Tucatinib is an investigational, oral, small molecule tyrosine kinase inhibitor that is highly selective for the kinase domain of HER2, without significant inhibition of EGFR. Recently, HER2CLIMB (NCT02614794), a pivotal, randomized, international, double-blind trial that evaluated tucatinib or placebo in combination with trastuzumab and capecitabine in patients with HER2+ metastatic breast cancer (MBC) with or without brain metastases, after progression with trastuzumab, pertuzumab, and ado-trastuzumab emtansine (T-DM1), showed superior progression-free and overall survival in patients treated on the tucatinib arm. Adverse events in the tucatinib arm were primarily low grade and occurred at rates similar to what was observed in the placebo arm. In this report, we characterize the in vitro and in vivo activity of tucatinib alone and in combination with T-DM1 in HER2-amplified preclinical models. In vitro assays demonstrate that tucatinib potently suppresses HER2-meditated signaling pathways, including phosphorylation of HER2, HER3, AKT and ERK. Ex vivo analysis of tucatinib-treated xenografts shows similar suppression of signal transduction pathways. Tucatinib inhibits the proliferation of HER2+ breast cancer (BC) cell lines in vitro with single digit nanomolar potencies, but was inactive in blocking cell proliferation of BC cell lines lacking amplified HER2. In HER2-amplified BC cell line derived- and patient-derived xenograft models, tucatinib suppresses growth of tumors as a single agent and shows improved activity when combined with T-DM1. To evaluate activity and drug penetration in HER2+ metastatic CNS tumors, we developed and characterized a red-shifted luciferase-expressing BT-474 BC cell line which was stereotaxically implanted into the brain. In vivo bioluminescence imaging was used to evaluate the efficacy of tucatinib and T-DM1 in CNS tumors as single agents and in combination. Histological assessment of treated tumors suggests that tucatinib can penetrate the blood-brain tumor barrier to suppress HER2 signaling. These data are further supported by a drug penetration study of CNS-implanted BT-474 tumors. Quantitative autoradiography analysis demonstrates that 14C-labeled tucatinib penetrates the CNS tumor mass with drug concentration levels exceeding measurements in normal brain regions. These data demonstrate that tucatinib is a uniquely selective and highly potent inhibitor of HER2 signaling, with activity against HER2+ breast cancer xenograft and brain metastasis models. Tucatinib is effective against implanted CNS tumors, suggesting that therapeutically relevant drug concentrations are achievable. These preclinical findings are consistent with clinical data showing tucatinib activity in patients with HER2+ MBC with brain metastases and support the continued development of tucatinib in patients with HER2+ BC.

Citation Format: Devra J. Olson, Anita Kulukian, Janelle D. Taylor, Margo C. Zaval, Albina Nesterova, Kelly M. Hensley, Michelle L. Ulrich, Nicole S. Stevens, Scott R. Peterson. Preclinical characterization of tucatinib in HER2-amplified xenograft and CNS implanted tumors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1962.