Cytokine-associated drug toxicity in human hepatocytes is associated signaling network dysregulation

Idiosyncratic drug hepatotoxicity is a major problem in pharmaceutical development due to poor prediction capability of standard preclinical toxicity assessments and limited knowledge of its underlying mechanisms. Findings in animal models have shown that adverse effects of numerous drugs with idiosyncratic hepatotoxicity in humans can be reproduced in the presence of coincident inflammatory cytokine signaling. Following these observations, we have recently developed an in vitro drug/inflammatory cytokine co-treatment approach that can reproduce clinical drug hepatotoxicity signatures -particularly for idiosyncratic drugs -in cultured primary human hepatocytes. These observations have suggested that drug-induced stresses may interact with cytokine signaling to induce hepatic cytotoxicity, but the hepatocyte signaling mechanisms governing these interactions are poorly understood. Here, we collect high-throughput phosphoprotein signaling and cytotoxicity measurements in cultured hepatocytes, from multiple human donors, treated with combinations of hepatotoxic drugs (e.g. trovafloxacin, clarithromycin) and cytokines (tumor necrosis factor-α, interferon-γ, interleukin-1α, and interleukin-6). We demonstrate, through orthogonal partial least-squares regression (OPLSR) modeling of these signal-response data, that drug/cytokine hepatic cytotoxicity is integratively controlled by four key signaling pathways: Akt, p70 S6 kinase, MEK–ERK, and p38–HSP27. This modeling predicted, and experimental studies confirmed, that the MEK–ERK and p38–HSP27 pathways contribute pro-death signaling influences in drug/cytokine hepatic cytotoxicity synergy. Further, our four-pathway OPLSR model produced successful prediction of drug/cytokine hepatic cytotoxicities across different human donors, even Corresponding author Douglas A. Lauffenburger Building 16, Room 343 Massachusetts Institute of Technology 77 Massachusetts Avenue Cambridge, MA, 02139, USA 617-252-1629 (tel), 617-258-0204 (fax), [email protected]. 6Present address: Molecular Imaging Program at Stanford, Stanford University School of Medicine, Stanford, California, USA 7Present address: Department of Mechanical Engineering, National Technical University of Athens, Athens, Greece 8Present address: Merrimack Pharmaceuticals, Cambridge, Massachusetts, USA 9These authors contributed equally to this work. Author contributions B.D.C. and L.G.A., with assistance from T.H., performed hepatocyte experiments and phosphoprotein and toxicity data collection and compendium construction. B.D.C. performed all data modeling and wrote the manuscript. B.D.C., L.G.A., and D.A.L. designed and interpreted the study and edited the manuscript. B.S.H., P.K.S., and L.G.G. assisted in designing and interpreting the study and editing the manuscript. NIH Public Access Author Manuscript Mol Biosyst. Author manuscript; available in PMC 2011 July 1. Published in final edited form as: Mol Biosyst. 2010 July 15; 6(7): 1195–1206. doi:10.1039/b926287c. N IH PA Athor M anscript N IH PA Athor M anscript N IH PA Athor M anscript though signaling and cytotoxicity responses were both highly donor-specific. Our findings highlight the critical role of kinase signaling in drug/cytokine hepatic cytotoxicity synergies and reveal that hepatic cytotoxicity responses are governed by multi-pathway signaling network balance.