Lenalidomide and Bortezomib: Targeting Osteoclastogenesis, Osteoclast Survival Factors, and Bone Remodeling Markers in Multiple Myeloma.
Authors: Breitkreutz Iris , Raab Marc S. , Vallet Sonia , Hideshima Teru , Raje Noopur , Mitsiades Constantine , Chauhan Dharminder , Munshi Nikhil C. , Richardson Paul G. , Anderson Kenneth C. 
Published: October 2019

Abstract
Osteolytic bone disease in Multiple Myeloma (MM) is caused by enhanced osteoclast (OCLs) activation and inhibition of osteoblast function. Lenalidomide and bortezomib have shown promising anti-MM effects, and bortezomib has inhibitory effects on OCLs. However, the effect of lenalidomide on OCLs in MM and how bortezomib interferes with osteoclastogenesis is unknown. Here we investigated the effect of lenalidomide and bortezomib on human OCLs. Peripheral blood mononuclear cells (PBMC) from MM patients 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. OCLs were identified by flow cytometric analysis using anti-αVβ3 integrin. Lenalidomide and bortezomib inhibited OCL differentiation indicated by a decrease in αVβ3-integrin (lenalidomide at 0μM: median 69.3%; range 28.9 – 89.0%; at 2μM: median 50.4%; range 21.5 – 64.2%; at 10μM: median 39.2%; range 33.6 – 47.5%) (bortezomib at 0nM: median 69.3%; range 28.9 – 89.0%; at 2nM: median 35.0%; range 11.0 – 79.0%; at 5nM: median 11.5%; range 5.5 – 8.8%; p<0.05). Tartrate-resistant acid phosphatase (TRAP) staining was performed to identify OCLs and confirm OCL activity. Both drugs decreased TRAP -positive cells (lenalidomide at 0μM: median 70.5%; range 50.0 – 84.0%; at 2μM: median 47.0%; range 31.0 – 77.0%; at 10μM: median 32.5%; range 14.0 – 44.0%; p<0.05) (bortezomib at 0nM: median 70.5%; range 50.0 – 84.0%; at 2nM: median 26.0%; range 11.0 – 39.0%; p<0.01; at 5nM: median 17.0%; range 13.0 – 34.0%; p<0.001). 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. Both lenalidomide and bortezomib inhibited bone resorption in a dose-dependent manner. Using western blot analysis, we identified PU.1 and pERK as major targets of lenalidomide, and NFATc1 as a target of bortezomib, resulting in inhibition of osteoclastogenesis. Furthermore, downregulation of cathepsin K, essential for resorption of the bone collagen matrix, was also noted. We demonstrated a significant decrease of growth and survival factors including MIP-1α, IL-6, B-cell activation factor (BAFF), and a proliferation-inducing ligand (APRIL). Importantly, in serum from patients with refractory of relapsed MM treated with lenalidomide, the essential bone remodeling factor RANKL, as well as the RANKL/Osteoprotegerin (OPG) ratio, were significantly reduced. The median OPG at baseline was significantly lower (median 8.8pg/ml, range 7.7–12.6pg/ml) than after treatment (median 10.4pg/ml, range 8.5–15pg/ml) (p<0.05). Lenalidomide also significantly inhibited the secretion of RANKL in patient’s serum (baseline: median 17pg/ml, range 9.0–36pg/ml; two months after therapy: median 4.2pg/ml, range 2.5–36pg/ml) (p<0.05). The ratio of RANKL/OPG was significantly higher before than after therapy (baseline: median 2.2pg/ml, range 1.1–3.0pg/ml; two months after therapy: median 0.5pg/ml, range 0.3–2.6pg/ml) (p<0.05). We conclude that both agents specifically target key factors in osteoclastogenesis, thereby inhibiting OCL differentiation and function.