The activation of Bruton's tyrosine kinase (Btk), a member of the Tec family of tyrosine kinases regulates B-cell development and activation, and plays a key role in antibody production. Interestingly, Btk and the other Tec kinase family, Tec, regulate OC differentiation via Receptor Activator of Nuclear Factor κ B (RANK) signaling. Moreover, patients with X-linked agammaglobulinemia (XLA) who harbor Btk null mutations have impaired OC function.
Here we show that, a potent and specific inhibitor of Btk, AVL-292, inhibits OC function in MM patients. AVL-292 is a highly selective, covalent Btk inhibitor that potently silences Btk enzymatic activity (IC50 < 0.5nM) with high selectivity towards Btk with lack of significant inhibition against other kinases involved in BCR signaling (Syk, Lyn). We examined the mechanism of action of AVL-292 in the context of OCs function. OC derived from MM patient monocytes were assayed with or without AVL-292. Interestingly, OC function was inhibited in the presence AVL-292 as demonstrated by pit formation assay. However, mRNA expression of Cathepsin K and TRAP, markers of OC differentiation, were increased in the presence of AVL-292. These data suggest AVL-292 inhibits OC function without affecting the OC differentiation.
It has been shown that BTK and Tec regulation of OC differentiation is related to calcium (Ca2+) signaling by increasing Ca2+ flux through direct phosphorylation of phospholipase C2 (PLCγ2). To delineate the mechanism of action of the BTK inhibitor against OC function, the RANK signaling proteins were detected by western blotting and Ca2+ concentration was measured by flourescence. AVL-292 inhibited phosphorylation of the BTK substrate, PLCγ2 in OCs. This was associated with an inhibition of intracellular Ca2+ release by AVL-292 which otherwise increased with RANKL stimulation in OCs.
Given the effect of AVL-292 on RANK signaling we next evaluated its effect on Proline-rich tyrosine Kinase 2 (Pyk2) and c-Src. Pyk2 plays a role in OC activation and localizes to the sealing zone in OC. RANK signaling activates c-Src, which phosphorylates Pyk2. Moreover c-Src controls OC bone resorption by regulating actin organization via cortactin. Interestingly, AVL-292 inhibited c-Src and Pyk2 phosphorylation. Furthermore, AVL-292 inhibited cortactin protein and mRNA expression, and upregulated c-Cbl protein (E3 ubiquitin ligase for c-Src) expression in OCs derived from MM patient monocytes.
These data demonstrate that the novel BTK inhibitor AVL-292 inhibits OC function through inhibition of OC sealing zone formation. These results suggest a potential novel mechanism of Btk inhibition with AVL-292 on osteoclast function and therefore bone resorption.
Evans:Celgene: Employment. Singh:Celgene: Employment. Westlin:Celgene: Employment. Raje:Onyx: Consultancy; Celgene: Consultancy; Millenium: Consultancy; Acetylon: Research Funding; Amgen: Research Funding; Eli-Lilly: Research Funding.
Asterisk with author names denotes non-ASH members.