Background.Targeting HER2 gene amplification is one of the great achievements in oncology resulting in the use of a wide array of anti-HER2 agents in the clinic. Unfortunately, 20% of patients still relapse with secondary organ metastasis and are currently incurable. While only 1.6% of primary non-HER2-amplified ER+ breast cancers harbor HER2 mutations, 6-10% of all metastatic breast cancers harbor HER2 mutations, suggesting their causal role in contributing to metastasis. The clinical value of HER2 activating mutations is being tested with the pan-HER tyrosine kinase inhibitor neratinib in phase II clinical trials (NCT01670877, NCT01953926, and NCT02465060). To date, neratinib has elicited only modest responses in ER+ breast cancer, often rapidly followed by progression. This study characterizes HER2-mutant induced resistance to endocrine therapy or neratinib induced resistance and resulting metastasis, in order to determine a more effective rational therapeutic approach for treating ER+ HER2-mutant breast cancer. Methods.HER2 mutation frequency and its impact on patient outcome was determined using METABRIC primary breast cancer (BC) and MSK IMPACT metastatic BC ER+ sequencing studies. Effects of estrogen (E2), fulvestrant, or neratinib on cell growth and HER2 signaling were examined on ER+/ILC cells (MM134) stably expressing HER2/WT, HER2/S310F, and HER2/L755S. Cell growth was measured using CellTiter-Glo and HER2 signaling was analyzed by western blot analysis. Additionally, the effect of these ER+/ILC and IDC HER2 mutations on tumor growth and endocrine or neratinib treatment resistance was determined using fat pad injections and MIND xenografts in NOD-scid gamma mice. Results.We searched ER+ sequencing datasets and identified HER2 mutations that are highly enriched in ER+ ILC as compared to ER+ IDC. These activating HER2 mutations in ER+ ILC are associated with early relapse and poor overall survival. Moreover, we are finding that ILC patients harboring the recurrent HER2/L755S mutation have worse overall survival compared to non-mutant HER2 ILC. MM134 cells expressing HER2/S310F and HER2/L755S show increased cell growth, strongly activated autophosphorylation of HER2, and increased downstream signaling (pMAPK and pAKT) as compared to cells expressing HER2/WT upon treatment with fulvestrant (1μM). Three clinically relevant in vivo models including ILC HER2/L755S Mammary INtraDuctal (MIND) xenografts, IDC HER2/L755S fat pad xenografts, and IDC HCI-003 (an ER+ patient-derived xenograft (PDX) naturally harboring the exon 20 activating HER2G778_P780 dup) exhibit fulvestrant and neratinib resistance and lung and ovary metastases. In addition, we find that HER2 mutations induced mTOR signaling. In contrast, however, the pan-HER drug poziotinib does potently inhibit tumor growth and organ-specific metastasis and perturbs mTOR activation in these models. Conclusion.We demonstrate that clinically associated HER2 mutations drive endocrine therapy or neratinib resistance and poor patient outcome in ER+ patients. Our data propose the use of the irreversible pan-HER TKI poziotinib for treating endocrine therapy or neratinib refractory ER+ HER2-mutant metastatic breast cancer.

Citation Format: Shyam. M Kavuri, Rashi Kalra, Ching Hui Chen, Junkai Wang, Ahmad Bin Salam, Lacey Dobrolecki, Alaina Lewis, Christina Sallas, Clayton Yates, Carolina Gutierrez, Balasubramanyam Karanam, Meenakshi Anurag, Bora Lim, Matthew Ellis. Distinct HER2 allele specific therapeutic response and preclinical efficacy of poziotinib in metastatic ER+ 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 P2-13-24.