Oral Presentation Annual Meetings of the Endocrine Society of Australia and Society for Reproductive Biology and Australia and New Zealand Bone and Mineral Society 2016

Anti-cancer agent melphalan modifies the bone microenvironment by increasing osteoclast formation (#153)

Ryan Chai 1 , Michelle McDonald 1 , Rachael Terry 1 , Natasa Kovacic 1 , Jenny Down 2 , Jessica Pettitt 1 , Sindhu Mohanty 1 , Shruti Shah 1 , Gholamreza Haffari 3 , Jiake Xu 4 , Matthew Gillespie 5 , Mike Rogers 1 , John Price 6 , Peter Croucher 1 , Julian Quinn 1
  1. Bone Biology, Garvan Institute of Medical Research, Sydney, NSW, Australia
  2. Bone Biology Group, Department of Human Metabolism, Medical School, University of Sheffield, Sheffield, United Kingdom
  3. Faculty of Information Technology, Monash University, Clayton, VIC, Australia
  4. School of Pathology and Laboratory Medicine, University of Western Australia, Nedlands, WA, Australia
  5. Faculty of Medicine and Health Sciences, Monash University, Clayton, VIC, Australia
  6. College of Health and Biomedicine , Victoria University, St Albans, VIC, Australia

Melphalan is a cytotoxic agent used to treat multiple myeloma (MM). We previously found that dormant tumour cells in bone can be activated by bone destruction, and that several anti-cancer agents increase formation of bone resorbing osteoclasts in vitro by cell stress response (CSR). To determine whether melphalan causes bone loss, we studied its effects on bone metabolism in vivo and in vitro.

Naive mice were injected with melphalan (7.5mg/kg) and bone structure, bone marrow and osteoclasts examined. Melphalan effects on osteoclast formation in bone marrow and RAW264.7 cells treated with RANKL were also determined. Melphalan effects on key regulators of osteoclast differentiation, CSR and RANKL expression in bone was studied by qRT-PCR, immunoblot and reporter assays.

Melphalan-treated mice showed a 2.5 fold increase in osteoclast numbers after 3 days. Trabecular bone volume and number were decreased after 14 days, by 50.9% and 46.3% respectively. Bone marrow cells from melphalan-treated mice had more immature macrophages, and yielded 2.1-fold more osteoclasts than vehicle-treated mice, following RANKL stimulation. Melphalan dose-dependently increased RANKL-dependent osteoclast formation in bone marrow cells and RAW264.7 cells in vitro; CSR inhibitor KNK437 greatly reduced these effects. Melphalan did not affect RANKL-induced NFkB or NFATc1 signals, but did increase MITF levels. Melphalan treatment also increased mRNA expression of osteoclast fusion-associated factors DC-stamp and OC-stamp. Consistent with this, fusion was increased in RAW264.7 cells with melphalan treatment in the absence of RANKL. In addition, melphalan marginally increased the RANKL to OPG mRNA ratio in bone and osteoblastic cells.

These data suggest melphalan causes bone loss by increasing osteoclast numbers through several mechanisms: enhancing their progenitor populations in bone marrow, upregulating osteoclastic genes; activation of CSR; and enhancing osteoclast fusion. Thus, while melphalan reduces tumour load, excessive use may drive bone loss and activate dormant MM cells.