Mechanisms of Resistance to Tyrosine Kinase Inhibitors in ROS1 Fusion-Positive Nonsmall Cell Lung Cancer.

ROS1 fusion-positive (ROS1+) nonsmall cell lung cancer (NSCLC) patients are highly sensitive to tyrosine kinase inhibitor (TKI) treatments. However, acquired TKI resistance remains the major hurdle preventing patients from experiencing prolonged benefits. 107 advanced or metastatic ROS1+ NSCLC patients who progressed on crizotinib and lorlatinib were recruited. Tissue and plasma samples were collected at baseline (N = 50), postcrizotinib (N = 91), and postlorlatinib (N = 21), which were all subject to the 139-gene targeted next-generation DNA sequencing. Molecular dynamics modeling was performed to investigate the effects of ROS1 mutations on binding to different TKIs. In patients with postcrizotinib and postlorlatinib samples, an accumulation of on- and off-target resistance alterations after multiple TKI treatments was observed. ROS1 G2032R and MET amplification were the most common on-target and off-target alterations, respectively. Patients with CD74-ROS1 and SLC34A2-ROS1 had longer progression-free survival (PFS) (P < 0.001) and higher rates of resistance mutations (on-target, P = 0.001; off-target, P = 0.077) than other ROS1 fusion variants following crizotinib treatment. Ten distinct on-target resistance mutations were detected after TKI therapies, of which 4 were previously unreported (ROS1 L2010M, G1957A, D1988N, L1982V). Molecular dynamics simulations showed that all 4 mutations were refractory to crizotinib, while G1957A, D1988N, and L1982V were potentially sensitive to lorlatinib and entrectinib. This study provided a comprehensive portrait of TKI-resistance mechanisms in ROS1+ NSCLC patients. Using in silico simulations of TKI activity, novel secondary mutations that may confer TKI resistance were identified and may support clinical therapeutic decision-making.

Zhao X ，Zhang X ，Chen H ，Bao H ，Wu X ，Wang H ，Bao H ，Pang J ，Wang S ，Wang J ... -  《-》

Spectrum of Mechanisms of Resistance to Crizotinib and Lorlatinib in ROS1 Fusion-Positive Lung Cancer.

Current standard initial therapy for advanced, ROS proto-oncogene 1, receptor tyrosine kinase fusion (ROS1)-positive (ROS1+) non-small cell lung cancer (NSCLC) is crizotinib or entrectinib. Lorlatinib, a next-generation anaplastic lymphoma kinase/ROS1 inhibitor, recently demonstrated efficacy in ROS1+ NSCLC, including in crizotinib-pretreated patients. However, mechanisms of lorlatinib resistance in ROS1+ disease remain poorly understood. Here, we assessed mechanisms of resistance to crizotinib and lorlatinib. Biopsies from patients with ROS1 + NSCLC progressing on crizotinib or lorlatinib were profiled by genetic sequencing. From 55 patients, 47 post-crizotinib and 32 post-lorlatinib biopsies were assessed. Among 42 post-crizotinib and 28 post-lorlatinib biopsies analyzed at distinct timepoints, ROS1 mutations were identified in 38% and 46%, respectively. ROS1 G2032R was the most commonly occurring mutation in approximately one third of cases. Additional ROS1 mutations included D2033N (2.4%) and S1986F (2.4%) post-crizotinib and L2086F (3.6%), G2032R/L2086F (3.6%), G2032R/S1986F/L2086F (3.6%), and S1986F/L2000V (3.6%) post-lorlatinib. Structural modeling predicted ROS1L2086F causes steric interference to lorlatinib, crizotinib, and entrectinib, while it may accommodate cabozantinib. In Ba/F3 models, ROS1L2086F, ROS1G2032R/L2086F, and ROS1S1986F/G2032R/L2086F were refractory to lorlatinib but sensitive to cabozantinib. A patient with disease progression on crizotinib and lorlatinib and ROS1 L2086F received cabozantinib for nearly 11 months with disease control. Among lorlatinib-resistant biopsies, we also identified MET amplification (4%), KRAS G12C (4%), KRAS amplification (4%), NRAS mutation (4%), and MAP2K1 mutation (4%). ROS1 mutations mediate resistance to crizotinib and lorlatinib in more than one third of cases, underscoring the importance of developing next-generation ROS1 inhibitors with potency against these mutations, including G2032R and L2086F. Continued efforts are needed to elucidate ROS1-independent resistance mechanisms.

Lin JJ ，Choudhury NJ ，Yoda S ，Zhu VW ，Johnson TW ，Sakhtemani R ，Dagogo-Jack I ，Digumarthy SR ，Lee C ，Do A ，Peterson J ，Prutisto-Chang K ，Malik W ，Hubbeling HG ，Langenbucher A ，Schoenfeld AJ ，Falcon CJ ，Temel JS ，Sequist LV ，Yeap BY ，Lennerz JK ，Shaw AT ，Lawrence MS ，Ou SI ，Hata AN ，Drilon A ，Gainor JF ... -  《-》

Crizotinib-Resistant ROS1 Mutations Reveal a Predictive Kinase Inhibitor Sensitivity Model for ROS1- and ALK-Rearranged Lung Cancers.

The identification of molecular mechanisms conferring resistance to tyrosine kinase inhibitor (TKI) is a key step to improve therapeutic results for patients with oncogene addiction. Several alterations leading to EGFR and anaplastic lymphoma kinase (ALK) resistance to TKI therapy have been described in non-small cell lung cancer (NSCLC). Only two mutations in the ROS1 kinase domain responsible for crizotinib resistance have been described in patients thus far. A patient suffering from a metastatic NSCLC harboring an ezrin (EZR)-ROS1 fusion gene developed acquired resistance to the ALK/ROS1 inhibitor crizotinib. Molecular analysis (whole-exome sequencing, CGH) and functional studies were undertaken to elucidate the mechanism of resistance. Based on this case, we took advantage of the structural homology of ROS1 and ALK to build a predictive model for drug sensitivity regarding future ROS1 mutations. Sequencing revealed a dual mutation, S1986Y and S1986F, in the ROS1 kinase domain. Functional in vitro studies demonstrated that ROS1 harboring either the S1986Y or the S1986F mutation, while conferring resistance to crizotinib and ceritinib, was inhibited by lorlatinib (PF-06463922). The patient's clinical response confirmed the potency of lorlatinib against S1986Y/F mutations. The ROS1 S1986Y/F and ALK C1156Y mutations are homologous and displayed similar sensitivity patterns to ALK/ROS1 TKIs. We extended this analogy to build a model predicting TKI efficacy against potential ROS1 mutations. Clinical evidence, in vitro validation, and homology-based prediction provide guidance for treatment decision making for patients with ROS1-rearranged NSCLC who progressed on crizotinib. Clin Cancer Res; 22(24); 5983-91. ©2016 AACR.

Facchinetti F ，Loriot Y ，Kuo MS ，Mahjoubi L ，Lacroix L ，Planchard D ，Besse B ，Farace F ，Auger N ，Remon J ，Scoazec JY ，André F ，Soria JC ，Friboulet L ... -  《-》

CRISPR/Cas9-edited ROS1 + non-small cell lung cancer cell lines highlight differential drug sensitivity in 2D vs 3D cultures while reflecting established resistance profiles.

The study of resistance-causing mutations in oncogene-driven tumors is fundamental to guide clinical decisions. Several point mutations affecting the ROS1 kinase domain have been identified in the clinical setting, but their impact requires further exploration, particularly in improved pre-clinical models. Given the scarcity of solid pre-clinical models to approach rare cancer subtypes like ROS1 + NSCLC, CRISPR/Cas9 technology allows the introduction of mutations in patient-derived cell lines for which resistant variants are difficult to obtain due to the low prevalence of cases within the clinical setting. In the SLC34A2-ROS1 rearranged NSCLC cell line HCC78, we knocked-in through CRISPR/Cas9 technology three ROS1 drug resistance-causing mutations: G2032R, L2026M and S1986Y. Such variants are located in different functional regions of the ROS1 kinase domain, thus conferring TKI resistance through distinct mechanisms. We then performed pharmacological assays in 2D and 3D to assess the cellular response of the mutant lines to crizotinib, entrectinib, lorlatinib, repotrectinib and ceritinib. In addition, immunoblotting assays were performed in 2D-treated cell lines to determine ROS1 phosphorylation and MAP kinase pathway activity. The area over the curve (AOC) defined by the normalized growth rate (NGR_fit) dose-response curves was the variable used to quantify the cellular response towards TKIs. Spheroids derived from ROS1G2032R cells were significantly more resistant to repotrectinib (AOC fold change = - 7.33), lorlatinib (AOC fold change = - 6.17), ceritinib (AOC fold change = - 2.8) and entrectinib (AOC fold change = - 2.02) than wild type cells. The same cells cultured as a monolayer reflected the inefficacy of crizotinib (AOC fold change = - 2.35), entrectinib (AOC fold change = - 2.44) and ceritinib (AOC fold change = - 2.12) in targeting the ROS1 G2032R mutation. ROS1L2026M cells showed also remarkable resistance both in monolayer and spheroid culture compared to wild type cells, particularly against repotrectinib (spheroid AOC fold change = - 2.19) and entrectinib (spheroid AOC fold change = - 1.98). ROS1S1986Y cells were resistant only towards crizotinib in 2D (AOC fold change = - 1.86). Overall, spheroids showed an increased TKI sensitivity compared to 2D cultures, where the impact of each mutation that confers TKI resistance could be clearly distinguished. Western blotting assays qualitatively reflected the patterns of response towards TKI observed in 2D culture through the levels of phosphorylated-ROS1. However, we observed a dose-response increase of phosphorylated-Erk1/2, suggesting the involvement of the MAPK pathway in the mediation of apoptosis in HCC78 cells. In this study we knock-in for the first time in a ROS1 + patient-derived cell line, three different known resistance-causing mutations using CRISPR/Cas9 in the endogenous translocated ROS1 alleles. Pharmacological assays performed in 2D and 3D cell culture revealed that spheroids are more sensitive to TKIs than cells cultured as a monolayer. This direct comparison between two culture systems could be done thanks to the implementation of normalized growth rates (NGR) to uniformly quantify drug response between 2D and 3D cell culture. Overall, this study presents the added value of using spheroids and positions lorlatinib and repotrectinib as the most effective TKIs against the studied ROS1 resistance point mutations.

Terrones M ，Deben C ，Rodrigues-Fortes F ，Schepers A ，de Beeck KO ，Van Camp G ，Vandeweyer G ... -  《Journal of Translational Medicine》

Acquired G2032R Resistance Mutation in ROS1 to Lorlatinib Therapy Detected with Liquid Biopsy.

Jóri B ，Falk M ，Hövel I ，Weist P ，Tiemann M ，Heukamp LC ，Griesinger F ... -  《-》