Gene Expression Ratios Lead to Accurate and Translatable Predictors of DR5 Agonism across Multiple Tumor Lineages

RESEARCH ARTICLE Gene Expression Ratios Lead to Accurate and Translatable Predictors of DR5 Agonism across Multiple Tumor Lineages Anupama Reddy1*¤a, Joseph D. Growney1, Nick S. Wilson1¤b, Caroline M. Emery1¤c, Jennifer A. Johnson1, Rebecca Ward1, Kelli A. Monaco1, Joshua Korn1, John E. Monahan1, Mark D. Stump1, Felipa A. Mapa1, Christopher J. Wilson1, Janine Steiger2, Jebediah Ledell2, Richard J. Rickles2, Vic E. Myer1, Seth A. Ettenberg1¤d, Robert Schlegel1, William R. Sellers1, Heather A. Huet1☯¤d, Joseph Lehár1☯¤e 1 Novartis Institutes for Biomedical Research, Cambridge, MA, United States of America, 2 Horizon CombinatoRx, Cambridge, MA, United States of America OPEN ACCESS Citation: Reddy A, Growney JD, Wilson NS, Emery CM, Johnson JA, Ward R, et al. (2015) Gene Expression Ratios Lead to Accurate and Translatable Predictors of DR5 Agonism across Multiple Tumor Lineages. PLoS ONE 10(9): e0138486. doi:10.1371/ journal.pone.0138486 Editor: Surinder K. Batra, University of Nebraska Medical Center, UNITED STATES Received: January 6, 2015 Accepted: August 30, 2015 Published: September 17, 2015 Copyright: © 2015 Reddy 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. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This study was funded by Novartis Institutes for Biomedical Research. All authors were employed by Novartis Institutes for Biomedical Research during the course of the study. Novartis Institutes for Biomedical Research provided support in the form of salaries for authors, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of the authors are articulated in the ‘author contributions’ section. ☯ These authors contributed equally to this work. ¤a Current address: Duke University Medical Center, Durham, NC, United States of America ¤b Current address: Agenus Inc., Lexington, MA, United States of America ¤c Current address: Biomed Valley Discoveries, Kansas City, MO, United States of America ¤d Current address: Unum Therapeutics, Cambridge, MA, United States of America ¤e Current address: Google Life Sciences, Mountain View, CA, United States of America * Abstract Death Receptor 5 (DR5) agonists demonstrate anti-tumor activity in preclinical models but have yet to demonstrate robust clinical responses. A key limitation may be the lack of patient selection strategies to identify those most likely to respond to treatment. To overcome this limitation, we screened a DR5 agonist Nanobody across >600 cell lines representing 21 tumor lineages and assessed molecular features associated with response. High expression of DR5 and Casp8 were significantly associated with sensitivity, but their expression thresholds were difficult to translate due to low dynamic ranges. To address the translational challenge of establishing thresholds of gene expression, we developed a classifier based on ratios of genes that predicted response across lineages. The ratio classifier outperformed the DR5+Casp8 classifier, as well as standard approaches for feature selection and classification using genes, instead of ratios. This classifier was independently validated using 11 primary patient-derived pancreatic xenograft models showing perfect predictions as well as a striking linearity between prediction probability and anti-tumor response. A network analysis of the genes in the ratio classifier captured important biological relationships mediating drug response, specifically identifying key positive and negative regulators of DR5 mediated apoptosis, including DR5, CASP8, BID, cFLIP, XIAP and PEA15. Importantly, the ratio classifier shows translatability across gene expression platforms (from Affymetrix microarrays to RNA-seq) and across model systems (in vitro to in vivo). Our approach of using gene expression ratios presents a robust and novel method for constructing translatable biomarkers of compound response, which can also probe the underlying biology of treatment response. PLOS ONE | DOI:10.1371/journal.pone.0138486 September 17, 2015 1 / 18 Gene Expression Ratios Predict Response to DR5 Agonist Competing Interests: This study was funded by Novartis Institutes for Biomedical Research. The authors were employees of Novartis while the research for this manuscript was being conducted. There are no patents, products in development or marketed products to declare. This does not alter the authors' adherence to all the PLOS ONE policies on sharing data and materials. Introduction Death Receptor 5 (DR5, TNFRSF10B), a receptor for Apo2L ligand or Tumor Necrosis Factor (TNF)–Related Apoptosis Inducing Ligand (Apo2L/TRAIL), signals through apoptotic pathways to induce cell death [1]. Multiple therapeutic agonists of DR5, including antibodies and recombinant Apo2L/TRAIL, have been developed and evaluated clinically in unselected patient populations [2]. Despite significant anti-tumor activity in preclinical models, efficacy in clinical settings has been disappointing. While these agents have been generally well tolerated, durable responses to monotherapy have been reported in only a few patients [3,4]. Thus, new approaches to targeting and predicting response to DR5 activation are needed to improve upon current therapeutics. Activation of DR5 leads to recruitment of the adaptor protein Fas-associated death domain (FADD) to the intracellular death domain of DR5 to form the death inducing signaling complex (DISC) [1]. Once bound in the DISC, initiator caspase-8 is cleaved, resulting in activation of downstream effector caspases (i.e., caspase-3 and -7) to drive the extrinsic apoptotic program. In so-called type II cells, caspase-8 cleaves BID to induce mitochondrial-dependent, intrinsic apoptotic signaling [5]. Several inhibitory proteins, such as c-FLIP, which negatively regulates caspase-8, and the IAP and Bcl-2 protein families keep the apoptotic program in check [1]. The heterogeneous expression of these and other pro-survival signaling factors suggests that multiple molecular features might contribute to mediating the response to DR5-mediated apoptosis [6], highlighting the complexity of predicting response. To date, DR5 targeting efforts have focused primarily on bivalent antibodies which depend on secondary Fc-mediated crosslinking for activity by immune cells [7–9]. We previously reported a novel, more potent tetravalent DR5 agonist Nanobody, DR5Nb1-tetra [10]. Nanobodies are a class of therapeutic proteins derived from the variable domains (VHH) of heavy chain-only antibodies that occur naturally in camelidae family [11]. In addition to increased valency, DR5Nb1-tetra induces apoptosis independent of exogenous cross-link (...truncated)