Abstract P1-13-17: Hyperactivation of the EGFR pathway is associated with resistance to tucatinib in HER2-positive breast cancer models
Authors: Liao Fu-Tien , Gordon Tia , Liu Chia Chia , Selenica Pier , Zhu Yingjie , Patel Juber , Nanda Sarmistha , Qin Lanfang , Fu Xiaoyong , Gazzo Andrea , Marra Antonio , Blanco-Heredia Juan , Weigelt Britta , Reis-Filho Jorge , Osborne C. Kent , Rimawi Mothaffar , Schiff Rachel , Veeraraghavan Jamunarani 
Published: March 2023

Abstract
Background: The HER2-specific tyrosine kinase inhibitor (TKI) tucatinib (Tuca) recently approved for advanced HER2+ breast cancer is making a move towards the early setting. Given its growing use, resistance is inevitable, as observed in the HER2CLIMB study, where only one patient with brain metastasis remained progression free after 2 years on Tuca. Driven by the prevailing lack of knowledge about the mechanisms of resistance, in this study, we sought to define these mechanisms and identify treatment strategies to overcome them. We previously reported (SABCS 2021) that our BT474 TucaR models acquired EGFR amplification and showed elevated levels of phosphorylated (p) and total (t) EGFR, pHER2, pHER3, and downstream pAKT and pS6. Since the HER pathway is activated by ligands, here we aim to assess if hyperactivation of EGFR via high levels of its ligands is an alternative mechanism of Tuca resistance. Materials and Methods: Our recently developed HER2+ BT474 (ATCC and AZ) cell models with acquired resistance to Tuca (TucaR) developed through long-term exposure to gradually increasing doses of Tuca and their naïve parental (P) were used. Genomic (DNA-seq), transcriptomic (RNA-seq), and proteomic (western blot) characterization were performed. Changes in cell growth and migration were assessed by methylene blue and Incucyte wound healing assays, respectively. Results: RNA-seq analysis demonstrated that the levels of TGFα was significantly higher in our BT474 TucaR models compared to P cells. Our results now demonstrate that exogenous supplementation of EGF to BT474-P cells rescues the Tuca-mediated inhibition of pEGFR, pHER2, and the downstream pAKT, pERK, and pS6 levels. Exogenous EGF was also found to reduce the levels of apoptosis, as assessed by cleaved PARP, mitigating the Tuca-induced cell death. Exogenous EGF or TGFα rendered naïve BT474 and SKBR3 cells resistant to Tuca while neratinib, a pan-HER TKI, effectively inhibited this ligand-driven cell growth. We previously showed that the HER signaling reactivation observed in our EGFR-amplified TucaR cells was inhibited by the EGFR-specific TKI gefitinib (Gef) (SABCS 2021) and that the TucaR cells displayed enhanced migratory capabilities (AACR 2022). Here, we demonstrate that in addition to curbing the growth of TucaR cells, Gef, either alone or together with Tuca, also markedly reverts the migration of the TucaR cells. Knockdown (KD) of EGFR but not HER2 selectively and substantially inhibited the migration of the TucaR cells. KD of EGFR also had a marked cell killing effect on only the TucaR cells, whereas HER2 KD inhibited the growth of P but not TucaR cells. Our findings are consistent with the notion that while the P cells are functionally dependent on HER2, in TucaR cells the survival dependence could be rewired to rely primarily on the hyperactive EGFR signaling. Genomic analysis further revealed that in addition to EGFR amplification, the AZ TucaR cells also acquired a gain of YES1, a src family receptor tyrosine kinase implicated in cancer cell growth, invasion, and metastasis. Functional studies using 2 siRNAs, however, showed that YES1 KD had no effect on the growth of TucaR cells, and the migration of both TucaR and P cells was equally affected by YES1 KD, precluding the potential role of YES1 in driving the resistant and enhanced migratory phenotypes. Conclusions: Hyperactivation of the EGFR pathway via amplification of EGFR or increased expression of its ligands confers resistance to Tuca, which may be overcome using dual/pan-HER TKIs or the combination of potent EGFR and HER2 inhibitors. Given the rapidly evolving treatment landscape of HER2+ breast cancer and biomarkers of resistance, our novel findings have potentially crucial therapeutic implications and suggest that rationally sequencing the currently available TKIs may be clinically important.
Citation Format: Fu-Tien Liao, Tia Gordon, Chia Chia Liu, Pier Selenica, Yingjie Zhu, Juber Patel, Sarmistha Nanda, Lanfang Qin, Xiaoyong Fu, Andrea Gazzo, Antonio Marra, Juan Blanco-Heredia, Britta Weigelt, Jorge Reis-Filho, C. Kent Osborne, Mothaffar Rimawi, Rachel Schiff, Jamunarani Veeraraghavan. Hyperactivation of the EGFR pathway is associated with resistance to tucatinib in HER2-positive breast cancer models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P1-13-17.