Elsevier

Bone

Volume 53, Issue 2, April 2013, Pages 487-496
Bone

Original Full Length Article
In vivo and in vitro effects of a novel anti-Dkk1 neutralizing antibody in multiple myeloma

https://doi.org/10.1016/j.bone.2013.01.012Get rights and content

Abstract

Over-expression of the protein Dickkopf-1 (Dkk1) has been associated with multiple myeloma bone disease. Previous reports with the use of anti-Dkk1 neutralizing Ab directed strategies have demonstrated a pro-anabolic effect with associated anti-myeloma activity in 2 in vivo mouse models. However new insights on the role of the wnt pathway in osteoclasts (OC) are emerging and the potential effect of a neutralizing Ab to Dkk1 in osteoclastogenesis remains to be elucidated. In order to better define the effect of an anti-Dkk1 neutralizing Ab on osteoclastogenesis and myeloma, we studied a novel anti-Dkk1 monoclonal Ab in our preclinical models. In vivo data confirmed the pro-anabolic and anti-MM effect. In vitro data in part confirmed the in vivo observation, suggesting an indirect anti-MM effect secondary to inhibition of osteoclastogenesis and thus the interaction between MM and bone microenvironment. However, when studies on osteoclastogenesis were extended to samples derived from MM patients, we observed a variable response to anti-Dkk1 treatment without correlation to expression of surface receptors for Dkk1 in OCs suggesting potential heterogeneity in the efficacy of such a strategy. In conclusion, Dkk1 is a promising target for the treatment of both MM and bone disease, and ongoing clinical studies will help elucidate its efficacy.

Highlights

► A novel anti-Dkk1 neutralizing antibody inhibits myeloma cell growth and inhibits bone resorption in a SCID-hu mouse model. ► Anti-Dkk1 monoclonal Ab affects OC differentiation in vitro. ► The anti-MM effect is mediated by the effect of anti-Dkk1 Ab in the context of the bone microenvironment.

Introduction

Ninety percent of multiple myeloma (MM) patients manifest osteolytic bone lesions. These can evolve to pathological fractures in 40–50% of the cases [1], spinal cord compression or require radiation therapy. Several studies have demonstrated that bone damage is consequent to the uncoupling of osteoclasts (OC) and osteoblasts (OB), ultimately leading to enhanced OC activity and blunted OB function with a consequent imbalance favoring bone resorption. A wide range of cytokines has been implicated in increased OC formation, including interleukin-6 (IL-6), IL-3, macrophage inflammatory protein-1 alpha (mip1α), upregulation of Receptor Activator for Nuclear Factor κ B Ligand (RANKL), and decreased expression of osteoprotegerin (OPG) [2]. In addition to enhanced bone destruction, bone deposition is also impaired secondary to OB dysfunction. Several factors affect osteoblastogenesis in MM patients including increased IL-3, IL-7, hepatocyte growth factor (HGF) and proteins involved in the wingless/int (wnt) pathway: secreted frizzled related proteins (sFRPs) and Dickkopf-1 (Dkk1) protein [3].

Dkk1 is one of 4 proteins that negatively regulates canonical wnt/β-catenin signaling. While activation of wnt pathway mediates the translocation of nonphosphorylated β-catenin into the nucleus with consequent activation of T-cell transcription factor (TCF)/lymphoid enhancer-binding factor (LEF), the binding of Dkk1 to LRP5/6 and kremen receptors ultimately induces β-catenin degradation by the proteasome [4]. Wnt pathway is active both in embryonic as well as in adult life. It is involved in cell proliferation, motility and apoptosis [5] and in embryos it regulates the development of head and limbs [6] as well as heart formation [7]. Wnt pathway is also implicated in regulation of the hematopoietic stem cell niche [8]; and in adult life it maintains bone homeostasis and bone mass, enhancing OB differentiation and function [4]. Dkk1 not only affects osteoblastogenesis through the modulation of OPG/RANKL ratio, but also affects osteoclastogenesis indirectly [4]. More interestingly, given that OC and OB are an integral part of the bone marrow microenvironment, the effect of wnt/Dkk1 antagonism may affect tumor growth negatively via impact on the BM milieu.

Deregulation of wnt pathway is implicated in oncogenesis and metastasis of several types of cancers [5]. Overexpression of Dkk1 has been described as a negative prognostic factor in patients with non-small cell lung cancer and esophageal squamous cell carcinoma [9], and aberrant activation of wnt pathway is involved in hepatocellular carcinoma [10], breast cancer [5], and other types of solid tumors. In MM patients, Dkk1 has been related to the extent of bone involvement. In 2003, Tian and colleagues [11] demonstrated that a subset of MM patients had increased serum levels of Dkk1 compared to MGUS, which correlated with the extent of bone disease. These observations have been confirmed by others [12], [13], and gone further to show decreased levels of serum Dkk1 in MM patients responsive to anti-myeloma treatment [14], [15].

Since Dkk1/wnt pathway is involved in cancer and bone pathophysiology, and the interaction of cancer and bone microenvironment is crucial to the progression of MM, Dkk1 may represent a potential target for treatment. Previous publications [16], [17], [18] demonstrated that neutralization of Dkk1 in in vivo mouse models of MM had a bone anabolic effect, with inconsistent results on OC and myeloma growth. Moreover wnt/Dkk1 pathway has recently been confirmed to be active in OC [19] although its role in osteoclastogenesis is poorly understood. The aim of this study was to evaluate a novel anti-Dkk1 neutralizing monoclonal Ab and to elucidate its effect in MM and the bone microenvironment, in particular in OCs.

Section snippets

Anti-Dkk1 neutralizing Ab

A mouse variable domain/rat IgG1 kappa chimeric monoclonal neutralizing Dkk1 Ab and isotype control were provided by Eli Lilly and Company [20]. The Ab cross reacts with both human and mouse Dkk1 protein.

Severe combined immunodeficient-hu mouse model (SCID-hu)

The SCID-hu MM mouse model was generated as previously described [18], in accordance with the animal protocol approved by the Institutional Animal Care and Use Committee. A total of 8 CB-17 severe combined immunodeficient (SCID) mice (Taconic) were implanted subcutaneously with human fetal long

Anti-Dkk1 neutralizing antibody inhibits tumor growth in a SCID-hu mouse model with anabolic effect in mice femurs

To address the effect of a novel anti-Dkk1 neutralizing Ab on both tumor burden and bone structure, SCID-hu mice were implanted with human fetal bone chips and injected with INA6 human MM cell line. Seven of eight mice (3 in the control group, and all 4 mice in the treated group) developed tumor, as demonstrated by an increase in shuIL6R levels. shuIL6R levels decreased after 4 weeks of treatment, suggesting an inhibitory effect of the anti-Dkk1 monoclonal Ab on INA6 MM cell growth (30.9 ± 10.5 vs

Conclusion and discussion

MM is a treatable but incurable hematological cancer associated with high morbidity related to bone involvement. The characteristic osteolytic lesions are the result of enhanced osteoclastogenesis and impaired osteoblastogenesis; moreover OC and OB, as part of the bone microenvironment also play a pivotal role in MM growth and progression. The observation that overexpression of Dkk1 in a subset of MM patients is associated with bone disease makes Dkk1 a promising target to improve bone quality

Acknowledgments

SZ is the recipient of the Brian Durie International Myeloma Foundation junior fellow award. NR is a LLS Clinical Scholar and recipient of the Claflin Distinguished Scholar Award.

Conflicts of interest

S.K. and L.S. are employees at Eli Lily and Company, Indianapolis; N.R is on the advisory board of Celgene, Amgen, Millenium and Novartis. NR has research funding from Acetylon.

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