Panel Of Resistant Cell Lines Research Articles

Abstract 3924: Aurora B, a potential new target in non-T790M lung cancer cells with acquired resistance to anti-EGFR therapy, is effectively blocked by the MET/AXL/FGFR inhibitor S49076

Abstract Background: Non-small cell lung cancer (NSCLC) tumors harboring mutations in the EGF receptor (EGFR) ultimately relapse to therapy with EGFR tyrosine kinase inhibitors (TKIs). S49076 is a potent ATP-competitive tyrosine kinase inhibitor of MET, AXL/MER and FGFR1/2/3 currently in a phase I clinical trial in combination with Gefinitib in NSCLC resistant to EGFR inhibitors. Methods: We generated 6 resistant lines by treating EGFR-mutated, TKI sensitive PC9 cells with increasing concentrations of gefitinib (GR1-5) or erlotinib (ER). The six lines conserved the exon 19 deletion but the T790M resistance mutation emerged in two of them (GR1, GR4), which remained sensitive to osimertinib. Six additional cell lines resistant to osimertinib were derived from GR1 and GR4 by exposure to increasing concentrations of the inhibitor. The 12 resistant cell lines were genotyped and characterized for AXL, GAS6, MET, FGFR1 and FGFR2 expression by qRT-PCR, immunohistochemistry and Western blotting. The effects of S49076 and the aurora kinase B (AURKB) inhibitor barasertib were analyzed by MTT, flow cytometry and immunocytochemistry. Western blotting and ELISA of key signal transduction proteins and gene silencing was used to gain insight in the mechanism of action of the drug. Results: Several mechanisms associated with resistance to EGFR TKIs were represented in our panel of resistant cell lines and they frequently co-occurred, including AXL/GAS6 and FGFR1 overexpression, MET activation or emergence and loss of the p.T790M. When using in vitro models, we discovered that the cell lines of our panel were relatively insensitive to single-agent treatment with inhibitors of AXL, MET or FGFR1. In contrast, resistant cells not harboring the p.T790M were sensitive in vitro and also in xenografts to S49076, a drug targeting AXL, MET and FGFR1/2/3. At higher doses (above 300 nM in cultured cells), S49076 blocks AURKB, which could therefore be considered as a further target of the compound. Since partial silencing of AURKB rendered cells resistant to S49076, we investigated the role of this protein. We found that the levels of phospho-histone H3 (pH3), but not Ki67, were increased in all the EGFR TKI resistant cells of our panel, revealing a widespread AURKB activation not related to increased cell proliferation. Treatment with S49076 or barasertib down-regulated pH3 and induced G1/S arrest and polyploidy. In cells harboring the p.T790M, polyploid cells underwent senescence. However, in absence of the resistance mutation, polyploidy was followed by cell death. Conclusions: AURKB is a novel target in non-T790M NSCLC with acquired resistance to first and third line EGFR TKIs. Multitargeted agents such as S49076, which inhibit not only AXL, MET or FGFR1 but also AURKB, might be more effective in this setting than agents targeting the receptor tyrosine kinases alone. Citation Format: Jordi Bertran-Alamillo, Valérie Cattan, Marie Schoumacher, Jordi Codony-Servat, Frederique Cantero, Ruth Roman, Sonia Rodriguez, Ana Gimenez-Capitan, Josep Castellvi, Cristina Teixido, Rafael Rosell, Miguel A. Molina-Vila. Aurora B, a potential new target in non-T790M lung cancer cells with acquired resistance to anti-EGFR therapy, is effectively blocked by the MET/AXL/FGFR inhibitor S49076 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3924.

Molecular Landscape of Acquired Resistance to Targeted Therapy Combinations in BRAF-Mutant Colorectal Cancer.

Although recent clinical trials of BRAF inhibitor combinations have demonstrated improved efficacy in BRAF-mutant colorectal cancer, emergence of acquired resistance limits clinical benefit. Here, we undertook a comprehensive effort to define mechanisms underlying drug resistance with the goal of guiding development of therapeutic strategies to overcome this limitation. We generated a broad panel of BRAF-mutant resistant cell line models across seven different clinically relevant drug combinations. Combinatorial drug treatments were able to abrogate ERK1/2 phosphorylation in parental-sensitive cells, but not in their resistant counterparts, indicating that resistant cells escaped drug treatments through one or more mechanisms leading to biochemical reactivation of the MAPK signaling pathway. Genotyping of resistant cells identified gene amplification of EGFR, KRAS, and mutant BRAF, as well as acquired mutations in KRAS, EGFR, and MAP2K1 These mechanisms were clinically relevant, as we identified emergence of a KRAS G12C mutation and increase of mutant BRAF V600E allele frequency in the circulating tumor DNA of a patient at relapse from combined treatment with BRAF and MEK inhibitors. To identify therapeutic combinations capable of overcoming drug resistance, we performed a systematic assessment of candidate therapies across the panel of resistant cell lines. Independent of the molecular alteration acquired upon drug pressure, most resistant cells retained sensitivity to vertical MAPK pathway suppression when combinations of ERK, BRAF, and EGFR inhibitors were applied. These therapeutic combinations represent promising strategies for future clinical trials in BRAF-mutant colorectal cancer. Cancer Res; 76(15); 4504-15. ©2016 AACR.