Abstract 880: Mapping the metastatic colorectal cancer phospho-proteome for predicting response to cetuximab
Authors: Beekhof Robin , Labots Mariette , Knol Jaco C. , Schelfhorst Tim R.A. , van Grieken Nicole , Piersma Sander R. , Pham Thang V. , Bertotti Andrea , Trusolino Livio , Verheul Henk M.W. , Jiménez Connie R. 
Published: July 2016

Abstract
Introduction: The discovery of the key role of the epidermal growth factor receptor (EGFR) and its downstream signaling effectors in the pathophysiology of colorectal cancer (CRC) has resulted in the clinical use of targeted therapies in the treatment of metastatic CRC (mCRC). However, clinical benefit to EGFR blockade is observed in only a subgroup of CRC patients wild type for KRAS andNRAS. Genomic characterization of patient derived xenograft (PDX) models of mCRC have revealed additional resistance mechanisms to the EGFR MAb cetuximab including amplification of MET, HER2 and mutations in ERBB2, EGFR, FGFR1, PDGFRA, and MAP2K1 (Bertotti et al. 2011, 2015). However, not all resistance is understood and the biochemical signaling correlates of sensitivity and resistance remain incompletely understood. Phosphoproteomics allows for comprehensive protein phosphorylation profiling, and thereby may shed new insights into the biology of CRC and the molecular basis of cetuximab response.
Aim: To identify aberrant signaling pathways, predictive biomarkers, and alternative drug targets in genomically-annotated CRC PDX models by mass spectrometry-based phosphoproteomics.
Methods: We performed LC-MS/MS based phosphoproteome profiling (van der Mijn et al. 2015; Piersma et al. 2015) on untreated CRC PDX models lacking KRAS, NRAS or BRAF mutation (n = 20). For comparison, KRAS, BRAF or NRAS-mutant and MET or HER2 amplified models (n = 21) were analyzed. Phosphopeptides were captured by phosphotyrosine antibodies and titanium dioxide. MS/MS data was searched using MaxQuant. Phosphoproteome data (ion intensities and spectral counts) were correlated with drug sensitivity. Dedicated bioinformatics was used to identify hyperactive kinases and signaling pathways in individual tumors.
Results: The dataset contained 13.418 tyrosine-, serine- and threonine-phosphorylated peptides derived from 4588 proteins, including 237 phosphorylated kinases. Unsupervised hierarchical clustering of phosphopeptide intensities revealed subclusters enriched for sensitive and resistant models. Among cetuximab-resistant models, HER2 and MET were identified as hyperphosphorylated and hyperactive driver kinases, confirming the genomic annotation. Sensitivity to combination treatment of both EGFR and driver kinases was observed previously, providing proof-of-principle of the use of phosphoproteomics for the identification of driver kinases.
Conclusion/Future: This project is uncovering the phosphoproteomic landscape of colorectal tumors and will yield a better understanding of signaling pathways involved, with an emphasis on identifying activated drug targets and predictive biomarkers in this disease, in relation to KRAS/NRAS/BRAF status and response to anti-EGFR therapy.
Citation Format: Robin Beekhof, Mariette Labots, Jaco C. Knol, Tim R.A. Schelfhorst, Nicole van Grieken, Sander R. Piersma, Thang V. Pham, Andrea Bertotti, Livio Trusolino, Henk M.W. Verheul, Connie R. Jiménez. Mapping the metastatic colorectal cancer phospho-proteome for predicting response to cetuximab. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 880.