Metastatic colorectal cancer (mCRC) harboring a BRAF(V600E) mutation is associated with poor prognosis and limited treatment options. Clinical trials targeting BRAF with MEK ± EGFR in 2- and 3-drug combinatorial therapy regimens have produced modest response rates and progression-free survival, with early development of resistance in most patients. Considerable efforts to study the mutational landscape or gene expression profiles or synthetic lethal genetic interactions of BRAF(V600E) mCRC have remained relatively ineffective at pinpointing novel therapeutic susceptibilities. This suggests that intrinsic resistance of BRAF(V600E) mCRC to combinatorial treatments is not driven by individual genetic dependencies, but is rather mediated by the concerted upregulation of multiple, parallel signaling pathways that cooperate to circumvent therapeutic effectiveness. To characterize which signaling circuits are rewired by BRAF and EGFR-targeted therapy, we used our new high throughput kinase activity mapping (HT-KAM) platform (Coppé et al 2019 Nature Cell Biology), which can reveal druggable kinase dependencies in cancer cell lines and tumor biospecimens by directly measuring the phospho-catalytic activity of kinases using their biological peptide targets as phospho-sensors. Out of the concerted, reprogrammed kinase circuits we identified with HT-KAM, we found a highly conserved, cell-autonomous, SRC kinases-relayed, COX2-PGE2-GNAS-driven inflammatory program that functions independently of the commonly studied BRAF-MEK-ERK / EGFR / PDPK1-AKT1 pathways. Specifically, we found that SRC family kinases are catalytically activated upon single and combination treatment with BRAF ± MEK ± EGFR inhibitors. We validated the specificity and potency of this BRAF/EGFR-independent vulnerability using genetic interventions (via shRNA or CRISPR) and drug treatments (adding dasatinib or saracatinib) in cell survival and colony formation assays across >15 BRAF(V600E) cell lines, and in patient tumor-derived xenograft (PDX) mouse models. Immunohistochemistry performed on patient tumors resistant to targeted therapies, and on residual PDX tumors after BRAF and MEK treatments, further reiterated SRC activation. Mechanistically, the activation of SRC kinases was induced by an autocrine PGE2-regulated GNAS-activation loop that COX2-inhibitors celecoxib or valdecoxib reversed both in vitro and in vivo. In PDX models, addition of celecoxib significantly improved tumor growth inhibition, and systematically outperformed 2- and 3-drug targeted therapy regimens tested in clinical trials without increasing toxicity (manuscript in preparation). In conclusion, we demonstrate that SRC-signaling is at the nexus of an autonomous inflammatory program with pro-tumorigenic activities, which may explain why BRAF(V600E) colorectal tumors develop resistance to current therapies. Moreover, targeting COX2 presents a promising new clinical strategy to restore therapeutic sensitivity in patients.

Citation Format: Chloe E Atreya, Ana Ruiz-Saenz, Changjun Wang, Bo Pan, Courtney A Dreyer, Diede Brunen, Anirudh Prahallad, Danislav Spassov, Dana J Steffen, Byron C Hann, Scott R VandenBerg, Silvio Gutkind, Mark M Moasser, Laura J van 't Veer, Jean-Philippe Coppe. A reversible SRC-relayed COX2-inflammatory program drives therapeutic resistance in BRAF(V600E) colorectal tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr B023. doi:10.1158/1535-7163.TARG-19-B023