Abstract 2646: Proteomic approaches to study cetuximab resistance in <i>RAS/BRAF</i> wild type colorectal cancer
Authors: Alexandros Georgiou, Adam Stewart, George Vlachogiannis, Lisa Pickard, Nicola Valeri, David Cunningham, Steven R. Whittaker, Udai Banerji
Journal: Cancer Research (2019)

Abstract Background: Cetuximab is a licensed anti-EGFR antibody treatment for RAS/BRAF wild type (WT) metastatic colorectal cancer (CRC) but is only effective in a proportion of these patients. Established mechanisms of resistance include primary and secondary activating RAS mutations. However, these cannot explain a proportion of resistant cases. We investigated the differences in early adaptive signaling in cetuximab sensitive and resistant cells using phospho(p)-proteomics in order to elucidate p-proteomic signatures of response, understand resistance mechanisms and suggest future therapeutic strategies. Methods: We used a panel of 12 cell lines (6 cetuximab sensitive and 6 resistant cell lines), cells isolated from CRC patients’ ascites (n=4) and patient derived organoids (n=2). All CRC samples used were RAS/BRAF WT. Following 1 and 4h exposure to cetuximab, a multiplex antibody-based platform was used to simultaneously quantify changes in 60 p-proteins. Key results were validated with Western blotting. Proliferation assays and 2-week colony assays were used to test potential cetuximab combinations. Statistical analysis included logistic regression, T tests and Spearman’s correlation Results: Following 1 and 4h exposure in sensitive cell lines there was downregulation of pHER2 and other intracellular effector p-proteins that function within MAPK, PI3K and JAK-STAT pathways. In contrast, in resistant cell lines at 1h there was a significantly lower degree of pHER2 downregulation and upregulation of pHER3 (p&amp;lt;0.05). Following 4h exposure, despite comparable pEGFR inhibition, in resistant cells there was statistically significant lack of downregulation/upregulation of known effector p-proteins of the PI3K pathway and also upregulation of other phosphorylated receptor tyrosine kinase (RTKs). When compared to sensitive cell lines, in resistant cells there were also statistically significant increase of pPDGFRα, pTIE2 and pIRS1. Other tyrosine kinases: pVEGFR, pFGFR1 and pSRC were also upregulated in ≥50% of resistant cell lines. The upregulation/lack of downregulation of the PI3K pathway and upregulation of other pRTKs were confirmed in cetuximab resistant patient derived cells and organoid cultures. We then investigated the combination of cetuximab with: PI3K inhibitor (i), AKTi, mTORi and IGF1Ri. The combination of cetuximab with PI3Ki pictilisib was able to sensitize to cetuximab in the resistant cell lines and organoid cultures with an effect that ranged from additive to synergistic. Conclusion: RAS/BRAF WT sensitive and resistant CRC cells have distinct p-proteomic signatures following 1 and 4h cetuximab exposure. In resistant cells, early adaptive signaling involves upregulation of RTKs and the PI3K pathway. The combination of cetuximab and pictilisib showed a modest benefit, however, overlapping toxicity may render this difficult to deliver clinically. Citation Format: Alexandros Georgiou, Adam Stewart, George Vlachogiannis, Lisa Pickard, Nicola Valeri, David Cunningham, Steven R. Whittaker, Udai Banerji. Proteomic approaches to study cetuximab resistance in RAS/BRAF wild type colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2646.