P2.14-56 Osimertinib Overcomes Alectinib Resistance Caused by Amphiregulin in a Leptomeningeal Carcinomatosis Model of EML4-ALK Lung Cancer

Central nervous system (CNS) metastasis, such as brain metastasis and leptomeningeal carcinomatosis (LMC), occurs in 20–40% of all patients with cancer. Anaplastic lymphoma kinase (ALK) is a clinically validated drug target and ALK rearrangements are found in approximately 3-5% of non-small cell lung cancer (NSCLC). ALK tyrosine kinase inhibitor (TKI) shows dramatic clinical efficacy in ALK-rearranged NSCLC patients, and the second-generation ALK-TKI alectinib is effective against CNS metastasis of ALK-rearranged NSCLC. However, the patients with ALK-rearrangement acquire resistance to alectinib over time and develop recurrent LMC metastasis. This study aimed to clarify the mechanism of resistance to alectinib in LMC and seek a novel therapeutic strategy. Alectinib-resistant cell line (A925L/AR) was established by continuous treatment with alectinib in the LMC mouse model inoculated with the alectinib-sensitive human lung cancer cell line, A925LPE3, which harbors the EML4-ALK gene fusion. The tumor level was measured by in vivo imaging system. To clarify the mechanism of alectinib resistance, tumor cell culture supernatants, patient cerebrospinal fluid (CSF), and patient serum were measured using ELISA kits for EGFR ligands. A925L/AR cells were moderately resistant to various ALK-TKIs, such as alectinib, crizotinib, ceritinib, and lorlatinib, compared with parental cells in vitro. A925L/AR cells acquired resistance through epidermal growth factor receptor (EGFR) activation due to overexpression of its ligand, amphiregulin. EGFR-TKIs and anti-EGFR antibodies re-sensitized A925L/AR cells to alectinib in vitro. In the LMC model with A925L/AR cells, combined treatment with alectinib and an EGFR-TKI, such as erlotinib and osimertinib, successfully controlled LMC progression. Imaging mass spectrometry showed accumulation of EGFR-TKIs in the tumor lesions. Moreover, notably high amphiregulin levels were detected in the cerebrospinal fluid from ALK-rearranged NSCLC patients with alectinib-resistant LMC compared with those in EGFR-mutated NSCLC patients with EGFR-TKI-resistant LMC or patients without LMC. We demonstrated that EML4-ALK lung cancer cells acquired moderate resistance to alectinib in the leptomeningeal space due to amphiregulin-triggered EGFR activation. Moreover, combined use of alectinib and EGFR-TKIs, including the third-generation inhibitor osimertinib, could overcome resistance in the LMC model. Our findings may provide rationale for clinical trials to investigate the effects of novel therapies dual-targeting ALK and EGFR in ALK-rearranged NSCLC with alectinib-resistant LMC.