Comparative oncology approach to drug repurposing in osteosarcoma.

BACKGROUND:Osteosarcoma is an orphan disease for which little improvement in survival has been made since the late 1980s. New drug discovery for orphan diseases is limited by the cost and time it takes to develop new drugs. Repurposing already approved FDA-drugs can help overcome this limitation. Another limitation of cancer drug discovery is the lack of preclinical models that accurately recapitulate what occurs in humans. For OS using dogs as a model can minimize this limitation as OS in canines develops spontaneously, is locally invasive and metastasizes to the lungs as it does in humans. METHODS:In our present work we used high-throughput screens to identify drugs from a library of 2,286 FDA-approved drugs that demonstrated selective growth inhibition against both human and canine OS cell lines. The identified lead compound was then tested for synergy with 7 other drugs that have demonstrated activity against OS. These results were confirmed with in vitro assays and an in vivo murine model of OS. RESULTS:We identified 13 drugs that demonstrated selective growth inhibition against both human and canine OS cell lines. Auranofin was selected for further in vitro combination drug screens. Auranofin showed synergistic effects with vorinostat and rapamycin on OS viability and apoptosis induction. Auranofin demonstrated single-agent growth inhibition in both human and canine OS xenografts, and cooperative growth inhibition was observed in combination with rapamycin or vorinostat. There was a significant decrease in Ki67-positive cells and an increase in cleaved caspase-3 levels in tumor tissues treated with a combination of auranofin and vorinostat or rapamycin. CONCLUSIONS:Auranofin, alone or in combination with rapamycin or vorinostat, may be useful new treatment strategies for OS. Future studies may evaluate the efficacy of auranofin in dogs with OS as a prelude to human clinical evaluation

FDA-approved compounds that show inhibition of osteosarcoma cell proliferation.

<p>FDA-approved compounds that show inhibition of osteosarcoma cell proliferation.</p

Auranofin, in combination with vorinostat or rapamycin, significantly reduces tumor growth of KHOS/NP and Abrams cells in mice.

<p><b>A-D.</b> Tumor formation assays in nude mice subcutaneously injected with KHOS/NP (1X10⁶ (<b>A</b>, <b>B</b>) or Abrams (1.5X10⁶ (<b>C</b>, <b>D</b>)) OS cells. When tumors reached 3 mm in diameter, mice were intraperitoneally injected with DMSO or auranofin (AF), along with vorinostat (VST (<b>A</b>, <b>C</b>), or rapamycin (RPM (<b>B</b>, <b>D</b>). Tumor sizes were three-dimensionally measured twice a week. Graphs showing sizes of tumors formed in mice (top). Note that the results of DMSO and auranofin alone in (<b>C)</b> were also used in (<b>D)</b>, since experiments in (<b>C)</b> and (<b>D)</b> were performed at the same time. Error bars: means ± S.D. (n = 5 animals for each group in (<b>A</b> and <b>B)</b>; n = 4 animals each group in (<b>C</b> and <b>D</b>)). **, P < 0.01; Student’s <i>t</i> test. NS: Not significant. Representative images of tumors formed in mice at day 21 (bottom). (<b>E)</b> Representative images of immunohistochemistry for Ki67 and cleaved caspase-3 using KHOS/NP tumors treated with DMSO or indicated drugs (magnification, 40X). Scale bars, 200 μm.</p

Primary screening of FDA-approved library identifies auranofin as a potential drug for OS therapy.

<p><b>A.</b> A diagram of the primary screening to select auranofin. <b>B.</b> Auranofin chemical structure (top) and concentration-response curves of auranofin cytotoxic effects on human (MG-63 and KHOS/NP) and canine (Abrams and D17) OS cells (bottom). Graph also includes IC₅₀ values for each cell line. Error bars: means ± S.D. from 3-independent experiments.</p

Auranofin shows synergistic effects with vorinostat and rapamycin on the viability of MG-63 cells.

<p>MG-63 cells were treated with various concentrations of auranofin along with vorinostat (<b>A</b>) or rapamycin (<b>B</b>) for 48 hours, and the cytotoxicity was determined. Representative Bliss independence plots are shown on the left. Summaries of all combination indices (numbers) calculated by Chou-Talalay plots using data obtained from varying combination pairs of auranofin and vorinostat or rapamycin (right).</p