Abstract 4999: Signaling-associated complexes to define targetable vulnerabilities in lung cancer
Authors: Smith Matthew , Licata Thomas , Bai Yun , Zhang Guolin , Vuaroqueaux Vincent , Fiebig Heinz-Herbert , Haura Eric B. 
Published: August 2015

Abstract
Lung cancer is the leading cause of cancer related death in the U.S. Despite successes targeting tyrosine kinase drivers such as EGFR and EML4-ALK, identification of patients who will benefit from emerging targeted therapy regimens remains a challenge. This is exemplified by the disappointing results in the recent Phase III evaluation of ornatuzumab in combination with erlotinib in advanced non-small cell lung cancer (NCT01456324), targeting both MET and EGFR in biomarker-defined patient populations. We have previously shown that proximity ligation assay (PLA) technology can be harnessed to evaluate EGFR pathway activation by annotating the presence of EGFR in complex with its major signaling adaptor GRB2. Presence of these “signaling-associated complexes” correlates with EGFR activity, reveals erlotinib pharmacodynamics and associates with improved outcomes to EGFR-directed therapies. Here, we show that PLA technology can also be harnessed to assess MET signaling activity in lung cancer. We have found that MET is constitutively associated with GRB2 in MET-amplified lung cancer cell lines and this interaction is abrogated by the MET-specific tyrosine kinase inhibitor PHA665,752 (MET TKI) as measured by biochemical approaches and PLA. In parallel experiments, siRNA-mediated knockdown of GRB2 and 100nM MET TKI both lead to dramatic reductions in cell viability, indicating that this interaction is critical in MET-driven cells. Conversely, GRB2 knockdown has no effect in MET-amplified H1648 cells which are also completely resistant to MET TKI. In MET¬-amplified patient-derived xenograft models of lung cancer we observe MET:GRB2 complexes in regions that also stain strongly using pMET(Y1234/5) immunohistochemistry (IHC). However, only a subset of regions exhibiting strong staining for total MET by IHC had MET:GRB2 complexes or pMET. Surprisingly, we have observed a very low frequency (2%) of lung adenocarcinoma patients (N = 169) with MET:GRB2 signaling complexes, even among high MET-expressing tumors, defined by MET IHC. These data suggest that MET:GRB2 is critical in the amplified setting, but may be less important in stromal-induced MET signaling. Consistent with this, GRB2 knockdown in non-amplified A549 and H292 cells fails to impair HGF-induced increases in pAKT and pERK. We are currently conducting mass spectrometry experiments to decipher differences between MET complexes in the amplified vs. ligand-stimulating setting and updated results will be presented. We suggest that only a subset of high MET-expressing cells has evidence of MET in complex with GRB2, potentially explaining the relatively low response rates observed with MET-directed therapies in lung cancer. We believe our approach of combining biochemical, PLA and mass spectrometry-based techniques will also be applicable to the development of improved biomarker strategies for newly emerging targets such as FGFR1.
Citation Format: Matthew Smith, Thomas Licata, Yun Bai, Guolin Zhang, Vincent Vuaroqueaux, Heinz-Herbert Fiebig, Eric B. Haura. Signaling-associated complexes to define targetable vulnerabilities in 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 4999. doi:10.1158/1538-7445.AM2015-4999