Additive Benefits of Radium-223 Dichloride and Bortezomib Combination in a Systemic Multiple Myeloma Mouse Model

Abstract

Osteolytic bone disease is a hallmark of multiple myeloma (MM) mediated by MM cell proliferation, increased osteoclast activity, and suppressed osteoblast function. The proteasome inhibitor bortezomib targets MM cells and improves bone health in MM patients. Radium-223 dichloride (radium-223), the first targeted alpha therapy approved, specifically targets bone metastases, where it disrupts the activity of both tumor cells and tumor-supporting bone cells in mouse models of breast and prostate cancer bone metastasis. We hypothesized that radium-223 and bortezomib combination treatment would have additive effects on MM. In vitro experiments revealed that the combination treatment inhibited MM cell proliferation and demonstrated additive efficacy. In the systemic, syngeneic 5TGM1 mouse MM model, both bortezomib and radium-223 decreased the osteolytic lesion area, and their combination was more effective than either monotherapy alone. Bortezomib decreased the number of osteoclasts at the tumor-bone interface, and the combination therapy resulted in almost complete eradication of osteoclasts. Furthermore, the combination therapy improved the incorporation of radium-223 into MM-bearing bone. Importantly, the combination therapy decreased tumor burden and restored body weights in MM mice. These results suggest that the combination of radium-223 with bortezomib could constitute a novel, effective therapy for MM and, in particular, myeloma bone disease.

Keywords: 5TGM1 mouse model; bortezomib; dexamethasone; multiple myeloma; myeloma bone disease; osteoblast; osteoclast; radium-223; systemic model; targeted alpha-therapy.

Figure 1

IC₅₀ isobologram for the combination effect of radium-223 dichloride (Ra-223) and bortezomib on the proliferation of 5GTM1 MM cells. CI, combination index.

Figure 2

Radium-223 and bortezomib in mono- or combination treatment inhibit tumor-induced bone destruction in the 5TGM1 myeloma mouse model with established bone disease. Mice were treated with vehicle, radium-223 dichloride (Ra-223; 330 kBq/kg, single i.v. injection), or bortezomib (1 mg/kg, i.p., twice a week). (a) Representative X-ray images of mice hind limbs analyzed for osteolytic lesions at sacrifice on study day 35. One animal shown from each group. Osteolytic areas are indicated by white arrows. (b) Total osteolytic lesion area in bone determined from both hind limbs at sacrifice using X-ray images and image analysis software. Horizontal lines represent the 5th, 25th, 50th, 75th, and 95th percentiles and crosses indicate mean values. * p < 0.05, ** p < 0.01, and *** p < 0.001 compared to vehicle; ## p < 0.01 compared to radium-223 monotherapy; §§ p < 0.01 compared to bortezomib monotherapy.

Figure 3

Effect of radium-223 and bortezomib in mono- or combination treatment on the number of osteoclasts and osteoblasts in the 5TGM1 myeloma mouse model. Representative histology images of tartrate-resistant acid phosphatase (TRAP) stained sections after treatment with (a) vehicle control, (b) radium-223 dichloride (Ra-223; 330 kBq/kg, single i.v. injection), (c) bortezomib (1 mg/kg, i.p., twice a week), or (d) their combination. Osteoclasts at the tumor–bone interface are indicated by red arrows. Scale bar indicates 50 μM and is representative for all images. (e) Number of osteoclasts at the tumor–bone interface. (f) Number of osteoblasts at bone surface. * p < 0.05, ** p < 0.01 and *** p < 0.001 compared to vehicle; ## p < 0.01 compared to radium-223 monotherapy; §§§ p < 0.001 compared to bortezomib monotherapy.

Figure 4

Effect of radium-223 and bortezomib in mono- or combination treatment on serum IgG2b levels and body weight in the 5TGM1 myeloma mouse model. Mice were treated with vehicle, radium-223 dichloride (Ra-223; 330 kBq/kg, single i.v. injection), or bortezomib (1 mg/kg, i.p., twice a week) as single agents or in combination. (a) Relative serum IgG2b paraprotein levels during the study (change from day 25 to day 34). Treatment start is indicated with a dotted line. (b) Serum IgG2b levels (absolute values) at the end of the study on day 34. (c) Relative mouse body weights during the study (change from day 27 to day 34). Treatment start (day 26) is indicated with a dotted line. (d) Mouse body weights (absolute values) at the end of the study on day 34. (e) Number of mice with paraplegia during the study. Horizontal lines in (b,d) represent the 5th, 25th, 50th, 75th, and 95th percentiles; crosses indicate mean values. * p < 0.05 compared to vehicle.

Figure 5

Effect of radium-223, bortezomib, and dexamethasone combination treatment on serum IgG2b levels and osteolytic lesion area in the 5TGM1 myeloma mouse model. Mice were treated with vehicle, radium-223 dichloride (Ra-223; 330 kBq/kg, single i.v. injection), and a combination of radium-223 (330 kBq/kg, single i.v. injection) with either bortezomib (1 mg/kg, i.p., twice a week) or bortezomib and dexamethasone (1 mg/kg, i.p., QD, 5 days on/2 days off). (a) Serum IgG2b levels (absolute values) at the end of the study on day 34. (b) Total osteolytic lesion area in bone determined from both hind limbs at sacrifice using X-ray images and image analysis software. Horizontal lines represent the 5th, 25th, 50th, 75th, and 95th percentiles and crosses indicate mean values. ** p < 0.01 and *** p < 0.001 compared to vehicle.

Figure 6

Effect of radium-223 and bortezomib in mono- or combination treatment on apoptosis, necrosis, and fibrosis in tumor-bearing tibias. Mice were treated with vehicle, radium-223 dichloride (Ra-223; 330 kBq/kg, single i.v. injection), or bortezomib (1 mg/kg, i.p., twice a week) as single agents or in combination. (a) Representative histological images (400x magnification) of MGT-stained sections of mouse tibias after treatment with radium-223 in combination with bortezomib. Arrows indicate apoptotic cells, the white circle indicates fibrotic area (i.e., evidence of past necrosis, lymphocytes, fibroblasts, and remaining tumor cells), and black circles indicate necrotic area. Scalebars indicate 100 μm and 50 μm in the upper and lower images, respectively. (b) Number of apoptotic cells relative to tumor area (n/mm²) analyzed from TUNEL-stained sections. (c) Necrotic tumor area relative to total tumor area analyzed from TUNEL-stained sections. Horizontal lines in (b,c) represent the 5th, 25th, 50th, 75th, and 95th percentiles and crosses indicate mean values. * p < 0.05 and *** p < 0.001 compared to vehicle; # p < 0.05 compared to radium-223 monotherapy; § p < 0.05 compared to bortezomib monotherapy.

Figure 7

Radium-223 incorporation into the bone matrix. The mice bearing 5TGM1 MM were treated with radium-223 dichloride (Ra-223; 330 kBq/kg, single i.v. injection) as a single agent or in combination with bortezomib (1 mg/kg, i.p., twice a week). Total activity (cpm/mg) of hind limbs at sacrifice on day 35. Horizontal lines in box plots represent the 5th, 25th, 50th, 75th, and 95th percentiles and crosses indicate mean values. ## p < 0.01 compared to radium-223 monotherapy.