Abstract C110: Cancer associated fibroblast-derived IGF-binding proteins augment osimertinib activity in EGFR-mutant NSCLC cells
Authors: Rix Lily L. Remsing , Sumi Natalia J. , Bryant Annamarie T. , Li Xueli , Welsh Eric A. , Fang Bin , Kuenzi Brent M. , Antonia Scott J. , Marusyk Andriy , Lovly Christine M. , Koomen John M. , Haura Eric B. , Rix Uwe 
Published: March 2020

Abstract
Background: Cancer associated fibroblasts (CAFs) are an integral part of the tumor microenvironment, which is implicated in tumor growth, metastasis and drug resistance. However, we also observed non-canonical CAF-driven sensitization to targeted drug treatment in specific, but not all, resistant EGFR mutant lung cancer cells. Elucidation of the underlying mechanisms may identify novel biomarker or drug combination approaches. Methods: Viability of EGFR-mutant, gefitinib resistant PC9GR cells in co-culture or in the presence of CAF conditioned medium (CM) was monitored by live-cell imaging using the IncuCyte system or via CellTiterGlow (CTG, Promega), respectively. Long term viability assays were analyzed by crystal violet staining. Gene expression differences of CAFs vs normal activated fibroblasts (NAFs) were determined by microarrays. Secreted proteins in the CM were identified by proteomics. Signaling changes were monitored by RTK array, phosphoproteomics and Western blot. Loss- and gain-of-function experiments were performed using siRNA, small molecule inhibitors, or addition of recombinant human (rh) proteins. Drug combinations were evaluated by CTG, crystal violet and ex vivo 3-D PDX assays. Results: Gene expression and secretome analysis of CAFs vs NAFs identified differential expression of secretory molecules, in particular IGF-binding proteins (IGFBPs), which regulate IGF1R signaling, a pathway linked to EGFR inhibitor resistance. RTK arrays and phosphoproteomics showed enhanced inhibition of IGF1R and ERK phosphorylation by osimertinib in the presence of CAF CM. Consistently, combination of IGF1R and EGFR inhibitors closely mimicked the effect of EGFR inhibition in the presence of CAF CM. CM from CAFs where IGFBPs were silenced by siRNA was less sensitizing, while rhIGFBPs conversely mimicked CM sensitizing effects. CAF CM vs NAF CM further reduced AKT and ERK phosphorylation upon EGFR inhibition. The combination effect of EGFR and IGF1R inhibition has been shown in several cell lines and ex vivo PDX models as well as with several different drug combinations. Conclusion: We found CAF-mediated drug sensitization in EGFR-mutant lung cancer, which involves the IGF1R signaling axis. IGFBPs secreted from CAFs attenuate compensatory signaling leading to improved EGFR inhibitor efficacy. This result highlights tumor suppressive effects of CAFs competing with their otherwise tumor promoting effects and adds to the growing evidence that eliminating CAFs in an undifferentiated way may be detrimental to cancer therapy. Rather, we show that mechanistic understanding of these suppressive pathways can lead to improved drug combinations that mimic these effects and may delay the onset of resistance.
Citation Format: Lily L. Remsing Rix, Natalia J. Sumi, Annamarie T. Bryant, Xueli Li, Eric A. Welsh, Bin Fang, Brent M. Kuenzi, Scott J. Antonia, Andriy Marusyk, Christine M. Lovly, John M. Koomen, Eric B. Haura, Uwe Rix. Cancer associated fibroblast-derived IGF-binding proteins augment osimertinib activity in EGFR-mutant NSCLC cells [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 C110. doi:10.1158/1535-7163.TARG-19-C110