Abstract PD3-09: HER2 L755S mutation is acquired upon resistance to lapatinib and neratinib and confers cross-resistance to tucatinib and trastuzumab in HER2-positive breast cancer cell 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: February 2021

Abstract
Background: The role of HER2 mutations in anti-HER2 resistance is gaining more importance in HER2-positive (+) breast cancer (BC). The common HER2 L755S mutation is further enriched in metastatic lesions compared to primary tumors. Despite their mounting significance, effective therapies for HER2-amplified/mutant tumors are lacking. We recently reported that acquired resistance to lapatinib (Lap)-containing regimens is mediated by HER2 L755S, which could be overcome by the irreversible pan-HER tyrosine kinase inhibitor (TKI) neratinib (Nrb). However, less is known about the role of L755S in resistance to new generation TKIs, and the clinically implementable therapeutic strategies to overcome it. Materials and Methods: Our recently developed HER2+ BT474 cell models with acquired resistance to Lap (LapR) or Nrb (NrbR), developed through long-term exposure to increasing doses of the respective drug, and their naïve parental (P) counterparts were used. The resistant derivatives and their cognate P cells were subjected to proteomic (Reverse phase protein array (RPPA) and western blot) and transcriptomic (RNA-seq) characterization. For drug efficacy studies, change in cell growth was assessed using the in situ imaging-based high-throughput IncuCyte system. Results: Proteomic profiling of the resistant models and their P equivalents revealed partial restoration of HER2 phosphorylation and downstream signaling in the LapR and NrbR derivatives. Consistent with activated mTOR signaling observed in the resistant cells, we detected reduced levels of phospho (p)-RAPTOR S792, which is otherwise essential to inhibit the mTOR complex 1 (mTORC1). In addition, p-P38MAPK T180/Y182 levels were reduced. RNA-seq analysis revealed the presence of HER2 L755S mutation in the LapR and NrbR derivatives, but not in P cells, suggesting that the HER signaling reactivation could be attributed to acquisition of HER2 L755S. Interestingly, the NrbR cells co-harbor other pathogenic mutations in key BC related genes, the therapeutic and functional significance of which is being investigated. Importantly, the NrbR derivatives were cross-resistant to Lap and the monoclonal antibody trastuzumab (T). Next, we determined the efficacy of Nrb and the HER2-selective TKI tucatinib (Tuca), both recently approved for metastatic HER2+ BC, either alone or in combination with T. Nrb effectively inhibited the growth of LapR cells, although a higher dose (IC50: ~50nM) was required to inhibit the growth compared to that needed for naïve P cells (~IC50: ~2nM). When combined with T, Nrb was effective in inhibiting the LapR cell growth, though the inhibitory effect may very well be driven entirely by Nrb. On the other hand, the resistant derivatives were cross-resistant to Tuca, both as a single-agent and in combination with T. We then evaluated the efficacy of the antibody drug conjugate TDM1 and the irreversible pan-HER TKI poziotinib. In contrast to the high sensitivity of P cells to both these agents, a spectrum of effect was observed in the NrbR derivatives, with responses ranging from partial growth inhibition by poziotinib to complete response with TDM1, suggesting their therapeutic potential against tumors harboring HER2 mutations. Conclusions: Our findings suggest that HER2 mutations, particularly HER2 L755S, that emerge under the pressure of potent HER2-targeted therapy may confer cross-resistance to other single agent or combination HER2-targeted therapy. This holds important therapeutic implications in light of current treatment landscape. An in-depth molecular characterization of our resistant models to determine the differential gene expression and mutational profile is ongoing to gain additional mechanistic insights and to guide discovery of other actionable targets.
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 acquired upon resistance to lapatinib and neratinib and confers cross-resistance to tucatinib and trastuzumab in HER2-positive breast cancer cell models [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD3-09.