Background:Treatment with ALK tyrosine kinase inhibitors elicits profound initial antitumor responses in ALK fusion-positive patients with lung adenocarcinoma. However, patients invariably develop acquired resistance to ALK inhibitors. Although the recent functional genetic studies characterized the mechanism of resistance to ALK inhibition, epigenetic mechanism of acquired resistance is poorly understood.

Method: We established in vivo and in vitro models of acquired resistance to ceritinib and crizotinib using H3122 and H2228 cells. For in vivo model, mice with established H3122-derived tumors were treated with ceritinib to derive ceritinib-resistant tumors. To investigate the epigenetic mechanisms of resistance, we performed methylated DNA binding domain sequencing (MBD-seq) for DNA methylation and chromatin immunoprecipitation-sequencing (ChIP-seq) for histone modifications associated with enhancers (H3K4me1 and H3K27ac) of H3122 and H3122 ceritinib-resistant (LR) cells. We also performed in situ hybridization of miR-34 and miR-449 in patient samples.

Results: Epithelial-to-mesenchymal transition with AXL activation was found in multiple in vitro and in vivo ALK-rearranged lung cancer models with acquired resistance to ceritinib. Genome and epigenome-wide analysis identified that miR-34a and miR-449a are decreased with loss of H3K27ac, while AXL and mesenchymal genes are increased in the process of acquired resistance. We confirmed that miR-34a and miR-449 were involved in AXL-dependent epithelial-mesenchymal transition (EMT) by treating mimics and inhibitors of miR-34a and miR-449a. In addition, pharmacological inhibition of AXL or ectopic expression of miR-34a or miR-449a restored sensitivity to ceritinib in H3122-LR cells. Histone deacetylase (HDAC) inhibitor, panobinostat, synergistically induced anti-proliferative effects with ceritinib in resistant cells. Treatment with panobinostat led to increased expression of miR-449a with increased H3K28ac signal in H3122 LR cells. Panobinostat also reversed EMT phenotype by downregulating N-cadherin and upregulating E-cadherin expression. Additionally, combination of panobinostat and ceritinib induced enhanced antitumor efficacy in acquired resistant xenograft models. To clinically validate our preclinical findings, we measured the expression of miR-34a and miR-449 by in situ hybridization in 5 matched ALK-rearranged lung cancers obtained from individuals both before and after ALK inhibitor treatment. We detected decreased expression of miR-34a and miR-449 in 3 of 5 samples (60%).

Conclusion: Our findings demonstrate that H3K27ac remodeling is a crucial event in ceritinib resistance and inhibition of both ALK and HDAC could prevent or overcome acquired resistance to ALK inhibitors in individuals with ALK-rearranged lung cancer.

Citation Format: Sun Min Lim, Mi Ran Yun, Kyoung Ho Pyo, Seong Keun Kim, Seon-Kyu Kim, Yong Sung Kim, Mirang Kim, Byoung Chul Cho. Epigenetic downregulation of miRNA-34a and miRNA-449 induce epithelial-mesenchymal transition and acquired resistance to ceritinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3346. doi:10.1158/1538-7445.AM2017-3346