Abstract 4171: Combined AXL/FN14 inhibition sensitize drug-resistant NSCLC in vitro and in vivo
Authors: Suresh Dhananjay , Zambre Ajit , Mukherjee Soumavo , Ghoshdastidar Shreya , Schnabel Jennifer L. , Chapman Sarah , Leevy W. Matthew , Upendran Anandhi , Kannan Raghuraman 
Published: July 2017

Abstract
Lung cancer is the number one cause of cancer-related deaths in both men and women with a median survival time of 8-10 months’ post treatment. Non Small Cell Lung Cancer (NSCLC) accounts for 80% of the lung cancers and the treatment plan is determined based on the active mutations (EGFR, ALK/ROS, and KRAS) present in the tumor. Patients bearing EGFR mutation initially respond to targeted tyrosine kinase inhibitor (TKI) therapy and after 10-14 months of treatment acquire TKI resistance. Consequently, the median PFS for NSCLC patients with EGFR mutation is only 9.5 months. The reason for this acquired drug resistance is not yet fully understood. Recent studies have reported oncogenes such as AXL could be responsible for TKI resistance. Therefore, understanding the mechanism of drug resistance is key in developing a solution to overcome the problem. For this study, we first examined the resistance mechanism and developed a biodegradable targeted nanoparticle based solution to systematically investigate the role of AXL in resistant NSCLC cell lines. In this study we (1) downregulated AXL using siRNA and separately (2) knocked out the AXL gene using crRNA (CRISPR) and treated with TKI. Our results show that AXL is responsible for activation of several EMT related proteins and upregulation of mTOR pathway. We believe the upregulation of these proteins is essential for cancer cells to switch pathways for proliferation and regulating miRNAs linked to mutations. Our results further confirm that AXL is responsible for regulating MMP-2 that is associated with cell invasion. Based on data from multiple cell lines such as H820 and A549, we demonstrate that AXL upregulation is responsible for resistance independent of EGFR activating mutations. The elucidated pathway for drug resistance was further confirmed in cell lines generated by knocking-out the AXL gene. Interpretation for AXL signaling and drug resensitization was confirmed by Western blotting, Zymography, Invasion & Migration assay, Apoptosis assay and MTT toxicity assay. We performed mRNA and miRNA analysis using qRT-PCR to understand gene expression post treatment. During the process of our investigation, we found that NSCLC cells undergo further survival cross talk with other biomarkers. Indeed, we report the first experimental evidence of a survival cross talk between AXL and FN14, a wound healing gene, that enhance cell survival post treatment. Down regulation of both AXL and FN14 dramatically reduced the IC50 of TKI. Based on the mechanism, we designed a gelatin nanoparticle that can carry both AXL and FN14 to deliver it in the tumor cell. The nanoparticle is targeted to tumor using EGFR-antibody and releases the silencing RNA within cytoplasm. We further demonstrated that dual-inhibition of AXL and FN14 in A549 mice xenografts showed tumor reduction compared to controls. In conclusion, inhibition of AXL and FN14 can maximize therapeutic response of TKI, wherein AXL is upregulated before or during drug treatment.
Citation Format: Dhananjay Suresh, Ajit Zambre, Soumavo Mukherjee, Shreya Ghoshdastidar, Jennifer L. Schnabel, Sarah Chapman, W. Matthew Leevy, Anandhi Upendran, Raghuraman Kannan. Combined AXL/FN14 inhibition sensitize drug-resistant NSCLC in vitro and in vivo [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 4171. doi:10.1158/1538-7445.AM2017-4171