Lenalidomide and Bortezomib Inhibit Osteoclast Differentiation and Activation in Multiple Myeloma: Clinical Implications.

Abstract Osteolytic bone disease in Multiple Myeloma (MM) is caused by enhanced osteoclast (OCL) activation and inhibition of osteoblast function. The proteasome inhibitor bortezomib (PS341, Velcade) has potent anti-myeloma activity with impressive clinical responses. A recent study indicated that bortezomib has inhibitory effects on OCL (ASH 2005, Abstract #2488). Lenalidomide (CC-5013, Revlimid) is an immunomodulatory derivative of thalidomide that has shown promising anti-MM effects in patients with relapsed or refractory MM (Richardson et. al, Blood Jul 06). Significantly, a phase I clinical trial showed that lenalidomide and bortezomib could achieve responses in the majority of patients with MM, refractory to either agent alone (ASH 2005, Abstract #365). However, the effect of lenalidomide on human OCL lineage is unknown. Here we investigated the effect of lenalidomide and bortezomib on human OCL. Peripheral blood mononuclear cells (PBMC) from MM patients (n=11) and healthy donors (n=5) were stimulated with receptor activator of NFk-B ligand (RANKL) (50ng/ml) and M-CSF (25ng/ml) for two weeks to induce OCL formation, in the presence or absence of lenalidomide or bortezomib. OCL were identified by flow cytometric analysis using anti-aVb3 integrin. Lenalidomide and bortezomib inhibited OCL differentiation in a dose-dependent manner (n=13, median control: 70.9% at 0 μM; 63% at lenalidomide 2μM and 45% at 10μM; 35% at bortezomib 2nM and 11% at 5nM). TRAP staining (tartrate-resistant acid phosphatase) was performed to identify OCL and confirm OCL activity. Lenalidomide, as well as bortezomib inhibited OCL in a dose-dependent manner, as evidenced by a marked decrease in TRAP+ cells. To assess bone resorption activity, OCL were cultured with dentine discs, in the presence or absence of lenalidomide and bortezomib, followed by light microscopic analysis and additional measurement of soluble collagen I fragments from the supernatant. Both lenalidomide and bortezomib inhibited bone resorption in a dose-dependent manner. We next asked whether mature OCL were affected. OCL were induced by cytokine stimulation for 3 weeks and treated for 72h, followed by flow cytometry. Neither lenalidomide nor bortezomib altered total number of aVb3 integrin-expressing mature OCL (n=6). In addition, OCL culture supernatants were collected, and two major MM growth and survival factors produced by OCL, B-cell activation factor (BAFF) and a proliferation-inducing ligand (APRIL), were measured by specific ELISA. Both lenalidomide and bortezomib strongly inhibited secretion of BAFF and APRIL. Finally, we determined whether lenalidomide and bortezomib inhibited expression of transcriptional factors important for OCL differentiation and survival. Cell lysates of CD14-expressing monocytic cells from normal donor PBMCs were subject to immunoblotting. Importantly, lenalidomide inhibited OCL differentiation by downregulation of PU.1 expression. These results therefore indicate, that lenalidomide and bortezomib inhibit OCL differentiation, thereby directly preventing the development of new osteolytic lesions. Moreover, BAFF and APRIL secretion by OCL is downregulated, thereby inhibiting MM cell survival in the bone marrow microenvironment.