Abstract 757: Activation of receptor-interacting serine/threonine protein kinase-2 (RIP2K) via EGFR-mediated CRAF transactivation induces the acquired resistance to Dabrafenib in BRAF V600E mutant non-small cell lung cancer
Authors: Jang Kangwon , Sohn Jinyoung , Kim Sung-Moo , Kim Kyoung Jin , Cho Byoung Chul 
Published: August 2015

Abstract
Background: A BRAF V600E mutation found in approximately 1.3% of non-small cell lung cancer (NSCLC) leads to constitutive activation of mitogen-activated protein kinase (MAPK) pathway. Therapeutic targeting of BRAF V600E mutation in NSCLC with Dabrafenib have shown encouraging response rate. The majority of patients, however, will eventually develop resistance.
Methods: To identify acquired resistance mechanism to Dabrafenib, resistant cells were established by exposure of MV522 NSCLC cells harboring BRAF V600E mutations to stepwise increasing Dabrafenib concentrations. To understand the biological relevance of these clones, we performed Western blot analysis, receptor tyrosine kinase (RTK) array, cell viability assays, Real time-PCR, sanger-sequencing, siRNA transfection and immunoprecipitation in vitro. MV 522 cells and resistant cells were inoculated to nude mouse to assess the combination effect of BRAF and EGFR inhibitor and evaluate the expression of biomarker from in vitro assay using immunohistochemistry in vivo.
Results:The resistant clones displayed activation of epidermal growth factor receptor (EGFR)-RAS-CRAF signaling and achieved sustained activation of ERK1/2, but not MEK1/2, despite high concentration of Dabrafenib. Heparin-binding epidermal growth factor (HB-EGF) selectively enhanced cell viability in the presence of Dabrafenib in MV 522 cells, even though EGFR ligands (TGF-alpha, Epiregulin, and Amphiregulin, and HB-EGF) increased in GSR clones. Combination of Dabrafenib with EGFR inhibitors (gefitinib or cetuximab) effectively blocked transactivation of CRAF from BRAF via inhibition of EGFR-RAS signaling in resistant cells, but knockdown of CRAF by short interfering RNA (siRNA) did not affect ERK activation. Interestingly, inhibition of receptor interaction protein kinase 2 (RIPK2) by siRNA in combination with Dabrafenib abrogated tumor cell survival via inhibition of ERK1/2 in the resistant clones. Furthermore, combination of Dabrafenib with gefitinib or cetuximab effectively inhibited activation of RIPK2 and ERK1/2 in resistant clones. The enhancement of Dabrafenib sensitivity by combination of gefitinib was confirmed in vivo.
Discussion: Taken together, these findings suggest that the acquired resistance to Dabrafenib in BRAF V600E mutant NSCLC is uniquely mediated by EGFR-RAS-RIPK2-ERK signaling, bypassing MEK1/2, which may necessitate therapeutic strategies different from those of melanoma.
Citation Format: Kangwon Jang, Jinyoung Sohn, Sung-Moo Kim, Kyoung Jin Kim, Byoung Chul Cho. Activation of receptor-interacting serine/threonine protein kinase-2 (RIP2K) via EGFR-mediated CRAF transactivation induces the acquired resistance to Dabrafenib in BRAF V600E mutant non-small cell lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 757. doi:10.1158/1538-7445.AM2015-757