Abstract GS6-05: Gain-of-function kinase library screen identifies FGFR1 amplification as a mechanism of resistance to antiestrogens and CDK4/6 inhibitors in ER+ breast cancer
Authors: Formisano L , Lu Y , Jansen VM , Bauer JA , Hanker A , Gonzalez Ericsson P , Lee K-M , Nixon MJ , Guerrero-Zotano AL , Schwarz LJ , Sanders M , Sudhan D , Dugger TC , Cruz MR , Behdad A , Cristofanilli M , Bardia A , O'Shaughnessy J , Mayer IA , Arteaga CL 
Published: February 2018

Abstract
Background: CDK4/6 inhibitors have been approved in combination with endocrine therapy for treatment of ER+ metastatic breast cancer. The goal of this study was to discover mechanisms of resistance to ER antagonists alone and in combination with CDK4/6 inhibitors.
Results: To achieve this goal, we used lentiviral vectors to individually express 559 human kinase open reading frames (ORFs) in ER+ MCF7 human breast cancer cells treated with fulvestrant ± the CDK4/6 inhibitor ribociclib. In MCF7 cells treated with fulvestrant alone or with ribociclib, we identified 15 and 17 kinases associated with resistance, respectively. Ten of these kinases overlapped in both groups. In a secondary screen, MCF7 cells were stably transduced with V5-tagged lentiviruses expressing the positive 'hits' for treatment with fulvestrant/ribociclib. Five of 17 kinases (FGFR1, FRK, HCK, FGR, CRKL) were confirmed to induce resistance to fulvestrant/palbociclib and fulvestrant/ribociblib. Survey of TCGA for copy number alterations and/or expression of these 5 genes showed only FGFR1 to be amplified/overexpressed in ˜15% of ER+ breast cancers. Experiments in vitro showed that ER+/FGFR1-amplified (amp) MDA-134, CAMA-1 and HCC1500 human breast cancer cells and MCF7 cells stably transduced with FGFR1 were relatively resistant to estrogen deprivation, fulvestrant and fulvestrant/palbociclib compared to non-FGFR1 amp MCF7 cells. This resistance was abrogated by treatment with the FGFR tyrosine kinase inhibitor (TKI) lucitanib. Treatment with fulvestrant or palbociclib alone modestly delayed growth of ER+/FGFR1-amp breast cancer patient-derived xenografts (PDX) established in nude mice. However, addition of the FGFR TKI erdafitinib to fulvestrant/palbociclib resulted in marked PDX regression in all mice without associated toxicity and a complete cell cycle arrest measured by Ki67. Treatment of FGFR-amp cells with FGF-2 strongly induced CCND1 (cyclin D1) expression. Downregulation of CCND1 with CCND1 RNAi oligonucleotides restored sensitivity of FGFR1-amp cells to fulvestrant/palbociclib, thus phenocopying the effect of FGFR TKIs. Conversely, overexpression of CCND1 in MCF7 cells induced resistance to estrogen deprivation and to fulvestrant ± palbociclib. Finally, we examined next gen sequencing of cell free tumor DNA by Guardant360 in 34 patients before and after progression on CDK4/6 inhibitor. In 10/34 (29%) post-progression specimens, we detected alterations in the FGFR pathway: FGFR1 amplification (n=7), FGFR1 N546K (n=1), FGFR2 N549K (n=1), and FGFR2 V395D (n=1) activating mutations.
Conclusions: These data suggest aberrant FGFR signaling is a mechanism of resistance to anti-ER therapies ± CDK4/6 inhibitors. We posit overexpression of cyclin D1 induced by both FGFR signaling and ER transcription plays a role in drug resistance. Based on these findings we propose ER+/FGFR1 amplified breast cancers are endocrine resistant and should be candidates for treatment with combinations of ER and FGFR antagonists. Accordingly, we have initiated a phase Ib trial of fulvestrant, palbociclib and erdafitinib in patients with antiestrogen resistant ER+/HER2-negative breast cancer with FGFR1-4 amplification.
Citation Format: Formisano L, Lu Y, Jansen VM, Bauer JA, Hanker A, Gonzalez Ericsson P, Lee K-M, Nixon MJ, Guerrero-Zotano AL, Schwarz LJ, Sanders M, Sudhan D, Dugger TC, Cruz MR, Behdad A, Cristofanilli M, Bardia A, O'Shaughnessy J, Mayer IA, Arteaga CL. Gain-of-function kinase library screen identifies FGFR1 amplification as a mechanism of resistance to antiestrogens and CDK4/6 inhibitors in ER+ breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr GS6-05.