Abstract A125: Predication of lorlatinib resistance mechanisms and therapeutic strategies to overcome the resistance in ALK rearranged non-small cell lung cancer
Authors: Okada Koutaroh , Araki Mitsugu , Oh-hara Tomoko , Nishio Makoto , Okuno Yasushi , Fujita Naoya , Katayama Ryohei 
Published: March 2020

Abstract
Introduction: ALK fusion gene is found in 3-5% of NSCLC patients. As the resultant ALK fusion protein constitutively activates ALK tyrosine kinase that causes tumorigenesis, ALK tyrosine kinase inhibitors have been developed for the treatment of ALK rearranged cancer and currently five ALK-TKIs have been clinically applied. Now, alectinib is widely used in first-line therapy. However, most of the patients experience the emergence of alectinib resistance due to the secondary mutation in ALK kinase domain such as I1171N or G1202R. Lorlatinib, third generation ALK-TKI, has shown to be able to overcome any of the earlier generation ALK-TKI resistant single point mutants including G1202R and I1171N. Even though lorlatinib showed marked clinical responses in first or second generation ALK-TKI resistant patients with ALK secondary mutation, multiple lorlatinib resistance ALK compound mutations have been discovered and reported in 2016 and 2018 in clinic. But in approximately half of the lorlatinib resistant cases, the mechanisms were uncovered yet. To further understand the lorlatinib resistance mechanisms, we tried to discover the lorlatinib resistance mechanisms using cell line models and in vivo models, and to explore the therapeutic strategies to overcome the resistance. Experimental Procedure: We firstly explored the lorlatinib resistance mutations by performing ENU random mutagenesis screening using murine pro-B Ba/F3 cells expressing EML4-ALK-G1202R or -I1171N. Secondary, we established the lorlatinib resistant tumor in vivo, and analyzed the resistance mechanism. Finally, we obtained the lorlatinib resistant cells using TKI naïve ALK positive NSCLC patient derived JFCR028-3 cells by treating with the high concentration of lorlatinib (1000 nM or 3000 nM). Summary of the Data: We identified total 15 kinds of lorlatinib resistant EML4-ALK compound mutations with G1202R or I1171N from ENU mutagenesis or in vivo resistant model. We also found that, approximately half of these lorlatinib resistant mutations were re-sensitized to first or second generation ALK-TKIs, such as crizotinib or alectinib. The structural analysis using a computational simulation succeeded to predict the mechanisms how and why those identified compound mutations conferred the resistance to lorlatinib and the re-sensitivity to early generation TKIs. On the other hand, EML4-ALK-G1202R+L1196M compound mutation found in patients’ samples were resistant to all clinically applied ALK-TKIs. But we found that two BCR-ABL inhibitors, AG-957 and adaphostin, inhibited ALK-G1202R+L1196M directly. From the high dose lorlatinib exposed JFCR-028-3 cells, we independently established lorlatinib (1000 nM or 3000 nM) resistant cells after 3 months drug treatment. The growth of those established lorlatinib-resistant (LR) cells were not suppressed by lorlatinib as well as other ALK-TKIs, and they had no mutation in ALK kinase domain. We, therefore, hypothesized the activation of bypass pathway other than ALK, and pharmacologically explored the resistance mechanism to lorlatinib. As a result, we found several drugs such as dasatinib re-sensitized LR cells to lorlatinib. Conclusion: Our results imply that the clinical sequences after lorlatinib resistance using biopsy or liquid-biopsy might provide important information to choose the effective sequential ALK-TKI therapy. Further studies are still needed to uncover the unidentified lorlatinib resistance mechanisms.
Citation Format: Koutaroh Okada, Mitsugu Araki, Tomoko Oh-hara, Makoto Nishio, Yasushi Okuno, Naoya Fujita, Ryohei Katayama. Predication of lorlatinib resistance mechanisms and therapeutic strategies to overcome the resistance in ALK rearranged non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A125. doi:10.1158/1535-7163.TARG-19-A125