Inhibition Of Mutant Her2 Results In Synthetic Lethality When Combined With Er Antagonists In Er+/Her2 Mutant Human Breast Cancer Cells

Background: Human epidermal growth factor receptor 2 (ERBB2 or commonly known as HER2) missense mutations have been reported in 2-4% of breast cancers and occur primarily in the absence of HER2 gene amplification. Based on TCGA, approximately 60% of these tumors are hormone-dependent and express estrogen receptor (ER) a. Among ER+ breast cancers with HER2 missense mutations, more than 80% are in the HER2 kinase domain. We examined herein whether ER+/HER2 mutant breast cancer cells are resistant to anti-estrogen therapies and, thus, whether they should be treated with combined ER and HER2 inhibitors. Methods: Three common HER2 activating mutations (G309A, L755S, V777L) and wild type (WT) HER2 were incorporated into ER+ MCF7 cells using AAV-mediated homologous recombination. The isogenic incorporation of a heterozygous mutation more accurately represents primary human tumors as compared to transfection and overexpression of exogenous vectors. We examined cell viability and ER transcriptional activity, using an ERE-luciferase reporter, in response to estrogen deprivation and treatment with fulvestrant (a selective ER downregulator) and neratinib (an irreversible, pan-HER tyrosine kinase inhibitor), either alone or in combination. Signaling downstream mutant HER2 was examined by immunoblot analysis. In vivo anti-tumor efficacy of fulvestrant ± neratinib is currently being assessed in ovariectomized athymic mice bearing MCF7/HER2 V777L xenografts. Results: MCF7 cells containing HER2 kinase missense mutations (L755S and V777L), but not cells with HER2 WT or an extracellular domain mutation (G309A), were able to proliferate exponentially in estrogen-free medium. MCF7/HER2 L755S and MCF7/HER2 V777L were also resistant to 1 mM fulvestrant, despite fulvestrant9s ability to downregulate ER in these cells. Additionally, MCF7/HER2 L755S and MCF7/HER2 V777L showed increased levels of pERK and p70S6K. Treatment with 200 nM neratinib potently inhibited growth of MCF7/HER2 L755S and MCF7/HER2 V777L in estrogen-free conditions and resensitized them to fulvestrant while partially downregulating HER2 levels. Addition of 1 nM estradiol markedly rescued all three HER2 mutant cells from neratinib-induced cell death suggesting that the inhibition of both ER and mutant HER2 is required for tumor cell apoptosis. Using ERE-luc reporter assays, neratinib did not inhibit basal or estrogen-induced ER transcriptional activity or ERα Ser118 phosphorylation, thus not supporting HER2 mutation-to-ER crosstalk in these genetically engineered cells. This result also suggests that the ER and HER2 mutant pathways can operate independently and it is the dual pathway inhibition that results in synthetic lethality. Conclusions: These data suggest that, in ER+ breast cancers and similar to HER2 gene amplification, HER2 kinase domain mutations induce resistance to antiestrogen therapies. Therefore, we propose simultaneous therapeutic targeting of both ER and HER2 signaling pathways is required for maximal inhibition of ER+ breast cancers also harboring HER2 activating mutations, as is currently being investigated in the phase II SUMMIT trial (NCT01953926). Citation Format: Croessmann S, Zabransky DJ, Cutler, Jr. RE, Lalani AS, Park BH, Arteaga CL. Inhibition of mutant HER2 results in synthetic lethality when combined with ER antagonists in ER+/HER2 mutant human breast cancer cells [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr PD2-05.