Abstract LB517A: The role of EGFR in resistance to tucatinib and its therapeutic implications
Authors: Veeraraghavan Jamunarani , Liao Fu-Tien , Gordon Tia , Selenica Pier , Nanda Sarmistha , Qin Lanfang , Zhu Yingjie , Patel Juber A. , Gazzo Andrea , Stossi Fabio , Mancini Michael A. , Gutierrez Carolina , Weigelt Britta , Reis-Filho Jorge S. , Osborne C. Kent , Rimawi Mothaffar F. , Schiff Rachel 
Published: June 2022

Tucatinib (Tuc) was recently approved for metastatic disease and is moving towards the early setting in HER2+ breast cancer (BC). Given the increasing clinical use of Tuc, resistance will likely soon emerge as a challenge. Here, we explore the yet unknown mechanisms of resistance to Tuc and identify treatment strategies to overcome it. Our recently developed models of BT474 (AZ and ATCC) with acquired resistance to Tuc (TucR) and their sensitive parental (P) were used. DNA-seq, RNA-seq, and RPPA/western blot were performed. Knockdown studies were performed using EGFR siRNA. Drug efficacy studies involved cell growth assays by imaging-based or methylene blue assays. We recently reported (SABCS 2021) that our BT474 TucR models acquired EGFR amplification. The TucR cells displayed elevated levels of phosphorylated (p) and total (t) EGFR, pHER2, pHER3, and downstream pAKT and pS6, which were substantially suppressed by the EGFR-specific tyrosine kinase inhibitor (TKI) gefitinib (Gef) or even further when combined with Tuc. Our new results demonstrate that EGFR knockdown selectively inhibits the growth and pHER2 levels in TucR vs P cells, supporting our hypothesis that heterodimerization of amplified EGFR with HER2 leads to higher pHER2 levels in TucR cells. We have recently also shown that TucR models were hypersensitive to Gef and this inhibition was further enhanced with Gef+Tuc, implying their survival dependence on EGFR. Here, we demonstrate that the TucR cells made resistant to 200nM Tuc maintain their resistant growth and elevated EGFR-dependent signaling even when exposed to 500nM, and can begrown as xenografts in the presence of clinically relevant dose of Tuc, emphasizing their true resistance via amplified EGFR. Importantly, both TucR models vs P cells were cross-resistant to trastuzumab but maintain partial sensitivity to TDM1. While the EGFR-specific antibody cetuximab (Cet) was partially effective as a single agent only in the ATCC model, it potently inhibited growth and induced cell killing in combination with Tuc in both models. A significantly greater inhibition in cell growth and survival was also observed when trastuzumab or TDM1 was combined with either Gef or Cet. Taken together, our results suggest that the activation of HER2 and the resistant growth and survival in the TucR models is completely dependent on the amplified EGFR, which we are currently further corroborating by additional mechanistic and xenograft studies. Whilst we have previously reported that resistance to lapatinib and neratinib confer cross-resistance to Tuc, our recent findings show that resistance to Tuc may be overcome using dual/pan-HER TKIs or the combination of potent EGFR and HER2 inhibitors. Overall, our novel findings hold crucial implications in light of the current treatment landscape of HER2+ BC and biomarkers of resistance, and places a particular emphasis on considerations to sequence currently available TKIs.

Citation Format: Jamunarani Veeraraghavan, Fu-Tien Liao, Tia Gordon, Pier Selenica, Sarmistha Nanda, Lanfang Qin, Yingjie Zhu, Juber A. Patel, Andrea Gazzo, Fabio Stossi, Michael A. Mancini, Carolina Gutierrez, Britta Weigelt, Jorge S. Reis-Filho, C. Kent Osborne, Mothaffar F. Rimawi, Rachel Schiff. The role of EGFR in resistance to tucatinib and its therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB517A.