TH and MW contributed equally to this work.
Multiple myeloma is caused by an accumulation of malignant plasma cells in the bone marrow. Myeloma is characterized by an osteolytic bone disease, caused by increased bone degradation and reduced bone formation. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)-β superfamily. BMP-signaling is important for both pre- and postnatal bone formation. Additionally, several BMPs induce growth arrest and apoptosis in myeloma cells. Thus, increasing BMP-signaling in myeloma patients may reduce tumor growth and restore bone formation. We therefore explored BMP4 gene therapy in a human-mouse model of multiple myeloma.
Calcium phosphate scaffolds with human mesenchymal stromal cells (MSCs) were implanted in RAG2-/-GC-/- mice and the MSCs were left to differentiate in vivo for 8 weeks to create a humanized bone microenvironment. Then, adeno-associated virus (AAV), AAV8-BMP4, which has tropism for liver cells and expresses murine Bmp4 under the control of the liver specific human α1-antitrypsin (hAAT1) promoter, were administered by tail-vein injection. Empty viral vectors, AAV8-CTRL, were used for the control group. After 2 weeks, when BMP4 was detectable in circulation, we injected fluorescently labelled KJON myeloma cells in 3 out of 4 scaffolds in each mouse. The KJON cells are hyperdiploid, have a relatively slow growth rate and rely on interleukin (IL)-6 supplementation in the absence of a supporting microenvironment, thus resembling primary human myeloma cells. Tumor growth was examined by weekly imaging until end-point, 6 weeks after tumor cell injection.
At end-point, serum levels of BMP4 in AAV8-BMP4 mice were in the range of 50-200 ng/mL, but not detectable in AAV8-CTRL mice. Strikingly, tumor growth as quantified by imaging was significantly reduced in AAV8-BMP4 mice compared with the AAV8-CTRL mice (p<0.01, 2-way ANOVA, Bonferroni post-test), suggesting that high levels of circulating BMP4 reduced tumor growth. Malignant plasma cells were not detected in the murine bone marrow or spleen as examined by imaging and flow cytometry, suggesting that the myeloma cells were confined to the humanized scaffolds. Myeloma cells isolated from tumors from both AAV8-BMP4 and AAV8-CTRL mice were still sensitive to BMP4 treatment in vitro, indicating that they did not acquire resistance to BMP4 during the experiment.
We hypothesized that increased circulating BMP4 would also be beneficial for bone in this model. Human bone is generated on the scaffolds by osteoblasts that differentiate from human MSCs seeded on the scaffolds before implantation. In this model, myeloma cells inhibit osteoblast differentiation and bone formation. Indeed, in AAV8-CTRL mice we found less bone in tumor cell-containing scaffolds compared with scaffolds without tumor cells (p<0.05, Kruskal-Wallis with Dunn's post-test). However, bone formation was not increased in tumor-containing scaffolds of AAV8-BMP4 mice. To delineate the effects of BMP4 overexpression on bone per se, without direct influence from the cancer cells, we examined the femurs by μCT. Surprisingly, the AAV8-BMP4 mice had significantly reduced trabecular bone volume (p=0.017), trabecular numbers (p=0.016), as well as significantly increased trabecular separation (p<0.001) compared with the AAV8-CTRL mice. Thus, high levels of circulating BMP4 seemed to inhibit murine trabecular bone formation. There was no difference in cortical bone parameters between the two groups.
Taken together, BMP4 gene therapy inhibited myeloma tumor growth, but also reduced trabecular bone formation in mice. Care should be taken when considering BMP4 as a therapeutic agent. If other BMPs that are also potent inhibitors of multiple myeloma cell survival and proliferation will have similar impact on bone remains to be investigated.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.