Genetic deletion of Sost or pharmacological inhibition of sclerostin prevent multiple myeloma-induced bone disease without affecting tumor growth

Abstract

Multiple myeloma (MM) causes lytic bone lesions due to increased bone resorption and concomitant marked suppression of bone formation. Sclerostin (Scl), an osteocyte-derived inhibitor of Wnt/β-catenin signaling, is elevated in MM patient sera and increased in osteocytes in MM-bearing mice. We show here that genetic deletion of Sost, the gene encoding Scl, prevented MM-induced bone disease in an immune-deficient mouse model of early MM, and that administration of anti-Scl antibody (Scl-Ab) increased bone mass and decreases osteolysis in immune-competent mice with established MM. Sost/Scl inhibition increased osteoblast numbers, stimulated new bone formation and decreased osteoclast number in MM-colonized bone. Further, Sost/Scl inhibition did not affect tumor growth in vivo or anti-myeloma drug efficacy in vitro. These results identify the osteocyte as a major contributor to the deleterious effects of MM in bone and osteocyte-derived Scl as a promising target for the treatment of established MM-induced bone disease. Further, Scl did not interfere with efficacy of chemotherapy for MM, suggesting that combined treatment with anti-myeloma drugs and Scl-Ab should effectively control MM growth and bone disease, providing new avenues to effectively control MM and bone disease in patients with active MM.

Figure 1. Genetic deletion of Sost decreases osteolysis and prevents bone loss induced by MM tumors without affecting tumor growth

(a) Experimental design (IT-intratibial injection). (b) Bone mineral density (BMD); *p≤0.05 vs wt/Scid mice. (c) Serum human Kappa light chain 4-wks after cell inoculation (n.d., not detected). (d) Tibia X-rays and number/area of osteolytic lesions (n.d., not detected) *p≤0.05 vs wt/Scid JJN3-injected mice. (e) MicroCT images and microarchitecture of proximal tibia cancellous bone; # p≤0.05 vs wt/Scid (saline); *p≤0.05 vs saline-injected mice. Saline/JJN3-injected: n=7/9 wt/Scid and n=6/10 Sost^−/−/Scid mice. Box plots: middle line in box represents the median, whiskers the 95% confidence interval of the mean, and circles are outliers from the 95% confidence interval. (BV/TV) is bone volume over tissue volume; (Tb.N) is trabecular number; (Tb.Th) is trabecular thickness and (Tb.Sp) is trabecular separation.

Figure 2. Genetic deletion of Sost prevents the decrease in bone formation induced by myeloma cells

(a) Region of interest analyzed: cancellous bone of the proximal tibia and dynamic histomorphometric indexes and representative images of labeled bone surfaces; saline/JJN3-injected: n=4/4 wt/Scid and n=5/6 Sost^−/−/Scid mice; # p≤0.05 vs wt/Scid (saline); *p≤0.05 vs saline injected mice. Static hystomorphometric quantification of osteoblasts (b) and osteoclasts (c) on bone stained with von Kossa and TRAPase; saline/JJN3-injected: n=7/9 wt/Scid and n=6/10 Sost^−/−/Scid mice; *p≤0.05 vs saline-injected mice. In figure b, red dotted lines indicate bone surfaces and yellow arrows point at osteoblasts. In figure c, black dotted lines indicate bone surfaces and yellow arrows point at TRAP positive osteoclasts. Abbreviations are as follows: Mineralizing surface over bone surface (MS/BS); mineral apposition rate (MAR); bone formation rate over bone surface (BFR/BS); osteoblast surface over bone surface (Ob.S/BS); osteoblast number over bone surface (Ob.N/BS); osteoclast surface over bone surface (Oc.S/BS); osteoblast number over bone surface (Oc.N/BS).

Figure 3. Treatment with Scl-Ab reduces the number of osteolytic lesions and increases cancellous bone mass in mice with established MM disease

(a) Experimental design. (b) Serum IgG2b at 8-wks; saline/5TGM1-injected: n=9/9 IgG and n=10/7 Scl-Ab; *p≤0.05 vs saline injected mice. (c) Hematoxylin/eosin staining of the tibia; asterisk indicates the MM tumors. (d) Images and quantification of Scl positive osteocytes (Scl +ve Ot) in cortical and cancellous bone; saline/5TGM1-injected: n=3/3 IgG and n=3/3 Scl-Ab; *p≤0.05 vs saline injected mice; red dotted lines indicate bone surfaces, red arrows point to Scl+ve Ot, and black arrows point to Scl-ve Ot. (e) Tibia X-rays and number of osteolytic lesions at 8-wks (n.d., not detected); saline/5TGM1-injected: n=9/9 IgG and n=10/7 Scl-Ab; *p≤0.05 vs 5TGM1-injected mice receiving IgG. (f) MicroCT images and bone microarchitecture; saline/5TGM1-injected: n=9/9 IgG and n=10/7 Scl-Ab; # p≤0.05 vs IgG-treated (saline) mice; *p≤0.05 vs saline-injected mice.

Figure 4. Scl-Ab treatment increases osteoblasts in mice with established MM disease

Static histomorphometry quantification of osteoblasts (a) and osteoclasts (b) in bone of mice treated with IgG or Scl-Ab; # p≤0.05 vs IgG-treated (saline) mice; *p≤0.05 vs saline-injected mice. Serum levels of P1NP (a) and CTX (b); # p≤0.05 vs IgG-treated (saline) mice; *p≤0.05 vs saline-injected mice. Saline/5TGM1-injected: n=9/9 IgG and n=10/7 Scl-Ab (c) Black dotted lines indicated bone surfaces, yellow arrows point at osteoblasts, and red arrows point at TRAP positive osteoclasts.

Figure 5. Scl-Ab does not alter the activity of anti-MM drugs

(a) Viability of human JJN3 and murine 5TGM1 myeloma cells treated with Bortezomib (BTZ), Dexamethasone (DEX) or the Notch inhibitor GSIXX alone, or in combination with Scl-Ab (b and c) for 48h; (n=4); *p≤ 0.05 vs. vehicle-treated cells (a) or vs. IgG-treated cells (b and c); bars represent mean ± SD. Similar results were observed in a separate experiment.

Figure 6. Model showing the effects of Scl and Scl-Ab therapy in bone colonized by myeloma cells

In healthy bone, Scl modulates bone mass by antagonizing Wnt signaling to inhibit bone formation and stimulate bone resorption (left panel). In MM-colonized bone overproduction of Scl contributes to bone loss and osteolysis by suppressing osteoblasts function and increasing osteoclast numbers (middle panel). Blockade of Scl using Scl-Ab increases osteoblast number and stimulates new bone formation, and results in modest decreases in bone resorption, thus improving MM-induced bone disease (right panel).