An Acidic Milieu Created In Myeloma-Osteoclast Interaction Enhances Tumor Growth, but Triggers Anti-Myeloma Activity of Reveromycin A, a Novel Anti-Resorptive Agent
Authors: Watanabe Keiichiro , Abe Masahiro , Cui Qu , Kawatani Makoto , Hiasa Masahiro , Nakano Ayako , Jinno Tadashi , Harada Takeshi , Fujii Shiro , Nakamura Shingen , Miki Hirokazu , Kagawa Kumiko , Takeuchi Kyoko , Ozaki Shuji , Tanaka Eiji , Osada Hiroyuki , Matsumoto Toshio 
Published: October 2019

Abstract
Abstract 454
Multiple myeloma (MM) develops and expands in the bone marrow, and causes devastating bone destruction by enhancing osteoclastic bone resorption in their close vicinity. In MM bone lesions, thus induced osteoclasts (OCs) in turn enhance MM cell growth and survival, thereby forming a vicious cycle between the progression of bone destruction and MM tumor expansion. Such cellular interactions create an acidic milieu not only through acids produced by OCs but also through a large amount of lactate by proliferating tumor cells (Warburg effect). Reveromycin A (RM-A), a small microbial metabolite, preferentially induces cellular apoptosis in an acidic milieu, and draws considerable attention as a novel anti-resorptive agent. In the present study, we explored whether an acidic condition induced by MM-OC interaction affects MM expansion and whether RM-A targets not only OCs but also such an acidic microenvironment to regress tumor expansion in MM. INA6 and RPMI8226 MM cells potently enhanced osteoclastogenesis and osteoclastic pit formation when cocultured with rabbit bone marrow cells on bone slices. Notably, large multinucleated OCs were almost completely disappeared and pit formation on bone slices was abolished upon the treatment with RM-A at concentrations as low as 100nM. The cocultures with rabbit bone marrow cells stimulated INA6 MM cell growth; RM-A at 1microM was however able to substantially decrease the MM cell viability in the cocultures after 12 hours, although RM-A at this concentration did not affect MM cell growth when MM cells were cultured alone at pH7.4. The suppression of INA6 MM cell viability by RM-A was obviously more potent than that under bisphosphonate treatment in which mature OCs and pits on bone slices similarly decreased in number, suggesting that the anti-MM effects of RM-A is not merely due to depletion of mature OCs. Blockade of acid release by the proton pump inhibitor concanamycin A abolished such RM-A effects. Because an acidic microenvironment increases cell permeability of RM-A to cause apoptosis, it is plausible that a highly acidic milieu created by OC-MM interaction allows RM-A to act on nearby MM cells as well as OCs. In order to clarify a role of tumor acidity in RM-A-triggered cell death, we examined the effects of RM-A on MM cell growth upon acidification with lactic acid. When lactic acid was added to media to adjust their pH to be 7.0 and 6.75, the growth of INA6 and RPMI8226 MM cells was enhanced up to 150 and 120%, respectively, after 24 hours compared to that at pH7.4. However, RM-A at 1microM induced cell death in these MM cells at pH7.0 (60-70% reduction of alive MM cells compared to those at pH7.4) and at pH6.75 (>90%), suggesting cytocidal effects of RM-A on lactate-producing MM cells densely proliferated in an acidic milieu. Because metoformin, anti-diabetic agent, up-regulates lactate production through stimulation of glycolysis, we next examined the effects of RM-A on MM cells in combination with metoformin. Metoformin dose-dependently enhanced lactate production by MM cells to decrease pH in their culture media over time; RM-A at 1microM showed potent cytotoxic effects on MM cells upon 24-hour preceded treatment with metoformin at 5 mM even when MM cells were started to be cultured at pH7.4, suggesting induction of anti-MM activity of RM-A with metoformin. Finally, in vivo RM-A effects were studied using INA6 MM cell-bearing SCID-rab mice. We injected RM-A sc at 4mg/kg twice daily for 18 days to the mice after confirming MM cell growth at 4 weeks after the MM cell inoculation. The RM-A treatment substantially decreased osteolytic lesions in X-ray and microCT images and MM tumor area in bone sections along with a reduction of INA6 cell-derived human soluble IL-6 receptor levels in mouse sera as a marker of MM tumor burden. These results collectively suggest that acidic microenvironment produced by MM-OC interaction enhances MM tumor progression but can trigger cytotoxic effects of RM-A on MM cells besides acid-producing OCs. RM-A may become a candidate for a novel therapeutic agent against MM with extensive bone resorption.

Disclosures:
No relevant conflicts of interest to declare.