Sequential ALK Inhibitors Can Select for Lorlatinib-Resistant Compound ALK Mutations in ALK-Positive Lung Cancer

Abstract

The cornerstone of treatment for advanced ALK-positive lung cancer is sequential therapy with increasingly potent and selective ALK inhibitors. The third-generation ALK inhibitor lorlatinib has demonstrated clinical activity in patients who failed previous ALK inhibitors. To define the spectrum of ALK mutations that confer lorlatinib resistance, we performed accelerated mutagenesis screening of Ba/F3 cells expressing EML4-ALK. Under comparable conditions, N-ethyl-N-nitrosourea (ENU) mutagenesis generated numerous crizotinib-resistant but no lorlatinib-resistant clones harboring single ALK mutations. In similar screens with EML4-ALK containing single ALK resistance mutations, numerous lorlatinib-resistant clones emerged harboring compound ALK mutations. To determine the clinical relevance of these mutations, we analyzed repeat biopsies from lorlatinib-resistant patients. Seven of 20 samples (35%) harbored compound ALK mutations, including two identified in the ENU screen. Whole-exome sequencing in three cases confirmed the stepwise accumulation of ALK mutations during sequential treatment. These results suggest that sequential ALK inhibitors can foster the emergence of compound ALK mutations, identification of which is critical to informing drug design and developing effective therapeutic strategies.Significance: Treatment with sequential first-, second-, and third-generation ALK inhibitors can select for compound ALK mutations that confer high-level resistance to ALK-targeted therapies. A more efficacious long-term strategy may be up-front treatment with a third-generation ALK inhibitor to prevent the emergence of on-target resistance. Cancer Discov; 8(6); 714-29. ©2018 AACR.This article is highlighted in the In This Issue feature, p. 663.

Figure 1. No single ALK mutations confer high level resistance to lorlatinib

A, for reference are shown previously reported IC₅₀ values of first-, second-, and third-generation ALK inhibitors on cellular ALK phosphorylation in Ba/F3 cells expressing non-mutant or mutant EML4-ALK (adapted from ref (12)). B, scheme of ENU mutagenesis screen using Ba/F3 cells. C, summary of the type and number of ALK kinase domain mutations identified in the mutagenesis screen using Ba/F3 cells harboring non-mutant EML4-ALK (either variant 1 or variant 3). Numerous crizotinib-resistant clones were identified, as shown in the right panel. In contrast, no lorlatinib-resistant clones were identified at comparable and clinically achievable drug concentrations, as shown in the left panel. Shown are combined data from two independent experiments.

Figure 2. Multiple different compound ALK mutations can cause resistance to lorlatinib

A, shown is a summary of the lorlatinib concentrations used in ENU mutagenesis screening of each Ba/F3 EML4-ALK mutant model. Grey wells indicate lorlatinib concentrations that were insufficient to prevent clonal outgrowth in long-term cell proliferation assays; screening was performed at lorlatinib concentrations that could prevent clonal outgrowth (see Supplementary Figure S2). The numbers of growing clones at each drug concentration in each model are shown. B, summary of compound ALK mutations identified in growing, lorlatinib-resistant clones after ENU mutagenesis. Each mutant EML4-ALK model is listed on the left. The x-axis depicts increasing concentrations of lorlatinib (from 50 to 1000 nM). Each pie chart depicts the secondary ALK mutation(s) that were identified and that led to lorlatinib resistance, as well as the relative abundance of each compound mutation. Shown are combined data of two independent experiments using Ba/F3 cells expressing mutant EML4-ALK (variant 1).

Figure 3. Functional validation of the lorlatinib-resistant ALK G1202R/L1196M compound mutant identified by ENU mutagenesis

A, cell viability assay of Ba/F3 cells expressing EML4-ALK variant 1, either non-mutant, single mutant (L1196M or G1202R), or compound mutant (G1202R/L1196M). Data are mean ± s.e.m. of three replicates. B, comparison of lorlatinib’s activity with that of other ALK inhibitors in the same Ba/F3 models. Shown are absolute IC₅₀ values. The compound mutant confers resistance to all generations of ALK inhibitors. Data are mean of three replicates. C, ALK phosphorylation in the same Ba/F3 models treated with lorlatinib, as assessed by immunoblotting of cell lysates. Lorlatinib potently suppresses ALK activation in non-mutant and single mutant EML4-ALK models, but fails to inhibit ALK in Ba/F3 cells expressing the compound mutant.

Figure 4. Summary of the genetic landscape of lorlatinib-resistant cancers

All clinical specimens underwent targeted NGS testing using either the MGH SNaPshot assay or the FoundationOne platform (see Supplementary Table S3). Shown here are known oncogenes (ALK, BRAF, EGFR, ERBB2, KRAS, and MET) and genes for which an alteration was detected in at least one sample. SNV, single nucleotide variant; indel, insertion or deletion; CNV, copy number variant.

Figure 5. Clonal evolution of resistance to sequential ALK targeted therapies

A, treatment course of patient MGH953. This patient received sequential first-, second-, and third-generation ALK inhibitors, with initial response and then relapse on each drug. At each progression event, the patient’s recurrent malignant pleural effusion was drained and a cytology block was prepared. The resistant cancers underwent SNaPshot NGS profiling, with the ALK sequencing results shown below the timeline. B, clonal analysis based on whole exome sequencing of MGH953 samples. The alectinib-resistant clone harboring ALK G1202R acquired an additional ALK L1196M mutation on the same allele, leading to clinical relapse on lorlatinib. C, treatment course of patient MGH086. This patient was also treated with sequential first-, second-, and third-generation ALK inhibitors, with initial response and then relapse on each drug. All five brigatinib-resistant specimens were excisions of a recurring left axillary nodal mass, while the post-lorlatinib specimen was an excisional biopsy of a growing subcutaneous metastasis. D, clonal analysis based on whole exome sequencing of MGH086 samples. Of note, we previously reported the results of clonal analysis up to the second brigatinib-resistant specimen (ref. 12). Here we have extended the clonal analysis with the addition of three brigatinib-resistant and one lorlatinib-resistant specimens (indicated in red). The dominant brigatinib-resistant clone harboring ALK E1210K/D1203N acquired an additional ALK G1269A mutation, leading to clinical relapse on lorlatinib. E, treatment course of patient MGH987. This patient received four sequential ALK inhibitors, including lorlatinib. Repeat biopsies were performed at the time of resistance to alectinib and lorlatinib; both resistant specimens were derived from a progressive liver metastasis. F, clonal analysis based on whole exome sequencing of MGH987 samples. The alectinib-resistant clone harboring ALK I1171N acquired an additional ALK L1198F mutation on the same allele.

Figure 6. Structural basis for lorlatinib resistance mediated by the compound ALK G1202R/L1196M mutant

A, co-crystal structures of non-mutant (blue) and ALK L1196M (green) tyrosine kinase domains bound to lorlatinib. B, aligned co-crystal structure of non-mutant ALK (blue) and model of the compound ALK G1202R/L1196M mutant (pink) based on MD simulations. The model comes from modifying the crystal structure of ALK L1196M with lorlatinib to replicate differences that were seen between the non-mutant and mutant kinase domains in MD simulations.