Bone disease is a hallmark of multiple myeloma (MM) and targeting osteoclasts (OC) to alleviate bone destruction is a component of the standard of care for MM. The activation of Bruton's tyrosine kinase (Btk), a member of the Tec family of tyrosine kinases, regulates B-cell activation and development and plays an important role in antibody production. Interestingly, Btk activation also occurs downstream of RANK signaling in OCs and its activation stimulates essential calcium signaling which plays an important role in OC function. Given this dual role of BTK in both B cell activation and osteoclastogenesis, we studied its role in the context of multiple myeloma (MM). Accordingly, we examined the efficacy of a potent and specific inhibitor of Btk (AVL-292) in OCs derived from MM patient monocytes.
AVL-292 is a highly selective, covalent Btk inhibitor that potently silences Btk enzymatic activity (IC50 < 0.5nM) and inhibits primary B cell proliferation and activation (EC50 ∼ 10nM). The compound showed high selectivity towards Btk when tested in a broad kinase panel of 62 kinases and importantly did not show significant inhibition against other kinases involved in BCR signaling (Syk, Lyn). OC derived from MM patient monocytes were assayed with or without AVL-292 for OC maturation by TRAP staining and functional activity by resorptive pit formation assay. OC function was inhibited in the presence of AVL-292 as determined by a decrease in pit formation. To delineate the mechanism of action of AVL-292 against OC function, the RANK signaling proteins were detected by western blotting and intracellular Ca2+ concentration was measured by fluorescence. AVL-292 inhibited phosphorylation of the Btk substrate, PLC γ2 in OCs. This was associated with an inhibition of intracellular Ca2+ release by AVL-292 which otherwise increased with RANKL stimulation in OCs. Although AVL-292 did not demonstrate direct cytotoxicity or inhibition of proliferation of MM cells, ongoing studies are confirming its activity in the context of co-cultures with accessory cells like OCs. These data demonstrate that the novel BTK inhibitor AVL-292 inhibits OC function through inhibition of Ca2+ mobilization through RANK signaling. These results suggest inhibition of Btk with AVL-292 has therapeutic potential for the treatment of myeloma related bone disease.
Evans:Avila Therapeutics: Employment, Equity Ownership. Singh:Avila Therapeutics: Employment, Equity Ownership. Raje:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Millenium: Membership on an entity's Board of Directors or advisory committees; Astra Zeneca: Research Funding; Amgen: Membership on an entity's Board of Directors or advisory committees; Acetylon: Research Funding.
Asterisk with author names denotes non-ASH members.