Abstract 335: In situ protein complexes as a measure of AXL signaling and inhibitor response in cell line models of lung cancer
Authors: Majumder Anurima , Bai Yun , Kinose Fumi , Lew Erin D. , Yokoyama Yumi , Murphy Danielle , Li Gary , Haura Eric B. 
Published: July 2017

Abstract
AXL kinase overexpression is associated with metastasis and poor prognosis in various cancers. Recent studies show that epithelial-mesenchymal transition (EMT) and associated activation of AXL is one of the mechanisms by which tumors develop resistance to targeted therapies, such as EGFR tyrosine kinase inhibitor (TKI) in lung cancer. These studies have made AXL an attractive drug target. However, the lack of genetic alterations in AXL and recent evidence suggesting that ligand secretion into the tumor microenvironment plays a key role in signaling and resistance in cancer cells, underscore the need for assays to visualize active AXL signaling associated complexes. In this study, we aimed to develop a Proximity Ligation Assay (PLA) to effectively measure AXL activation in tumor tissues in situ, and to use the assay to evaluate the pharmacodynamic effect of a novel AXL TKI, RXDX-106, in lung cancer models. To this end, several lung cancer cell lines were analyzed for total and phosphorylated AXL (pAXL) expression as well as effects of AXL TKI on downstream signaling. Co-immunoprecipitation studies were also conducted to identify adaptor proteins that form active signaling complexes with AXL kinase, and PLAs were developed to detect these complexes in situ.
H1299 and Calu1 cells have the highest pAXL among the cells screened. RXDX-106 potently inhibits pAXL and downstream pAKT, but does not affect pERK or cell viability, in these cell lines. We also observe an increase in total AXL in response to the RXDX-106. Co-immunoprecipitation shows RXDX-106-dependent reduction in AXL-PI3KR1 (Phosphoinositide-3-Kinase, Regulatory Subunit 1) interaction that is consistent with the drug-induced reduction in pAKT by western blot. Based on this observation, we developed PLA to detect active AXL:PI3KR1 and AXL:pY100 signaling complexes in these cells. H1299 and Calu1 cells have high basal AXL:PI3KR1 and AXL:pY100 PLA foci that are abrogated by RXDX-106, in both fresh fixed and formalin-fixed paraffin-embedded cells. As expected, the HCC827 cells, which lack ligand independent pAXL, do not show significant labeling by either PLA.
Overall, we demonstrate that RXDX-106 is a potent inhibitor of AXL kinase and its downstream PI3K/AKT signaling pathway. We also show that activated AXL signaling complexes can be annotated specifically by PLA. The data provides justification for the extension of our PLA as a tool to measure drug-targetable signaling complexes to identify new models of activated AXL and enrich for activated AXL in patient tissues.
Citation Format: Anurima Majumder, Yun Bai, Fumi Kinose, Erin D. Lew, Yumi Yokoyama, Danielle Murphy, Gary Li, Eric B. Haura. In situ protein complexes as a measure of AXL signaling and inhibitor response in cell line models of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 335. doi:10.1158/1538-7445.AM2017-335