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In KRAS-Mutated Colorectal Cancer Author Manuscript Published OnlineFirst on October 5, 2010; DOI: 10.1158/1535-7163.MCT-10-0376 AuthorPublished manuscripts OnlineFirst have been peeron Octoberreviewed and 5, 2010accepted as for10.1158/1535-7163.MCT-10-0376 publication but have not yet been edited. Identification of Predictive Markers of Response to the MEK1/2 Inhibitor Selumetinib (AZD6244) in KRAS-Mutated Colorectal Cancer John J. Tentler1, Sujatha Nallapareddy1, Aik Choon Tan1, Anna Spreafico1, Todd M. Pitts1, M. Pia Morelli1, Heather M. Selby1, Maria I. Kachaeva1, Sara A. Flanigan1, Gillian N. Kulikowski1, Stephen Leong1 John J. Arcaroli1, Wells A. Messersmith1 and S. Gail Eckhardt1 Author Affilliations: Division of Medical Oncology1, University of Colorado Denver Anschutz Medical Campus, Aurora, Colorado Running Title: Predictive Biomarkers for Selumetinib in CRC Key Words: MEK inhibitors, predictive biomarkers, colorectal cancer Abbreviations: CRC: colorectal cancer; MAPK: mitogen activated protein kinase; MEK: MAPK/ERK kinase; EGFR: epidermal growth factor receptor; FZD2: frizzled homolog 2 Financial Support: Research grants CA106349 (SGE); CA079446 (SGE), CA46934 (NCI Cancer Center Support Grant); AstraZeneca Inc. Research Agreement (AS, SGE); Cancer League of Colorado Seed Grant (JJT). Requests for reprints: John J. Tentler, Ph.D. 12801 E 17th Ave, MS 8117, Aurora, CO 80045. email: [email protected]; 1 1 Potential Conflicts of Interest: Research funding provided by AstraZeneca to AS, SGE. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research 1 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2010 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 5, 2010; DOI: 10.1158/1535-7163.MCT-10-0376 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Abstract Mutant KRAS activity leads to activation of the RAS/RAF/MEK/ERK pathway in approximately 44% of colorectal cancer (CRC) tumors. Accordingly, several inhibitors of the MEK pathway are under clinical evaluation in several malignancies including CRC. The goal of this study was to develop and characterize predictive biomarkers of response to the MEK1/2 inhibitor AZD6244 in CRC in order to maximize the clinical utility of this agent. Twenty-seven human CRC cell lines were exposed to AZD6244 and classified according to the IC50 value as sensitive (≤ 0.1 M) or resistant (> 1 M). All cell lines were subjected to immunoblotting for effector proteins, KRAS/BRAF mutation status, and baseline gene array analysis. Further testing was performed in cell line xenografts and KRAS mutant CRC human explants models to develop a predictive genomic classifier for AZD6244. The most sensitive and resistant cell lines were subjected to differential gene array and pathway analyses. Members of the Wnt signaling pathway were highly overexpressed in cell lines resistant to AZD6244 and appear to be functionally involved in mediating resistance by shRNA knockdown studies. Baseline gene array data from CRC cell lines and xenografts was used to develop a k-Top Scoring Pair (k-TSP) classifier which predicted, with 71% accuracy, which of a test set of patient- derived KRAS mutant CRC explants would respond to AZD6244, providing the basis for a patient-selective clinical trial. These results also indicate that resistance to AZD6244 may be mediated, in part, by upregulation of the Wnt pathway, suggesting potential rational combination partners for AZD6244 in CRC. Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research 2 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2010 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 5, 2010; DOI: 10.1158/1535-7163.MCT-10-0376 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Introduction Colorectal cancer (CRC) is the third leading cause of cancer related mortality in the United States (1). The response rates and overall survival in metastatic colon cancer has improved in the recent years with the introduction of chemotherapeutics such as oxaliplatin and irinotecan, as well as agents targeting the vascular endothelial growth factor and epidermal growth factor receptor pathways, such as bevacizumab and cetuximab, respectively (2-4). Despite these advances in treatment, advanced unresectable CRC remains incurable, resulting in over 50,000 deaths in the U.S (1). Therefore, novel individualized treatment options are needed to achieve improved rates of disease control, and hopefully, survival. The Ras-mitogen activated protein kinase (MAPK) pathway is a key signaling network which acts by transferring growth factor signals from cell surface receptors to the cytoplasm and nucleus through a sequential protein kinase cascade, including Raf, MAPK/ERK kinase (MEK), and ERK that ultimately regulate several cellular functions, including cell division, growth, survival, proliferation, differentiation, migration and cell death (5). This pathway has been shown to be constitutively activated in several cancers, leading to uncontrolled cell proliferation, resistance to apoptosis and association with an aggressive neoplastic phenotype (6). Constitutive activation of the MAPK pathway may also contribute to cancer cell resistance to chemotherapy in several types of human malignancies, including pancreatic, colon, lung, thyroid and breast cancers (7, 8). Because MEK1/2 is the exclusive substrate for RAF and the only substrate for MEK1/2 are ERK1/2, inhibitors of MEK inhibitors represent an attractive novel class of targeted anticancer therapeutics (9, 10). Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research 3 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2010 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 5, 2010; DOI: 10.1158/1535-7163.MCT-10-0376 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Several MEK inhibitors have been developed as clinical therapeutics and partial responses have been observed in patients with advanced melanoma and GI cancers, as well as prolonged stable disease in several tumor types (11-14). Phase I studies of MEK inhibitors have shown acceptable toxicity profiles at doses that inhibit MEK activity. Selumetinib (AZD6244; Astra Zeneca/ARRY-142886; Array BioPharma) (Figure S1) is a potent, orally-available, highly-specific non-ATP competitive MEK1/2 inhibitor with nanomolar activity against MEK in cell culture and mouse models. AZD6244 has undergone phase I and II clinical trials in a variety of tumor types with the best response reported as being prolonged disease stabilization (15, 16, 17). One of the most challenging aspects of anti-cancer therapeutics is that many patients exhibit inherent or acquired drug resistance. Despite intensive study, the molecular and genetic basis for drug resistance remains poorly understood. Therefore, a critical component of cancer drug development is the identification of biomarkers which may be used to predict responses to novel agents, thus facilitating individualization of therapy. Ideally, this should be performed concurrently with early clinical development to minimize the exposure of patients to potentially toxic agents that have little hope of conferring clinical benefit. This has unfortunately been best illustrated in numerous studies that retrospectively identified that CRC patients with tumors containing mutations in KRAS derived no benefit from the EGFR-targeted antibodies cetuximab and panitumumab (2, 4, 18). Clearly, the earlier development of patient selection strategies is needed to prevent similar occurrences in the future. The objective of this study was to utilize gene array technology to identify differentially expressed genes that could serve as potential biomarkers of sensitivity to Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. Copyright © 2010 American Association for Cancer Research 4 Downloaded from mct.aacrjournals.org on September 30, 2021. © 2010 American Association for Cancer Research. Author Manuscript Published OnlineFirst on October 5, 2010; DOI: 10.1158/1535-7163.MCT-10-0376 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited. AZD6244 in CRC. Additionally, gene and pathway analyses were conducted to determine if there were pathways associated with resistance to AZD6244. These results could be applied to clinical trials of AZD6244 in CRC and to identify rational combination strategies. Materials and Methods Drugs: The MEK 1/2 inhibitor AZD6244 (ARRY-142886; Array BioPharma) was provided by Astra Zeneca, Inc. and prepared as a 10 mmol/L stock solution in DMSO. The chemical structure for this compound is depicted in Figure S1. Cell culture. Twenty-seven human colon cancer cell lines were obtained from American Type Culture Collection (Manassas, Va). The GEO cells were a generous gift from Dr. Fortunato Ciardiello (Cattedra di Oncologia Medica, Dipartimento Medico-Chirurgico di Internistica Clinica e Sperimentale "F Magrassi e A Lanzara," Seconda Università degli Studi di Napoli,
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