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

Abstract

Purpose: 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.

Experimental design: Biopsies from patients with ROS1 ⁺ NSCLC progressing on crizotinib or lorlatinib were profiled by genetic sequencing.

Results: 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 ROS1^L2086F causes steric interference to lorlatinib, crizotinib, and entrectinib, while it may accommodate cabozantinib. In Ba/F3 models, ROS1^L2086F, ROS1^{G2032R/L2086F}, and ROS1^{S1986F/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%).

Conclusions: 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.

Figure 1.. Schematic of resistant biopsies from the ROS1 fusion-positive NSCLC cohort.

The schematic summarizes a total of 47 post-crizotinib and 32 post-lorlatinib biopsies analyzed from 55 patients included in the cohort. The timing of these biopsies for each patient are further delineated in Supplementary Figure S1. Pt, patient.

Figure 2.. Distribution of ROS1-dependent and ROS1-independent resistance.

(A) The frequency of ROS1 mutation(s) detected in each distinct crizotinib- or lorlatinib-resistant biopsy is demonstrated. If ROS1 mutation(s) were detected in only one of the paired plasma and tissue specimens, these mutation(s) were included. Of note, not all samples were tested for the presence of the ROS1 L2086F mutation. (B) The distribution of presumed resistance mechanisms identified in the post-crizotinib and post-lorlatinib biopsy cohort. amp, amplification; mut, mutation; lof, loss-of-function.

Figure 2.. Distribution of ROS1-dependent and ROS1-independent resistance.

(A) The frequency of ROS1 mutation(s) detected in each distinct crizotinib- or lorlatinib-resistant biopsy is demonstrated. If ROS1 mutation(s) were detected in only one of the paired plasma and tissue specimens, these mutation(s) were included. Of note, not all samples were tested for the presence of the ROS1 L2086F mutation. (B) The distribution of presumed resistance mechanisms identified in the post-crizotinib and post-lorlatinib biopsy cohort. amp, amplification; mut, mutation; lof, loss-of-function.

Figure 3.. Preclinical activity of ROS1 inhibitors against mutant ROS1 kinases.

(A) IC₅₀ values of crizotinib, entrectinib, lorlatinib, repotrectinib, cabozantinib, ceritinib, brigatinib, and taletrectinib, or ALK (not ROS1) inhibitor alectinib, in parental Ba/F3 cells and Ba/F3 cells expressing nonmutant or mutant CD74-ROS1. Data are from three replicates. (B) IC₅₀ values of crizotinib, entrectinib, lorlatinib, repotrectinib, and cabozantinib against nonmutant ROS1, ROS1^G2032R, or ROS1^L2086F-based mutant kinases. (C) Suppression of phospho-ROS1 and its downstream targets in Ba/F3 cells expressing nonmutant ROS1, ROS1^G2032R, or ROS1^L2086F treated with ROS1 inhibitors.

Figure 4.. Resistance to lorlatinib with a ROS1 L2086F mutation and subsequent treatment with cabozantinib.

(A) Left panel, X-ray co-crystal structure of the nonmutant ROS1 kinase domain bound to crizotinib (colored yellow) or lorlatinib (colored green). Right panel: Structural modeling of ROS1^L2086F mutant (phenylalanine colored pink with Connolly surface) bound to lorlatinib. (B) Treatment course of MGH0026. The patient had disease progression on lorlatinib. A lorlatinib-resistant lymph node was biopsied and analyzed by NGS (results shown below the timeline). Patient received a brief course of pemetrexed, discontinued for intolerability, before starting cabozantinib. (C) Representative computed tomography images before and after one month on cabozantinib, showing improved aeration but persistent right lung consolidation and loculated effusion, and slightly decreased left pleural effusion.