Abstract P4-01-02: A spectrum of secondary mutations in HER2 augment breast cancer cell growth and reduce neratinib sensitivity in HER2-mutant breast cancer
Authors: Marin Arnaldo Andres , Mamun Abdullah Al , Akamatsu Hiroaki , Ye Dan , Sudhan Dhivya , Brown Benjamin , Brewer Monica Red , Eli Lisa , Meiler Jens , Arteaga Carlos L , Hanker Ariella B 
Published: March 2022

Abstract
Activating mutations in HER2 (ERBB2) drive tumor growth and antiestrogen resistance in ~5% of metastatic ER+ breast cancers. Treatment with the irreversible HER2 tyrosine kinase inhibitor neratinib, in combination with fulvestrant, leads to objective responses in patients with HER2-mutant ER+ breast cancer. However, acquired resistance is common. The majority of HER2-mutant tumors progressing on neratinib-based therapy acquire secondary mutations in HER2. Except for the HER2T798I gatekeeper mutation, whether these secondary HER2 mutations are causal to neratinib resistance is not known. We hypothesized that secondary HER2 mutations augment HER2 pathway activation and promote neratinib resistance. We first studied acquired secondary HER2 mutations found in circulating tumor DNA (ctDNA) of 2 patients with breast cancer harboring the primary HER2L869R hotspot mutation. The following mutations were absent at baseline but detected in ctDNA at the time of progression on neratinib: S310Y, L755S, D769Y, and T798I (patient 1, partial response to neratinib/fulvestrant &gt;16 months); and T862A, T798I, S310F, and I767M (patient 2, stable disease &gt;7 months). We stably expressed each mutation alone or in combination with HER2L869R in MCF10A breast epithelial cells. All single mutations, with the exception of T862A and T798I, promoted growth factor-independent proliferation and growth in 3D Matrigel. Enhanced growth was blocked by treatment with 10 nM neratinib. Cells expressing HER2L869R together with S310F, I767M, and D769Y remained highly sensitive to neratinib (IC50 &lt;6 nM). In contrast, HER2L869R/L755S and HER2L869R/T862A (double-mutant) cells formed large acini in 3D Matrigel in the presence of neratinib, and exhibited a 3- to 5-fold higher IC50 than HER2L869R in 2D cell viability assays. Western blot analysis revealed that HER2L869R/L755S and HER2L869R/T862A cells required a higher concentration of neratinib (50-100 nM; 4 h) to block phosphorylation of HER2 and downstream targets AKT, ERK, and S6 relative to cells transduced with HER2L869R alone (10 nM neratinib). While HER2T862A alone did not increase HER2 pathway activation or growth factor-independent growth compared to wild type (WT) HER2, phosphorylation of HER2, AKT, ERK, and S6 was elevated in cells expressing HER2L869R/T862A relative to each single-mutant. Likewise, cells expressing HER2L869R/T862A formed larger and more invasive acini in 3D Matrigel. Computational modeling predicted that the active state of HER2 was stabilized in the HER2L869R/T862A double-mutant relative to that of either single mutant. The overall energetic barrier for the active to inactive state transition also increased for the double mutant compared to that of each single mutant. Molecular Mechanics Poisson-Boltzmann Surface Area (MMPBSA) simulations showed that neratinib binding affinity is higher for HER2L869R/T862Arelative to HER2L869R or HER2WT. Two secondary HER2 juxtamembrane domain mutations, E698del and P699L, were also acquired in HER2V777L-driven breast cancers that had progressed on neratinib. However, MCF10A cells co-transduced with V777L/E698del and with V777L/9669L remained highly sensitive to neratinib. Conclusions: In tumors with an oncogenic primary HER2 mutation, acquired secondary mutations in HER2L755S and HER2T862A reduced neratinib sensitivity. The HER2L869R/T862A double-mutant stabilizes the HER2 activation state and enhances HER2 signaling output which, in turn, was incompletely blocked by neratinib. Some but not all acquired secondary HER2 mutations enhance breast tumor growth and/or are causal to neratinib resistance.
Citation Format: Arnaldo Andres Marin, Abdullah Al Mamun, Hiroaki Akamatsu, Dan Ye, Dhivya Sudhan, Benjamin Brown, Monica Red Brewer, Lisa Eli, Jens Meiler, Carlos L Arteaga, Ariella B Hanker. A spectrum of secondary mutations in HER2 augment breast cancer cell growth and reduce neratinib sensitivity in HER2-mutant breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-02.