Mechanisms and clinical activity of an EGFR and HER2 exon 20-selective kinase inhibitor in non-small cell lung cancer

Abstract

Although most activating mutations of epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancers (NSCLCs) are sensitive to available EGFR tyrosine kinase inhibitors (TKIs), a subset with alterations in exon 20 of EGFR and HER2 are intrinsically resistant and lack an effective therapy. We used in silico, in vitro, and in vivo testing to model structural alterations induced by exon 20 mutations and to identify effective inhibitors. 3D modeling indicated alterations restricted the size of the drug-binding pocket, limiting the binding of large, rigid inhibitors. We found that poziotinib, owing to its small size and flexibility, can circumvent these steric changes and is a potent inhibitor of the most common EGFR and HER2 exon 20 mutants. Poziotinib demonstrated greater activity than approved EGFR TKIs in vitro and in patient-derived xenograft models of EGFR or HER2 exon 20 mutant NSCLC and in genetically engineered mouse models of NSCLC. In a phase 2 trial, the first 11 patients with NSCLC with EGFR exon 20 mutations receiving poziotinib had a confirmed objective response rate of 64%. These data identify poziotinib as a potent, clinically active inhibitor of EGFR and HER2 exon 20 mutations and illuminate the molecular features of TKIs that may circumvent steric changes induced by these mutations.

Fig. 1. Exon 20 insertion mutations induce de novo resistance to covalent and noncovalent TKIs

a, PFS of patients with classical EGFR mutations and exon 20 insertion mutations in EGFR demonstrating resistance to first-line therapy (log-rank P < 1.0 × 10⁻⁹). b, Schematic of EGFR and HER2 exon 20 insertions generated in a stable Ba/F3 model. c–h, Averaged dose response curves of cell viability of Ba/F3 cell lines expressing six different EGFR (c–e) and six different HER2 (f–h) exon 20 insertion mutations indicated in bold in b treated with first-, second-, or third-generation TKIs for 72 h. In c–h, the mean ± s.e.m. of the six cell lines is plotted for each concentration (n = 3 biologically independent experiments). i, 3D modeling of EGFR D770insNPG (green) and EGFR T790M (yellow). The NPG insertion is highlighted in pink; the P-loop is highlighted in red. Shifts of the P-loop (red arrow) and the α-C helix (blue arrow) into the binding pocket result in steric hindrance, reducing the size of the binding pocket. j, 3D modeling of HER2 A775insYVMA (blue) and HER2-WT (yellow). The YVMA insertion is highlighted in pink, and the P-loop is highlighted in red. Overall shifts of the P-loop (red arrow) and the α-C helix (blue arrow) into the binding pocket result in an overall reduction in the size of the binding pocket.

Fig. 2. Poziotinib potently inhibits EGFR and HER2 exon 20 insertion mutants

a,b, Dose-response curves showing the cell viability of Ba/F3 cell lines expressing EGFR (a) and HER2 (b) exon 20 insertion mutations that were treated with poziotinib for 72 h. The mean ± s.e.m. of each individual cell line is plotted for each concentration (n = 3 biologically independent experiments). c, Western blotting showing inhibition of p-EGFR and p-HER2 in Ba/F3 cell lines after 2 h of poziotinib treatment (n = 2 biologically independent experiments). β-actin was used as a loading control. Uncropped blots are available in Supplementary Fig. 10. d, Correlation of expression levels of Ba/F3 exon 20 insertions compared to sensitivity (n = 2 biologically independent experiments). The Pearson correlation coefficient of 13 biologically independent samples and the P value were determined using GraphPad Prism. e,f, Dose-response curves showing the cell viability of the patient-derived cell lines CUTO14 expressing EGFR A767dupASV (e) and YUL-0019 expressing EGFR N771delinsFH (f) that were treated with poziotinib or afatinib for 72 h (n = 3 biologically independent experiments). The mean ± s.e.m. of the experimental replicates is plotted for each concentration. g, IC₅₀ values of EGFR-mutant Ba/F3 cells normalized to the IC₅₀ values of Ba/F3 EGFR-T790M cell line after incubation with afatinib, osimertinib, rociletinib, or poziotinib for 72 h (n = 3 biologically independent experiments). Dot plots are representative of mean ± s.e.m. Values greater than 1 are indicative of less potent inhibition compared to T790M, whereas values less than one indicate more potent inhibition of exon 20 insertions compared to T790M. h, EGFR-T790M (yellow) with osimertinib (blue) has a very large binding pocket (h) compared to EGFR D770insNPG (green) with poziotinib (orange) (i). Steric hindrance induced by the NPG insertion is shown in red. Blue also indicates changes occurring in the drug binding pocket that compensate for the steric hindrance caused by exon 20 insertions.

Fig. 3. Poziotinib reduces tumor burden in mouse models bearing EGFR or HER2 exon 20 insertion mutants

a,b, Dot plots showing tumor volume of mice bearing EGFR D770insNPG (a) or HER2 A775insYVMA (b) that were treated daily with vehicle (EGFR, n = 5 mice; HER2, n = 4 mice), 20 mg/kg of afatinib daily (EGFR n = 4 mice), or 10 mg/kg of poziotinib daily (EGFR, n = 5 mice; HER2, n = 6 mice) for 4 weeks. The mean ± s.e.m. of percent change in tumor volume after 4 weeks of treatment is plotted. A two-sided Student’s t-test was used to calculate the P values; n.s., nonsignificant. c,d, Representative MRI images of EGFR-mutant (c) and HER2-mutant (d) GEMMs before and after 4 weeks of poziotinib treatment showing robust tumor regression. The red H marks the heart. e,f, Plots of tumor volume of each mouse (numbered Ex20 1–6) bearing EGFR D770insNPG (e; n = 4 biologically independent mice) or HER2 A775insYVMA (f; n = 6 biologically independent mice) treated with 10 mg/kg of poziotinib 5 d per week for 12 weeks showing that mice continue to respond to poziotinib treatment. g, Tumor area in nude mice in which cells from the YUL-0019 line were grown. Mice were treated with vehicle control (n = 6 biologically independent mice), 20 mg/kg afatinib (n = 6 biologically independent mice), 5 mg/kg poziotinib (n = 4 biologically independent mice), or 10 mg/kg poziotinib (n = 3 biologically independent mice). Mean ± s.e.m. of tumor area is plotted for each measurement. A multiple-comparisons test using the Holm–Sidak method was used to determine statistical significance between groups, and P values can be found in Supplementary Table 3. *P < 0.05, **P < 0.01, ***P < 0.001. h, Dot plots of tumor burden in mice bearing EGFR H773insNPH PDXs were treated with vehicle control (n = 6 biologically independent mice), 5 mg/kg poziotinib (n = 6 biologically independent mice), or 10 mg/kg poziotinib (n = 3 biologically independent mice). The mean ± s.e.m. of percent change in tumor volume after 4 weeks of treatment is plotted. A one-way ANOVA analysis was used in combination with Tukey’s test to determine statistical significance.

Fig. 4. Poziotinib inhibits EGFR exon 20 insertion mutants in patients with NSCLC

a, Waterfall plot of the first 11 patient responses on clinical trial NCT03066206. Objective partial responses are shown in green (n = 7), stable disease is shown in blue (n = 3), and unconfirmed response is shown in gray (n = 1). b, CT scan of a patient with EGFR S768I mutant NSCLC 1 d before poziotinib treatment and after 8 weeks of poziotinib therapy. The patient had previously been treated with both erlotinib and afatinib with progression and had a 50% reduction in the volume of target lesions after 4 weeks of poziotinib therapy. c, CT scans of a patient with EGFR D770delinsGY 1 d before and after 8 weeks of poziotinib treatment. Patient had been previously treated with afatinib and AP32788 with no response but had a 32% reduction in the volume of target lesions with poziotinib treatment. d, CT scans of a patient with EGFR S768dupSVD 1 d before and after 16 weeks of poziotinib treatment. The patient had a confirmed objective partial response as seen in the second scan.