Abstract P6-03-05: AXL-HER2 dimer as mechanism of anti-HER2 acquired resistance in HER2 amplified brest cancer models: A new step towards precision medicine
Authors: Adam-Artigues Anna , Tormo Eduardo , Cervera Raimundo , Rojo Federico , Pérez-Fidalgo Alejandro , Zazo Sandra , González-Alonso Paula , Hernándo Cristina , Martínez María Teresa , Gambardella Valentina , Poveda Jesús , Simón Soraya , Ortega Belén , Moragón Santiago , Rovira Ana , Albanell Joan , Burgués Octavio , Bermejo Begoña , Eroles Pilar , Lluch Ana , Cejalvo Juan Miguel 
Published: March 2020

Abstract
Background: Breast cancer (BC) is a heterogeneous disease. HER2+ BC represents between 15-20% of cases. Trastuzumab, a monoclonal antibody, has been successfully improved clinical benefits in both localized and advanced setting. Despite this evidence, many patients experience resistance to therapy. The objective of this study is to analyze AXL receptor as a potential mechanism of resistance and its implication as a prognostic factor. Methods: We used three breast cancer cell lines with acquired resistance to Trastuzumab. Resistant models were generated by treating parental cells (AU565, SKR3, BT474) with constant dose of Trastuzumab (15μg/mL) for 6 months. Cell viability was estimated by MTT assay. Proteins were assessed by Western blot and genes by qRT-PCR. AXL was downregulated by siRNA and a selective tyrosine kinase (TK) AXL inhibitor (TP-0903). Proteins interaction was assessed by immunoprecipitation and Duolink®PLA. The prognostic value of AXL was evaluated in primary tumor in a cohort of HER2+ BC patients treated with Trastuzumab plus chemotherapy from our center (n=33). Results: Acquired resistant cell lines (RCLs) maintained HER2 amplification. RCLs were more proliferative than sensitive parental cell lines. There was an important up-regulation of AXL (&gt;2.5 fold-change) and epithelial-mesenchymal transition markers (VIM, CDH2, CTNNB1 and FN1) in RCLs (p&lt;0.05). Sensitivity to Trastuzumab was restored by silencing AXL and by using TP-0903 both able to cause a decrease in cell viability and Trastuzumab IC50 (p&lt;0.05). AXL high expression was ligand independent, as GAS6 was not increased. Heterodimerization between AXL and HER2 was observed among our RCLs. In our cohort, AXL high expression was detected in patients who relapsed and it was associated with worse prognosis with a decrease of disease-free survival (p&lt;0.05). The worse prognostic role of AXL was validated in a public data set (p&lt;0.001). Conclusions: Our results suggest: 1) RCLs were more proliferative and showed mesenchymal-like enriched phenotype; 2) AXL-HER2 dimer was identified as a potential mechanism of secondary resistance to Trastuzumab; 3) genetic and pharmacological AXL inhibition restored sensitivity to Trastuzumab in in vitro models; 4) AXL expression was associated with worse prognosis in our HER2+ trastuzumab-treated BC patients and validated in silico. These results showed AXL as a prognostic factor and a potential therapeutic target in HER2+ patients with resistance to Trastuzumab and AXL overexpression.
Citation Format: Anna Adam-Artigues, Eduardo Tormo, Raimundo Cervera, Federico Rojo, Alejandro Pérez-Fidalgo, Sandra Zazo, Paula González-Alonso, Cristina Hernándo, María Teresa Martínez, Valentina Gambardella, Jesús Poveda, Soraya Simón, Belén Ortega, Santiago Moragón, Ana Rovira, Joan Albanell, Octavio Burgués, Begoña Bermejo, Pilar Eroles, Ana Lluch, Juan Miguel Cejalvo. AXL-HER2 dimer as mechanism of anti-HER2 acquired resistance in HER2 amplified brest cancer models: A new step towards precision medicine [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P6-03-05.