Abstract LB-C21: CXCR7 expression is necessary for the maintenance of mesenchymal phenotype in acquired EGFR TKI resistance in NSCLC

Abstract

Abstract Activating EGFR mutations in non-small lung cancer (NSCLC) confer sensitivity to reversible EGFR tyrosine kinase inhibitors (TKIs), including gefitinib and erlotinib. Despite promising initial response, acquired resistance develops mediated by the emergence of the secondary T790M mutation or by focal amplification of MET. An epithelial-to-mesenchymal transition (EMT) is clinically linked to NSCLCs with acquired EGFR TKI resistance. The exact mechanisms of EGFR TKI resistance with EMT phenotype remain elusive; therefore, we have engineered EGFR-mutated NSCLC cell lines with mesenchymal phenotype by stably depleting E-Cadherin or by overexpressing Snail or chronically exposing the cells to TGFβ1. The resulting mesenchymal cells are resistant to EGFR TKIs. We employed genomic analyses to identify that C-X-C chemokine receptor type 7 (CXCR7) is commonly overexpressed in the engineered cells with mesenchymal phenotype. We also discovered that CXCR7 is overexpressed in EGFR-mutated HCC4006 NSCLC cells grown resistant to gefitinib that developed a mesenchymal phenotype (HCC4006 Ge-R). To extend our findings to in vivo, we assessed if CXCR7 is overexpressed in mouse lung cancers driven by human EGFR exon19 deletion/T790M (TD) that initially respond to the EGFR mutant-specific irreversible TKI WZ4002 to promote tumor regression but later develops tumors with acquired resistance. We discovered that the murine tumors with acquired resistance to WZ4002 present mesenchymal phenotype and overexpress CXCR7. Stable depletion of CXCR7 in HCC4006Ge-R mesenchymal gefitinib resistant cells promoted gradual mesenchymal to epithelial transition. Sustained depletion of CXCR7 in HCC4006Ge-R cells resulted in inactivation of PI3K and MAPK pathways upon gefitinib treatment and greatly restored the sensitivity to gefitinib. Furthermore, the depletion of CXCR7 in HCC4006GeR cells resulted in the downregulation of mesenchymal transcription factors essential including TWIST, ZEB1 and ZEB2, suggesting but not proving that CXCR7 maintains mesenchymal phenotype. To determine if the resistance mechanisms to gefitinib with mesenchymal cells expressing CXCR7 evolve under the selective pressure of gefitinib or pre-exist prior to treatment, we sorted EGFR-mutated NSCLC cells with CXCR7 and mesenchymal markers to find less than 5% of the cells express CXCR7 with mesenchymal phenotype. Interestingly, ectopic expression of CXCR7 in EGFR-mutated cells was not sufficient to confer resistance to EGFR TKIs or to promote EMT. Taken together, we discovered that CXCR7 is necessary for the maintenance of EGFR TKI resistance with mesenchymal phenotype and the population of mesenchymal cells that overexpress CXCR7 pre-exists and is selected upon chronic EGFR TKI treatment. Citation Format: Jeffrey H. Becker, Yandi Gao, Ines Pulido, Eiki Kikuchi, Margaret Soucheray, Rutu Gandhi, Camilla L. Christensen, Fatima Al-shahrour, Kwok-Kin Wong, Julian Carretero, Takeshi Shimamura. CXCR7 expression is necessary for the maintenance of mesenchymal phenotype in acquired EGFR TKI resistance in NSCLC. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr LB-C21.