Acquired resistance to crizotinib from a mutation in CD74-ROS1

Abstract

Crizotinib, an inhibitor of anaplastic lymphoma kinase (ALK), has also recently shown efficacy in the treatment of lung cancers with ROS1 translocations. Resistance to crizotinib developed in a patient with metastatic lung adenocarcinoma harboring a CD74-ROS1 rearrangement who had initially shown a dramatic response to treatment. We performed a biopsy of a resistant tumor and identified an acquired mutation leading to a glycine-to-arginine substitution at codon 2032 in the ROS1 kinase domain. Although this mutation does not lie at the gatekeeper residue, it confers resistance to ROS1 kinase inhibition through steric interference with drug binding. The same resistance mutation was observed at all the metastatic sites that were examined at autopsy, suggesting that this mutation was an early event in the clonal evolution of resistance. (Funded by Pfizer and others; ClinicalTrials.gov number, NCT00585195.).

Figure 1. The CD74–ROS1 Translocation in the Patient's Lung Cancer before Treatment with Crizotinib

In Panel A, a cell block prepared from a malignant pleural effusion shows clusters of tumor cells in a micropapillary pattern (hematoxylin and eosin). In Panel B, a break-apart fluorescence in situ hybridization (FISH) assay with a 5′ ROS1 probe (green) and a 3′ ROS1 probe (red) shows the ROS1 rearrangement, as indicated by the presence of single isolated red 3′ ROS1 probes (arrowheads). The normal ROS1 locus is shown as unsplit red and green pairs of probes (arrows). The nuclei are stained with 4′,6-diamidino-2-phenylindole. In Panel C, Sanger sequencing of the product of reverse-transcriptase–polymerase chain reaction shows the fusion of CD74 exon 6 (red) to either ROS1 exon 34 (light blue) or exon 35 (dark blue). The predicted plasma-membrane orientations for each splice form are shown at right. The N-terminal (N) of CD74 is intracellular and contains a transmembrane (TM) domain. Only the major splice form (CD74 fused to ROS1 exon 34), which contains a second transmembrane domain, is expected to be pathogenic, as a result of the placement of the tyrosine kinase domain of the ROS1 C-terminal (C) in the intracellular compartment.

Figure 2. Identification of an Acquired ROS1 G2032R Mutation at the Time of Resistance to Crizotinib

Axial CT scans of the chest (Panel A) show the patient's disease burden before treatment, after a response to crizotinib, and at the time of crizotinib resistance. Sanger sequencing of RT-PCR products (Panel B) before and after treatment with crizotinib shows the acquired c.6094G→A mutation, which encodes for p.Gly2032Arg. (These coding and amino acid sequences are numbered in accordance with National Center for Biotechnology Information [NCBI] reference sequences CCDS5116 and NP_002935.2, respectively.) In all six malignant sites examined at autopsy (Panel C), the c.6094G→A ROS1 mutation was detected by means of Sanger sequencing of RT-PCR products. Genomic DNA sequencing of ROS1 exon 38 in the patient's grossly and microscopically normal liver tissue shows the nonmutated ROS1 sequence; the CD74–ROS1 fusion transcript could not be detected on RT-PCR in the normal liver. In the summary of autopsy findings at right, the presence of the G2032R mutation is indicated by a plus sign, and the absence of the G2032R mutation by a minus sign.

Figure 3. Mutation of Highly Conserved Glycine at Residue 2032 to Arginine and Resistance to Crizotinib through Steric Interference with Drug Binding

In Panel A, the alignments of amino acid sequences show that G2032 (red asterisk) is highly conserved among all 13 ROS1 paralogs and among many other clinically important tyrosine and serine–threonine kinases. Identical residues are highlighted in black, and conserved substitutions are highlighted in gray. Panel B shows the results of the transient transfection of 293T cells with expression plasmids containing either nonmutated CD74–ROS1 or G2032R CD74–ROS1. The transfected cells were treated with increasing concentrations of crizotinib (top) or TAE684 (bottom) for 6 hours. Lysates were prepared and Western blot analyses were performed with the use of the indicated antibodies. The 293T cells were also transfected with a plasmid-expressing green fluorescence protein as a negative control. GAPDH denotes glyceraldehyde-3-phosphate-dehydrogenase. In Panel C, in vitro enzymatic assays show a marked decrease in the ability of crizotinib to inhibit kinase activity for the G2032R ROS1 mutant as compared with the nonmutant. V_i and V_o are the initial reaction rates in the presence and absence of crizotinib, respectively. Panel D shows the crystal structure of crizotinib bound to the nonmutant ROS1 kinase domain (at left) and a model of the G2032R mutant (at right), in which there is a predicted steric clash with crizotinib. In this atom-coloring scheme, ROS1 carbon is green, crizotinib carbon purple, oxygen red, nitrogen blue, chlorine gold, and fluorine brown. For emphasis, residue 2032 is shown in a space-filling representation. The red asterisks indicate the L2026 gatekeeper residue of ROS1.