Resensitization to Crizotinib by the Lorlatinib ALK Resistance Mutation L1198F

Abstract

In a patient who had metastatic anaplastic lymphoma kinase (ALK)-rearranged lung cancer, resistance to crizotinib developed because of a mutation in the ALK kinase domain. This mutation is predicted to result in a substitution of cysteine by tyrosine at amino acid residue 1156 (C1156Y). Her tumor did not respond to a second-generation ALK inhibitor, but it did respond to lorlatinib (PF-06463922), a third-generation inhibitor. When her tumor relapsed, sequencing of the resistant tumor revealed an ALK L1198F mutation in addition to the C1156Y mutation. The L1198F substitution confers resistance to lorlatinib through steric interference with drug binding. However, L1198F paradoxically enhances binding to crizotinib, negating the effect of C1156Y and resensitizing resistant cancers to crizotinib. The patient received crizotinib again, and her cancer-related symptoms and liver failure resolved. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT01970865.).

Figure 1. Acquired Resistance to Lorlatinib and Resensitization to Crizotinib

Panel A shows the various treatments the patient received for metastatic anaplastic lymphoma kinase (ALK)–rearranged non–small-cell lung cancer as well as the duration of each treatment. Panel B shows computed tomographic (CT) images of the patient's metastatic liver disease before she received lorlatinib, during the time she had a response to lorlatinib, when the disease subsequently relapsed at 9 months, and during the patient's second response to crizotinib therapy. A radiologic second response to crizotinib was noted on the first restaging CT obtained after 8 weeks of treatment. Shown is the patient's second response after she had received crizotinib for 12 weeks. Panel C shows serial monitoring of total bilirubin levels and alkaline phosphatase levels before and after retreatment with crizotinib. To convert values for bilirubin to micromoles per liter, multiply by 17.1. The starting dose of crizotinib was 250 mg once daily, which was increased to 250 mg twice daily once the patient's liver-function tests showed improvement. Crizotinib treatment was initiated on day 1. Arrowheads indicate intermittent administration of low-dose vinorelbine, which was eventually discontinued because of neutropenia. The relative dose intensity (the proportion of administered doses relative to planned doses) of vinorelbine was 0.54. Panel D shows the results of fluorescence in situ hybridization (FISH) assays of tumor cells for ALK (left) and MET (right). The standard break-apart ALK FISH assay was used to screen the lorlatinib-resistant sample for ALK rearrangement and gene amplification. Split signals (white arrows) were observed in 30% of the cells, a finding consistent with ALK rearrangement; no amplification of ALK was detected. A dual-color FISH assay with a C-MET probe (Repeat-Free Poseidon C-MET [7q31] probe, Kreatech) and a copy-number control probe (centromere 7 [CEP7], Abbott–Vysis) were used to screen for MET amplification. The MET-to-CEP7 ratio (i.e., the ratio of red signals to cyan signals) was 1.0, indicating no amplification. Amplification of C-MET would appear as more red signals than cyan signals. Panel E shows clonal evolution of resistance to ALK inhibitors in the patient. This model is based on an analysis of whole-exome sequencing of pretreatment and resistant biopsy samples. A founder ALK C1156Y subclone was detectable at low frequency in the pretreatment tumor specimen. With crizotinib therapy, this subclone expanded to 50% of the tumorcell population and led to the patient's relapse. Lorlatinib was effective against the crizotinib-resistant tumor, but the C1156Y subclone acquired a second ALK mutation, L1198F. The double-mutant subclone (C1156Y–L1198F) was insensitive to lorlatinib and became the dominant subclone in the relapsed tumor. Selected mutations identifying each subclone are shown. Biopsies were performed where indicated. No biopsy was performed when the patient received intervening therapies between crizotinib and lorlatinib, so the proportion of subclones shown during this time period are extrapolated from the post-crizotinib sample.

Figure 2. Cellular and Biochemical Characterization of ALK C1156Y–L1198F

As shown in Panel A, Ba/F3 cells harboring wild-type echinoderm microtubule-associated protein-like 4 (EML4)–ALK or different mutant versions of EML4-ALK were treated with various ALK inhibitors for 48 hours. Cell survival was determined with the use of a CellTiter-Glo assay. Half-maximal inhibitory concentration (IC₅₀) values for the EML4-ALK mutants were determined and were normalized to the IC₅₀ for wild-type EML4-ALK. Ratios equal to 1 correspond to similar cellular potency between mutant and wild-type ALK proteins, whereas ratios less than 1 correspond to greater potency of the mutant as compared with wild-type ALK. As shown in Panel B, inhibition constants (K_i's) for binding of wild-type and mutant ALK kinases with various ALK inhibitors were determined. Shown are the binding affinities of ALK mutants relative to wild-type ALK. Ratios close to 1 correspond to similar binding affinities between mutant and wild-type ALK proteins, whereas ratios less than 1 correspond to greater affinity of the mutant relative to the wild-type kinase.

Figure 3. Structural Basis for Resistance to Lorlatinib and Sensitivity to Crizotinib Mediated by ALK C1156Y–L1198F

Nonphosphorylated ALK wild-type and mutant (C1156Y, L1198F, C1156Y–L1198F) kinase domains were co-crystallized with crizotinib or lorlatinib. Shown are the co-crystal structures of crizotinib bound to the single ALK C1156Y mutant (Panel A), lorlatinib bound to the single ALK C1156Y mutant (Panel B), and crizotinib bound to the ALK C1156Y–L1198F double mutant (Panel C). Panel D shows modeling of lorlatinib bound to the double ALK C1156Y–L1198F mutant, highlighting the steric clash between the phenylalanine residue and lorlatinib. L1122 is a g-loop leucine residue that creates a binding pocket with L1198F just above the piperidine and nitrile groups. The co-crystal structures of lorlatinib and crizotinib bound to ALK C1156Y and ALK C1156Y–L1198F are shown in Figure S6 in the Supplementary Appendix.