasma cell disorder characterised by bone marrow infiltration with clonal plasma cells that secrete monoclonal immunoglobulin which is detected in serum or urine samples. Bone disease is a well-known devastating complication. It has a significant impact to the quality of life and morbidity in multiple myeloma. The uncoupling effect of osteoblast and osteoclast activity is the major element in development of myeloma bone disease (MBD). Imaging techniques are used as the current standard method for detection of bony lesions. They have limitations as they cannot provide a real-time assessment of bone turnover. Early detection of relapse disease is crucial to allow preventative therapeutic intervention as it could significantly impact on quality of life. AimsBone biomarkers such as C-terminal telopeptide of type 1 collagen (CTX-1) and procollagen type 1 N-propeptide (P1NP) can be used as an early predictor marker for MBD relapses and a monitor for MBD at different stages of the disease. MethodsCTX-1 and P1NP were measured by chemiluminescent immunoassay on fasting plasma samples from 111 patients including newly diagnosed multiple myeloma (n=28), remission (n=34), relapses (n=22) and control (n=27). These were measured at regular intervals over a 30 month study period. Relapse disease was identified by conventional biomarkers like paraprotein and serum free light chains, and confirmed by imaging and bone marrow biopsy. In a subset of patients with disease relapse, the Mann-Whitney test was used to compare bone markers pre-relapse and at relapse. ResultsCTX-1 levels were significantly higher in newly diagnosed multiple myeloma compared to remission and control groups (p < 0.0001). In relapse group, CTX-1 rose significantly at the time of pre-relapse to relapse state (p=0.0001). A rise of ≥ 2.0 fold rise in the level of CTX-1 from remission to relapse disease was noted. The median time between the pre-relapse sample and relapse disease was 3 months (range 1-14 months). Most of them had new bone lesions at relapse. This proves that it has potential as an early predictor of relapse or progressive bone disease. A case showed CTX-1 level was the only biochemical parameter to rise significantly prior to relapse as compared to the other conventional biomarkers (ie. paraprotein and serum free light chain). As for P1NP, the rise in P1NP from pre-relapse to relapse was not significant (p=0.0810). ConclusionOsteoclast biomarker serum CTX-1 correlates accurately with the disease burden in newly diagnosed multiple myeloma patients as compared to the rest of the groups. It is a more sensitive early predictor of relapse/progressive disease than established biomarkers. It is a more robust marker than P1NP. The rise in P1NP goes against the theory that there is an uncoupling of bone turnover in MBD and requires further study. CTX-1 is more cost effective and accessible than imaging and should be used routinely when monitoring bone disease activity in multiple myeloma patients, facilitating early intervention when relapse occurs. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Abstract 5624: Anti-c-Met Nanobody®: A potential new drug in cancer treatment.

Abstract c-Met is the tyrosine kinase receptor of the hepatocyte growth factor (HGF). HGF-c-Met signaling is involved in a wide variety of human malignancies including colon, gastric, bladder, breast, ovarian, pancreatic, kidney, liver, lung, head and neck, thyroid, and prostate cancers, as well as sarcomas, hematological malignancies, melanoma and central nervous system tumors. In recent years, multiple agents have been developed directed to interfere at different levels in the HGF-c-Met signaling pathway, and some are currently being tested in Phase II and/or III clinical trials. Nanobodies® are therapeutic proteins based on the smallest functional fragments of heavy chain antibodies, occurring in the Camelidae family. Importantly, they retain the full antigen-binding capacity of the original heavy chain only antibodies and are highly stable. In this study, we examined the anti-cancer effects of an anti-c-Met Nanobody in vitro using human multiple myeloma as a model system. The anti-c-Met Nanobody comprises of two Nanobody moieties, one targeting c-Met and one binding to human serum albumin for half-life extension. HGF is thought to contribute to the pathogenesis of multiple myeloma in different ways, as HGF is a pivotal growth and pro-migratory factor and inhibits osteoblastogenesis in vitro suggesting that HGF may contribute to the development of myeloma bone disease. Elevated levels of HGF in serum of multiple myeloma patients are correlated with a poor prognosis. We show that the anti-c-Met Nanobody effectively inhibited the proliferation of ANBL-6 human multiple myeloma cells via inhibition of an HGF autocrine growth loop, as well as the proliferation of INA-6 cells induced by exogenously added HGF. In addition, the HGF-induced migration of INA-6 cells was completely and specifically blocked following treatment with the Nanobody at a concentration of 1 μM. The Nanobody also inhibited the HGF-induced adhesion of multiple myeloma cells to fibronectin, but did not affect stromal cell-derived factor-1 alpha-induced adhesion. Furthermore, the Nanobody abolished the inhibiting effect of HGF on bone morphogenetic protein-2-induced ALP-activity and the mineralization of human mesenchymal stem cells. Finally, we show that the Nanobody reduced the HGF mediated phosphorylation of the c-Met Tyrosine (Y) residues Y1349, Y1234/1235 and Y1003 and the phosphorylation of the downstream proteins MAPK and Akt in INA-6 cells. In conclusion, the anti-c-Met Nanobody inhibited c-Met signaling with high specificity and potency resulting in inhibition of multiple myeloma cell migration, proliferation and adhesion, and in blocking of the HGF mediated inhibition of osteoblastogenesis. Given the potential of Nanobodies to surpass drawbacks of antibodies, this anti-c-Met Nanobody might represent a potential novel therapeutic agent in the treatment of multiple myeloma and other cancers driven by HGF-c-Met signaling. Citation Format: Tobias S. Slørdahl, Tinneke Denayer, Siv Helen Moen, Therese Standal, Magne Børset, Cedric Ververken, Torstein B. Rø. Anti-c-Met Nanobody®: A potential new drug in cancer treatment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5624. doi:10.1158/1538-7445.AM2013-5624

Brain‐derived neurotrophic factor is a potential osteoclast stimulating factor in multiple myeloma

Multiple myeloma (MM) is characterized by accumulation of monoclonal plasma cells in the bone marrow and progression of lytic bone lesions. The mechanisms of enhanced bone resorption in patients with myeloma are not fully defined. We have previously identified the role of brain-derived neurotrophic factor (BDNF) in proliferation and migration of MM cells. In our study, we investigated whether BDNF was possibly involved in MM cell-induced osteolysis. We showed that BDNF was elevated in MM patients and the bone marrow plasma levels of BDNF positively correlated with extent of bone disease. In osteoclast formation assay, bone marrow plasma from patients with MM increased osteoclast formation and the effect was significantly blocked by neutralizing antibody to BDNF, suggesting a critical role for BDNF in osteoclast activation. Furthermore, the direct effects of recombinant BDNF on osteoclast formation and bone resorption support the potential role of BDNF in the MM bone disease. BDNF receptor TrkB was expressed by human osteoclast precursors and a Trk inhibitor K252a markedly inhibited osteoclast formation stimulated with BDNF, demonstrating that BDNF used TrkB for its effects on osteoclast. Finally, bone marrow plasma BDNF level positively correlated with macrophage inflammatory protein-1α and receptor activator of nuclear factor-κB ligand, two major osteoclast stimulatory factors in MM. These results support an important role for BDNF in the development of myeloma bone disease.

Laser-capture micro-dissection and gene array analysis to identify new molecules implicated in the development of myeloma bone disease

Laser-capture micro-dissection and gene array analysis to identify new molecules implicated in the development of myeloma bone disease

Inhibiting glycogen synthase kinase-3 (GSK-3) prevents the development of myeloma bone disease

Inhibiting glycogen synthase kinase-3 (GSK-3) prevents the development of myeloma bone disease

Osteoclast Gene Expression Profiling in Multiple Myeloma

Multiple myeloma (MM) is a fatal hematologic malignancy associated with clonal expansion of malignant plasma cells within the bone marrow and the development of a destructive osteolytic bone disease. The principal cellular mechanisms involved in the development of myeloma bone disease are an increase in osteoclastic bone resorption, and a reduction in bone formation. Myeloma cells (MMC) are found in close association with sites of active bone resorption, and the interactions between myeloma cells and other cells within the specialized bone marrow microenvironment are essential, both for tumor growth and the development of myeloma bone disease. In order to investigate the gene expression profile (GEP) of osteoclastic cells, we compare GEP of osteoclastic cells (7 samples) with normal B cells (7 samples), normal bone marrow plasma cells (7 samples), bone marrow stromal cells (5 samples), bone marrow CD3 cells (5 samples), CD14 cells (7 samples), CD15 cells (7 samples), CD34 cells (7 samples) and primary MMC (123 samples). Using SAM analysis, a set of 552 genes was overexpressed in osteoclasts compared to others cell subpopulations with a FDR ≤ 1% and a ratio ≥ 2. Osteoclasts specifically overexpressed genes coding for chemokines (CCL2, CCL7, CCL8, CCL13, CCL18, CXCL5 and CCL23) and MMC growth factors (IGF-1, APRIL and IL-10). Anti- IGF-1 receptor and TACI-Fc inhibit MMC growth induced by osteoclasts. Among the chemokines overexpressed by osteoclasts, the majority of them have a common receptor: CCR2 expressed by MMC. Anti-CCR2 MoAb inhibits migration of the CCR2+ HMCL in response to osteoclasts. Expression data of purified MMC were analyzed by supervised clustering of group with higher (CCR2high) versus lower (CCR2low) CCR2 expression level. Patients in the CCR2high group are characterized by a higher bone disease. A set of 176 genes was differentially expressed between CCR2high and CCR2low MMC. CCR2high displayed a gene signature linked to the dependency of MMC on the interactions with the BM osteoclastic subpopulation and the osteoclastic bone resorption.Taken together, our findings suggest addition of chemokine antagonists to current treatment regimens for MM should result in better therapeutic responses because of the loss of both the protective effect of the bone marrow environment on the MMC and the osteoclastic cells activity.

Increasing Wnt signaling in the bone marrow microenvironment inhibits the development of myeloma bone disease and reduces tumor burden in bone in vivo

There is increasing evidence to suggest that the Wnt signaling pathway plays a critical role in the pathogenesis of myeloma bone disease. In the present study, we determined whether increasing Wnt signaling within the bone marrow microenvironment in myeloma counteracts development of osteolytic bone disease. C57BL/KaLwRij mice were inoculated intravenously with murine 5TGM1 myeloma cells, resulting in tumor growth in bone and development of myeloma bone disease. Lithium chloride (LiCl) treatment activated Wnt signaling in osteoblasts, inhibited myeloma bone disease, and decreased tumor burden in bone, but increased tumor growth when 5TGM1 cells were inoculated subcutaneously. Abrogation of beta-catenin activity and disruption of Wnt signaling in 5TGM1 cells by stable overexpression of a dominant-negative TCF4 prevented the LiCl-induced increase in subcutaneous growth but had no effect on LiCl-induced reduction in tumor burden within bone or on osteolysis in myeloma-bearing mice. Together, these data highlight the importance of the local microenvironment in the effect of Wnt signaling on the development of myeloma bone disease and demonstrate that, despite a direct effect to increase tumor growth at extraosseous sites, increasing Wnt signaling in the bone marrow microenvironment can prevent the development of myeloma bone disease and inhibit myeloma growth within bone in vivo.

Targeting Wnt Signaling in Myeloma In Vivo; Differential Effects on Tumor Burden and Myeloma Bone Disease.

Multiple myeloma is characterized by uncontrolled proliferation of myeloma cells within the bone marrow and the development of a severe osteolytic bone disease. In addition to a well characterized increase in osteoclastic bone resorption, myeloma bone disease is associated with a reduction in bone formation. Osteoblast differentiation and bone formation are regulated in vivo by canonical Wnt signaling and activation of β-catenin. Therefore increasing Wnt signaling in the bone microenvironment in multiple myeloma may prevent the development of myeloma bone disease. In support of this, we have previously demonstrated that activation of Wnt signaling with lithium chloride (LiCl) in the 5TGM1 murine model of myeloma reduces tumor burden and osteolytic bone disease. However, we also found that LiCl treatment increased subcutaneous (s.c.) tumor growth. This suggests that the reduction in tumor burden within the bone microenvironment may be an indirect effect mediated through the effects of LiCl to prevent myeloma bone disease. The aim of the current study was to determine the effect of specific molecular blockade of Wnt signaling in myeloma cells in vivo. 5TGM1-GFP myeloma cells were transfected by electroporation with either myc-tagged dominant negative TCF4 (DNTCF4) or pcDNA. Following stable selection by culture in G418, expression of DNTCF4 was confirmed by western blot for myc. No difference was found in the growth rates of 5TGM1-pcDNA or 5TGM1-DNTCF4 in vitro. Treatment with LiCl or Wnt3A had no significant effect on cell viability in vitro, but significantly increased β-catenin activity, as measured by TOPFLASH activity in 5TGM1-pcDNA cells. This increase was not observed in 5TGM1-DNTCF4, confirming that expression of DNTCF4 blocked Wnt signaling induced by LiCl in 5TGM1 myeloma cells. C57Bl/KaLwRij mice were inoculated with 5TGM1-pcDNA or 5TGM1-DNTCF4 cells by either intravenous (i.v.) or s.c. injection. Mice were treated from time of tumor cell inoculation with 200mg/kg/day LiCl or vehicle control (d.H20) by oral gavage for 28 days. I.v. inoculation of myeloma cells resulted in a significant increase in serum IgG2bκ concentrations and the proportion of GFP-positive cells in the bone marrow. A significant reduction in trabecular bone volume was also observed. MicroCT analysis of the tibia demonstrated that LiCl significantly increased trabecular bone volume in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice. LiCl significantly decreased serum IgG2bκ concentrations in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice, with a greater effect in 5TGM1-DNTCF4 myeloma-bearing mice. FACS analysis of GFP-positive cells demonstrated that LiCl significantly reduced tumor burden in the bone marrow in both 5TGM1-pcDNA and 5TGM1-DNTCF4 myeloma-bearing mice. However, following s.c inoculation, LiCl significantly increased s.c. tumor volume of 5TGM1-pcDNA tumors, but had no effect on 5TGM1-DNTCF4 s.c. tumor volume. Taken together these results demonstrate that the effect of increasing Wnt signaling in myeloma is dependent upon the microenvironment. By specific inhibition of β-catenin activity in myeloma cells combined with systemic stimulation of the Wnt signaling pathway, our results suggest that increasing Wnt signaling in myeloma in vivo has dual effects; firstly to enhance myeloma growth directly, and secondly to enhance osteoblast differentiation and thus indirectly reduce tumor burden in bone, highlighting the importance of the bone marrow microenvironment in regulating myeloma growth and survival.

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.