Modulators of Sensitivity and Resistance to Inhibition of PI3K Identified in a Pharmacogenomic Screen of the NCI- 60 Human Tumor Cell Line Collection Kevin A. Kwei, Joffre B. Baker, Robert J. Pelham* Genomic Health, Inc., Redwood City, California, United States of America Abstract The phosphoinositide 3-kinase (PI3K) signaling pathway is significantly altered in a wide variety of human cancers, driving cancer cell growth and survival. Consequently, a large number of PI3K inhibitors are now in clinical development. To begin to improve the selection of patients for treatment with PI3K inhibitors and to identify de novo determinants of patient response, we sought to identify and characterize candidate genomic and phosphoproteomic biomarkers predictive of response to the selective PI3K inhibitor, GDC-0941, using the NCI-60 human tumor cell line collection. In this study, sixty diverse tumor cell lines were exposed to GDC-0941 and classified by GI50 value as sensitive or resistant. The most sensitive and resistant cell lines were analyzed for their baseline levels of gene expression and phosphorylation of key signaling nodes. Phosphorylation or activation status of both the PI3K-Akt signaling axis and PARP were correlated with in vitro response to GDC-0941. A gene expression signature associated with in vitro sensitivity to GDC-0941 was also identified. Furthermore, in vitro siRNA-mediated silencing of two genes in this signature, OGT and DDN, validated their role in modulating sensitivity to GDC-0941 in numerous cell lines and begins to provide biological insights into their role as chemosensitizers. These candidate biomarkers will offer useful tools to begin a more thorough understanding of determinants of patient response to PI3K inhibitors and merit exploration in human cancer patients treated with PI3K inhibitors. Citation: Kwei KA, Baker JB, Pelham RJ (2012) Modulators of Sensitivity and Resistance to Inhibition of PI3K Identified in a Pharmacogenomic Screen of the NCI-60 Human Tumor Cell Line Collection. PLoS ONE 7(9): e46518. doi:10.1371/journal.pone.0046518 Editor: Kristy L. Richards, University of North Carolina at Chapel Hill, United States of America Received May 30, 2012; Accepted September 2, 2012; Published September 28, 2012 Copyright: 2012 Kwei et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: Genomic Health, Inc. provided funding for this work. The funders had no role in study design, data collection and analysis, or preparation of the manuscript but approved the decision to publish. Competing Interests: Genomic Health, Inc. provided funding for this work. All the authors are paid employees of Genomic Health, Inc., own stock in the company, and are inventors named in an United States patent application (assigned to Genomic Health, Inc.) describing methods to identify biomarkers predictive of patient response to targeted chemotherapies, including GDC-0941 and NVP-BEZ235. This does not alter adherence by the authors or Genomic Health, Inc. to all PLOS ONE policies on sharing data and materials. No other competing interests, personal or financial, exist. * E-mail: rpelham@genomichealth.com Introduction The PI3K (phosphoinositide 3-kinase) signaling cascade is one of the most frequently de-regulated pathways in human cancer [1], resulting in aberrant cell proliferation and migration. Activation by upstream receptor tyrosine kinases stimulates PI3K-mediated conversion of phosphatidylinositol (4,5) diphosphate (PIP2) into the secondary messenger phosphatidylinositol (3,4,5) triphosphate (PIP3). PIP3 subsequently recruits 3-phosphoinositide dependent protein kinase 1 (Pdpk1) and the serine-threonine protein kinase Akt to the plasma membrane, culminating in the phosphorylation and activation of Akt. It is Akt which then mediates a cascade of phosphorylation events leading to the activation of downstream pathways promoting tumor survival and growth [1,2]. Perturba- tions in multiple components of the PI3K-Akt signaling axis have been observed in numerous tumor types, most notable of which are mutations in or amplifications of PIK3CA, the gene encoding the p110a catalytic subunit of the class I PI3K. Additional mutations include amplifications and activating mutations in PI3KCB, PDPK1 and AKT1 and loss of heterozygosity and mutational inactivation of PTEN, a negative regulator of PI3K- signaling [1–3]. The frequent activation of PI3K, and its downstream effectors, has prompted multiple independent efforts to develop selective inhibitors against the PI3K pathway as a chemotherapeutic strategy in cancer, and a number of these compounds are now in clinical development [2]. One of these PI3K inhibitors is GDC- 0941, an orally bio-available small molecule inhibitor that selectively targets all class I PI3K isoforms [4]. Several pre-clinical studies of GDC-0941, including one using a panel of 54 breast cancer cell lines, have demonstrated that it has potent anti-tumor activities in cell lines that harbor activating PI3K mutations [5–8]. Interestingly, these studies have also shown that mutation status alone does not predict sensitivity, as a number of cell lines lacking an activated PI3K are nevertheless sensitive to GDC-0941. These finding suggest that additional, and heretofore uncharacterized, factors may also modulate the cellular response to GDC-0941. A better understanding of biology that underlies sensitive versus resistance phenotypes has the potential to enhance the clinical utility of this and other drugs targeting the PI3K-Akt signaling axis, by facilitating the selection of those patients most likely to derive benefit. The goal of this study was to identify additional novel pathways and/or genes that confer GDC-0941 sensitivity or resistance PLOS ONE | www.plosone.org 1 September 2012 | Volume 7 | Issue 9 | e46518