RANKL blockade prevents and treats aggressive osteosarcomas

We are happy to respond to your concerns regarding our paper entitled “RANKL blockade prevents and treats aggressive osteosarcomas” published in Science Translational Medicine, Vol 7, issue 317 (317 ra 197) on Dec 9, 2015.
First, our goal was to generate a conditional deletion of Rank in osteoclasts, without causing osteopetrosis, and the inducible Mx1-Cre was ideal to allow deletion after the bones had matured. This promoter has been shown to target osteoclasts (1). The suggested inducible cathepsin K-Cre mice (2) only became available in 2012 after these particular experiments were done, and constitutive cathepsin K-Cre expressing mice can present additional unwarranted drawback due to unexpected germline deletion of gene in mice (3). With regard to off-target effects: RANK immunohistochemistry and TRAP staining showed that, although our treatment with pIpC induced extensive osteoclast inactivation ahead of tumorigenesis, there still existed many RANK positive cells within the bone(Fig S6). B) Although Mx1-Cre mediated Rank deletion might occur in skeletal stem and progenitors that give rise to osteoblasts, this is not of concern here because i) RANK is not expressed in normal osteoblasts, although it is ectopically expressed in some osteosarcomas; ii) we provided evidence that Col1α1-Cre mediated Rank deletion in the osteoblast lineage alone has no impact on tumor development or progression in the MOTO GEMM.
With regard to our pIpC induction regimen: To compare tumor development and progression between MOTO-Rank+/+ and MOTO-RankΔ/ΔOC mice, we required an observation window beyond 20 weeks of age. The dosing regimen of pIpC was thus optimized in a pilot study, where we found that a 13-week-treatment of pIpC (10 weeks of age onwards) was required for efficient Rank deletion in osteoclasts over 20 weeks. This was not achieved in the pilot study when using the previously reported regimen of 7 doses of pIpC over 2 weeks, as visualized by RANK and TRAP staining, due to ongoing osteoclastogenesis.
Second, RANK-Fc has been demonstrated as a specific and powerful inhibitor of RANKL function and PBS is widely used and accepted as the control in such studies. Furthermore: A) Although RANK-Fc is commonly referred to as a monoclonal antibody, it is a recombinant protein containing the murine extracellular domain of RANK fused to the Fc portion of murine IgG1 (provided by Amgen; actually incorrectly listed in our paper as fused to the human IgG1-Fc portion; now corrected with an erratum). Amgen previously tested this reagent against mammary tumors and spontaneous lung metastases in immune-competent MMTV-neu mice, using both PBS and IgG1 as controls (4). B) In our study, we demonstrated that RANK-Fc effects such as inactivated osteoclast differentiation and blocked tumorigenesis were accompanied by dramatically suppressed serum RANKL. Also, tumors occurred a few weeks after the discontinuation of RANK-Fc, accompanied by increased circulating RANKL and osteoclast re-activation. This clearly demonstrated that the RANK-Fc effects were delivered through RANKL blockade. C) Notably, RANK-Fc reduced spontaneous lung metastasis (Fig 7C) as well as experimental lung metastasis (Fig 7Biii), which was included precisely to deal with the concern raised in the final paragraph by Walkley and Martin.
References –
1. K. Tsuji-Takechi, T. Negishi-Koga, E. Sumiya, A. Kukita, S. Kato, T. Maeda, P. P. Pandolfi, K. Moriyama, H. Takayanagi, Stage-specific functions of leukemia/lymphoma-related factor (LRF) in the transcriptional control of osteoclast development. Proc Natl Acad Sci U S A 109, 2561-2566 (2012).
2. M. A. Sanchez-Fernandez, S. Sbacchi, M. Correa-Tapia, R. Naumann, J. Klemm, P. Chambon, S. Al-Robaiy, M. Blessing, B. Hoflack, Transgenic mice for a tamoxifen-induced, conditional expression of the Cre recombinase in osteoclasts. PLoS One 7, e37592 (2012).
3. C. L. Winkeler, R. D. Kladney, L. B. Maggi, Jr., J. D. Weber, Cathepsin K-Cre causes unexpected germline deletion of genes in mice. PLoS One 7, e42005 (2012).
4. E. Gonzalez-Suarez, A. P. Jacob, J. Jones, R. Miller, M. P. Roudier-Meyer, R. Erwert, J. Pinkas, D. Branstetter, W. C. Dougall, RANK ligand mediates progestin-induced mammary epithelial proliferation and carcinogenesis. Nature 468, 103-107 (2010).

We sincerely hope we addressed your concerns.

Rama Khokha