Abstract 4008: Activating HER2 (ERBB2) mutations lead to endocrine therapy resistance through S6K activation
Authors: Croessmann Sarah , Formisano Luigi , Dugger Teresa , Avogadri-Connors Francesca , Cutler Richard E. , Lalani Alshad S. , Arteaga Carlos L. 
Published: August 2018

Abstract
Background: HER2 missense mutations are present in 2-4% of breast cancers, mainly in ER+ tumors (cBioPortal). Interrogation of METABRIC data revealed that ER+ cancers with HER2 missense mutations treated with endocrine therapy are associated with decreased survival compared to ER+ tumors with wild type (WT) HER2.
Methods: The HER2 activating mutations G309A, L755S and V777L, and HER2WT were incorporated into ER+ MCF7 cells using AAV-mediated gene targeting. We examined cell viability and ER transcriptional activity (ERE-luciferase reporter) in response to estrogen deprivation and treatment with fulvestrant. Signaling downstream mutant HER2 was examined by phosphokinase arrays and immunoblot analyses. Clonogenic growth assays and immunoblot analysis of cells treated with targeted inhibitors were used to examine mechanisms of estrogen independent growth. Inhibitors included neratinib (pan-HER tyrosine kinase), everolimus (TORC1) and selumentinib (MEK). In vivo anti-tumor efficacy of fulvestrant ± neratinib ± everolimus were assessed in ovariectomized athymic mice bearing MCF7/HER2V777L xenografts.
Results: MCF7 cells containing HER2 kinase missense mutations (L755S and V777L), but not cells with HER2WTor an extracellular domain mutation (G309A), were able to proliferate exponentially in estrogen-free medium. Cells with these mutations were also resistant to 1 μM fulvestrant, despite fulvestrant's ability to downregulate ER transcriptional activity and ER protein levels, suggesting the resistance to antiestrogens was independent of ER activity. MCF7 cells with HER2L755S and HER2V777L, but not cells transduced with HER2WT or HER2G309A, showed elevated levels of p70S6K and pS6K in estrogen-deprived medium or upon treatment with fulvestrant. These changes were not associated with increased pAKT, suggesting the upregulation was independent of PI3K activation. Treatment with neratinib or everolimus, each in combination with fulvestrant, resensitized HER2L755S and HER2V777L cells to fulvestrant while also downregulating pS6K levels. Addition of selumetinib to fulvestrant did not enhance the weak antitumor effect of fulvestrant in the HER2 mutant cells or downregulate S6K. Mice bearing MCF7/HER2V777L xenografts treated with fulvestrant and neratinib or with fulvestrant and everolimus but not with fulvestrant alone were equipotent at inhibiting tumor growth, further suggesting that S6K activation is causally associated with resistance to endocrine therapy in ER+/HER2 mutant tumors. Interestingly, the triple combination (everolimus, neratinib, and fulvestrant) induced complete regression of HER2V777L tumors.
Conclusions: These data suggest that, in ER+ breast cancers, HER2 kinase mutations abrogate dependence on ER function through S6K kinase activation and targeting HER2 and/or S6K in combination with an ER antagonist exhibits significant antitumor effect.
Citation Format: Sarah Croessmann, Luigi Formisano, Teresa Dugger, Francesca Avogadri-Connors, Richard E. Cutler, Alshad S. Lalani, Carlos L. Arteaga. Activating HER2 (ERBB2) mutations lead to endocrine therapy resistance through S6K activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4008.