Abstract 1911: HER2 L755S mutation is associated with acquired resistance to lapatinib and neratinib, and confers cross-resistance to tucatinib in HER2-positive breast cancer models
Authors: Veeraraghavan Jamunarani , Mistry Ragini , Nanda Sarmistha , Sethunath Vidyalakshmi , Shea Martin , Mitchell Tamika , Anurag Meenakshi , Mancini Michael A. , Stossi Fabio , Osborne C. Kent , Rimawi Mothaffar F. , Schiff Rachel 
Published: September 2020

Abstract
Despite the availability of potent HER-targeted agents, de novo and acquired resistance is common and continues to pose a major challenge, especially in the advanced setting. Amassing evidence point to the importance of HER2 mutations, including the most common HER2 L755S mutation, in mediating anti-HER2 resistance. The HER2 L755S mutation, in particular, is observed to be enriched in metastatic lesions compared to primary breast tumors. The need for effective therapy to treat tumors harboring HER2 mutations prevails. We have previously reported that acquired resistance to lapatinib (L)-containing treatments, mediated by HER2 L755S, could be overcome by the recently FDA-approved irreversible pan-HER tyrosine kinase inhibitor (TKI) neratinib (N). While N has shown great promise in patients with HER2-mutant metastatic breast cancer, its efficacy is somewhat limited. More recently, tucatinib, a HER2-selective TKI, has been shown to be effective in HER2-positive (+) brain metastases. Its potency in the context of HER2 mutations, however, has not yet been fully studied. In this study, we used the HER2+ BT474-L resistant (LR) cells, harboring endogenous HER2 L755S mutation, and parental (P) cells to first determine whether tucatinib may be effective in overcoming resistance mediated by HER2 mutations. Our results showed that while N effectively inhibited the growth of LR cells, although at a dose higher than that needed to inhibit the growth of naïve P cells, tucatinib failed to inhibit the growth of LR cells. Our results suggest that HER2 L755S mutation may confer cross-resistance to tucatinib. To further study mechanisms of resistance to 2nd generation anti-HER2 agents, we recently developed cell models with acquired resistance to N, through long-term exposure of the BT474-P and LR cells to increasing doses of N. These cells were profiled by reverse phase protein array (RPPA) and western blot analysis, which revealed restoration of HER2 phosphorylation in the NR derivatives, despite being cultured in the continuous presence of N. Interestingly, RNA-seq analysis revealed the presence of HER2 L755S mutation in all the NR derivatives, but not in the P cells, suggesting that the reactivated HER2 signaling observed in NR cells could be attributed to the emergence/acquisition of HER2 L755S mutation. Furthermore, while the P cells were highly sensitive to tucatinib, L, and the monoclonal antibody trastuzumab (T), the NR derivatives were totally resistant to these agents, suggesting that N resistance may also confer cross-resistance to tucatinib, L, and T. Additional molecular characterization to examine differential gene expression and mutational profile of the resistant derivatives, as well as testing of novel anti-HER2 regimens and drug combinations targeting downstream mediators to overcome resistance, both in vitro and in vivo, is ongoing.
Citation Format: Jamunarani Veeraraghavan, Ragini Mistry, Sarmistha Nanda, Vidyalakshmi Sethunath, Martin Shea, Tamika Mitchell, Meenakshi Anurag, Michael A. Mancini, Fabio Stossi, C. Kent Osborne, Mothaffar F. Rimawi, Rachel Schiff. HER2 L755S mutation is associated with acquired resistance to lapatinib and neratinib, and confers cross-resistance to tucatinib in HER2-positive breast cancer models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1911.