Elsevier

Bone

Volume 60, March 2014, Pages 8-15
Bone

Original Full Length Article
The orally available Btk inhibitor ibrutinib (PCI-32765) protects against osteoclast-mediated bone loss

https://doi.org/10.1016/j.bone.2013.11.025Get rights and content

Highlights

  • Btk inhibitor ibrutinib suppresses osteoclastogenesis by inhibiting NFATc1.

  • Ibrutinib inhibits bone resorption of osteoclasts by targeting integrin pathway.

  • Oral administration of ibrutinib ameliorates RANKL-induced bone loss in mice.

  • This study reveals the efficacy of ibrutinib for the treatment of bone diseases.

Abstract

Bone-resorbing osteoclasts play an essential role in normal bone homeostasis, as well as in various bone disorders such as osteoporosis and rheumatoid arthritis. Previously we showed that the Tec family of tyrosine kinases is essential for the differentiation of osteoclasts and the inhibition of Btk is a promising strategy for the prevention of the bone loss in osteoclast-associated bone disorders. Here we demonstrate that an orally available Btk inhibitor, ibrutinib (PCI-32765), suppresses osteoclastic bone resorption by inhibiting both osteoclast differentiation and function. Ibrutinib downregulated the expression of NFATc1, the key transcription factor for osteoclastogenesis, and disrupted the formation of the actin ring in mature osteoclasts. In addition, genome-wide screening revealed that Btk regulates the expression of the genes involved in osteoclast differentiation and function in both an NFATc1-dependent and -independent manner. Finally, we showed that ibrutinib administration ameliorated the bone loss that developed in a RANKL-induced osteoporosis mouse model. Thus, this study suggests ibrutinib to be a promising therapeutic agent for osteoclast-associated bone diseases.

Introduction

Bone tissue homeostasis is tightly regulated by bone-forming osteoblasts and bone-resorbing osteoclasts [1], [2]. While osteoblasts contribute to the formation of the bone matrix by providing the necessary bone matrix proteins and minerals, osteoclasts degrade the bone by producing proteases for matrix protein digestion and protons for the dissolution of the minerals in bone. An imbalance in bone formation and resorption is the underlying cause of various bone disorders, and bone loss in osteoporosis and rheumatoid arthritis (RA) mainly results from enhanced osteoclastic bone resorption [3], [4], [5]. Thus, it is an attractive therapeutic strategy to protect bone tissues by the inhibition of osteoclastic bone resorption.

Osteoclasts are multinucleated cells that attach to the bone matrix by the use of an actin ring, an actin-rich structure, and degrade bone matrix by the secretion of protons and proteases into a space that forms between the osteoclasts and the bone surface through a specialized structure known as ruffled border membranes [6]. The differentiation of osteoclasts is regulated by three signals mediated by the colony stimulating factor 1 receptor (Csf1r, also known as c-Fms or M-CSF receptor), tumor necrosis factor receptor superfamily member 11a (Tnfrsf11a, also known as receptor activator of NFκB, RANK), and certain immunoglobulin (Ig)-like receptors, including OSCAR, TREM-2, SIRP1β and PIR-A [2]. While M-CSF receptor signaling supports cell proliferation and the survival of the osteoclast precursor cells during osteoclastogenesis [7], the differentiation process is activated by the RANK signal in cooperation with the Ig-like receptors. The RANK signal activates NFκB and Fos, both of which are transcription factors essential for the osteoclast differentiation [2]. Ig-like receptors transmit a signal to activate phospholipase Cγ (PLCγ) through their adaptors, immunoreceptor tyrosine-based activation motif (ITAM)-harboring molecules DAP12 and FcRγ [8]. PLCγ induces calcium oscillation, which leads to the activation of calcineurin, a Ca2 +/calmodulin-dependent phosphatase [9]. Finally, RANK and Ig-like receptor signals are integrated by the master transcription factor of osteoclastogenesis, nuclear factor of activated T-cells c1 (NFATc1), which induces the expression of molecules required for the bone-resorbing activity of osteoclasts, such as cathepsin K, MMP9, the chloride channel CLC-7 and the H+-ATPase subunits [9].

The tyrosine kinases Btk and Tec are expressed in B lymphocytes and myeloid lineages and play important roles in the development and function of these lineages [10]. Previously, we demonstrated that Btk and Tec are also expressed in osteoclast lineage cells, and Btk−/− Tec−/− mice exhibit severe osteopetrosis due to a defect in osteoclast differentiation [11]. These kinases are activated in response to RANKL stimulation, and interact with B-cell linker (BLNK) and lymphocyte cytosolic protein 2 (Lcp2, also known as SLP-76), which are phosphorylated downstream of the Ig-like receptors. The adaptor molecules also bind PLCγ to induce efficient phosphorylation by the kinases contained in the complexes. Thus, Btk and Tec are kinases that link RANK with the Ig-like receptors. Based on the respective phenotypes of Btk−/− and Tec−/− mice, it is suggested that Btk has a more important role in osteoclast differentiation than Tec. In addition, the broad-based Btk inhibitor LFM-A13 had a therapeutic effect on inflammatory bone destruction and bone loss in an osteoporosis model [11]. Thus, a Btk inhibitor is a promising drug for the bone diseases associated with the increased activity of osteoclasts.

The Btk inhibitor ibrutinib binds covalently to a specific cysteine residue (Cys 481) in the active site of Btk so as to inhibit its enzyme activity irreversibly [12]. Oral administration of ibrutinib leads to 24 hour target inhibition supporting once daily dosing [12], suggesting that ibrutinib is a first of its kind, orally available Btk inhibitor. It has already been demonstrated that ibrutinib has therapeutic effects against certain types of leukemia [13] and autoimmune diseases, such as arthritis [14], in mice. Importantly, in the RA models, ibrutinib treatment protected the bone destruction in the joints by targeting B lymphocytes, macrophages and mast cells, all of which are involved in the RA pathology [14]. Although the suppressive effects of ibrutinib on osteoclasts in vitro have been shown [15], it remains unclear whether ibrutinib has a similar effect on osteoclasts in vivo. In this study, we aimed to investigate the effect of ibrutinib on osteoclast differentiation and function both in vitro and in vivo in an effort to develop a novel strategy for the treatment of osteoclast-associated bone diseases.

Section snippets

In vitro kinase assay

In vitro kinase assay with ibrutinib was performed as described previously [12] with slight modifications. Briefly, serial dilutions of ibrutinib were incubated with various kinases in basic reaction buffer, 20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/ml BSA, 0.1 mM Na3VO4, 2 mM DTT, and 1% DMSO with poly(Glu, Tyr) as a substrate followed by the addition of 33P-ATP (specific activity 500 μCi/μl) into the reaction mixture to initiate the reaction. The kinase reaction was

Ibrutinib inhibits osteoclast differentiation

We previously reported that ibrutinib (PCI-32765) is a potent inhibitor of Btk [12]. To determine the biochemical activity of ibrutinib, we performed an in vitro kinase assay using various kinases with serial dilutions of ibrutinib. Since the drug is a covalent inhibitor with a high systemic clearance, only those kinases with a high homology to Btk and containing a cysteine residue aligned with Cys 481 in Btk, such as other Tec family kinases (Bmx, Txk and Tec), B lymphocyte kinase (Blk), and

Discussion

Previously we demonstrated that the tyrosine kinases Btk and Tec are essential for the osteoclast differentiation, and the Btk inhibitor LFM-A13 blocks osteoclast formation both in vitro and in vivo [11]. LFM-A13 was originally developed to block the kinase activity of Btk by binding to the catalytic site in its kinase domain [28], and exhibited Btk inhibitory activity along with Tec inhibitory activity [29]. However, since LFM-A13 is not orally available and has high cell toxicity, its usage

Conclusion

It was demonstrated that ibrutinib inhibits osteoclast differentiation and function in vitro by regulating the expression of osteoclast-associated genes. Furthermore, this study showed that oral administration of ibrutinib protects against bone loss in a mouse model of osteoporosis, suggesting that this Btk inhibitor is a potential therapeutic agent for certain osteoclast-related diseases, such as osteoporosis and RA.

Conflict of interest statement

B.Y.C. and J.J.B. are employees of Pharmacyclics Inc. and have a financial interest in ibrutinib. H.T. has received research support from Pharmacyclics Inc. The remaining authors declare no competing financial interests.

Acknowledgments

We thank A. Ishizaki-Yamamura, T. Oishi-Ando, Y. Ogiwara, H. Otsuka and T. Negishi-Koga for discussion and technical assistance. This work was supported in part by Pharmacyclics Inc. and a grant for ERATO, the Takayanagi Osteonetwork Project from the Japan Science and Technology Agency (H.T.); Grant-in-Aid for Young Scientist A and Challenging Exploratory Research from the Japan Society for the Promotion of Science, and grants from Kowa Life Science Foundation, Takeda Science Foundation and

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