Abstract P1-13-08: Extended adjuvant neratinib/fulvestrant blocks ER/HER2 crosstalk and maintains complete responses of ER+/HER2+ tumors following treatment with chemotherapy and anti-HER2 therapy
Authors: Sudhan DR , Schwarz LJ , Guerrero-Zotano AL , Nixon M , Formisano L , Croessmann S , Gonzalez Ericsson PI , Sanders ME , Balko JM , Avogadri-Connors F , Cutler RE , Lalani AS , Bryce R , Auerbach A , Arteaga CL 
Published: February 2018

Abstract
Background: Neratinib is a potent, irreversible pan-HER tyrosine kinase inhibitor. The phase III trial ExteNET showed improved disease-free survival in patients (pts) with HER2+ breast cancer treated with neratinib vs placebo after trastuzumab-based adjuvant therapy. The benefit from neratinib appeared to be greater in pts with ER+ tumors. Thus, we sought to elucidate mechanisms that may explain the benefit from extended adjuvant therapy with neratinib in pts with ER+/HER2+ breast cancer using a human-in-mouse model that simulates the clinical outcomes seen in ExteNET.
Results: Mice with established ER+/HER2 amplified MDA-361 tumors were treated with trastuzumab (tz) + paclitaxel (pac) for 4 weeks, and then randomized to fulvestrant (fulv) ± neratinib for 4 weeks. All MDA-361 tumors exhibited a prompt and marked reduction in volume after tz/pac treatment; 10 mice achieved a complete response (CR) before receiving 'extended adjuvant' therapy with fulv (n=5) or neratinib/fulv (n=5). A CR was maintained with neratinib/fulv following tz/pac. However, mice treated with fulv alone, relapsed rapidly (p&lt;0.05 at week 8) despite of a complete downregulation of tumor ER levels. In a second experiment, nude mice with established MDA-361 xenografts were treated with pertuzumab/tz/pac for 4 weeks. Following a CR, mice were randomized to neratinib/fulv vs. fulv. Again, mice treated with neratinib/fulv maintained a CR, while mice in the fulv alone arm exhibited tumor progressions within a week. In three ER+/HER2+ cell lines (MDA-361, BT474 and UACC893) but not in ER+/HER– MCF7 cells, treatment with neratinib induced ER reporter transcriptional activity whereas treatment with fulv resulted in an increase in HER2 phosphorylation, suggesting compensatory crosstalk between the ER and HER2 pathways. To further understand the molecular basis of this crosstalk, MDA-361 tumor-bearing mice were treated with either fulv, neratinib or the combination for 7 days, after which tumors were harvested and analyzed using a Nanostring breast cancer ER panel consisting of 196 ER-regulated genes. Compared to vehicle or fulv-treated tumors, tumors treated with neratinib alone and neratinib/fulv showed marked downregulation of cyclin D1 mRNA expression. Similarly, in MDA-361, BT474 and UACC893 cells but not in MCF7 cells, only neratinib/fulv downregulated cyclin D1, P-AKT and P-ERK. Finally, treatment with neratinib/fulv but not fulv alone reduced cyclin D1 transcriptional reporter activity and cyclin D1 protein levels, and induced cell cycle arrest, suggesting double blockade is required to overcome compensatory crosstalk between ER and amplified HER2.
Conclusions: Neratinib/fulv but not fulv alone maintained complete responses of ER+/HER+ tumors following treatment with tz/pac or pertuzumab/tz/pac, reminiscent of the results in ExteNET. Neratinib treatment promoted ER transcriptional activity whereas ER downregulation with fulv was associated with increased HER2 signaling. In ER+/HER2+ breast cancer cells and tumors, neratinib/fulv synergistically inhibited growth, cyclin D1 expression, and AKT and MAPK activation, thus providing a plausible mechanism to explain the results in the ExteNET trial.
Citation Format: Sudhan DR, Schwarz LJ, Guerrero-Zotano AL, Nixon M, Formisano L, Croessmann S, Gonzalez Ericsson PI, Sanders ME, Balko JM, Avogadri-Connors F, Cutler RE, Lalani AS, Bryce R, Auerbach A, Arteaga CL. Extended adjuvant neratinib/fulvestrant blocks ER/HER2 crosstalk and maintains complete responses of ER+/HER2+ tumors following treatment with chemotherapy and anti-HER2 therapy [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-13-08.