Biochemical analysis of EGFR exon20 insertion variants insASV and insSVD and their inhibitor sensitivity

Abstract

Somatic mutations in the epidermal growth factor receptor (EGFR) are a major cause of non-small cell lung cancer. Among these structurally diverse alterations, exon 20 insertions represent a unique subset that rarely respond to EGFR tyrosine kinase inhibitors (TKIs). Therefore, there is a significant need to develop inhibitors that are active against this class of activating mutations. Here, we conducted biochemical analysis of the two most frequent exon 20 insertion variants, V769_D770insASV (insASV) and D770_N771insSVD (insSVD) to better understand their drug sensitivity and resistance. From kinetic studies, we found that EGFR insASV and insSVD are similarly active, but have lower K_{m, ATP} values compared to the L858R variant, which contributes to their lack of sensitivity to 1st-3rd generation EGFR TKIs. Biochemical, structural, and cellular studies of a diverse panel of EGFR inhibitors revealed that the more recently developed compounds BAY-568, TAS6417, and TAK-788 inhibit EGFR insASV and insSVD in a mutant-selective manner, with BAY-568 being the most potent and selective versus wild-type (WT) EGFR. Cocrystal structures with WT EGFR reveal the binding modes of each of these inhibitors and of poziotinib, a potent but not mutantselective inhibitor, and together they define interactions shared by the mutant-selective agents. Collectively, our results show that these exon20 insertion variants are not inherently inhibitor resistant, rather they differ in their drug sensitivity from WT EGFR. However, they are similar to each other, indicating that a single inhibitor should be effective for several of the diverse exon 20 insertion variants.

Keywords: EGFR inhibitors; X-ray crystallography; enzymology; lung cancer.

Fig. 1.

Biochemical inhibition curves for EGFR exon 20 insertion inhibitors. (A–F) Concentration–response curves for inhibition of WT EGFR and the L858R, L858R/T790M, insASV, insSVD, and insNPG variants by the indicated inhibitor. Data were normalized to enzyme activity when no inhibitor is present, and data points are shown as mean ± SD, n = 3. The experiment was performed three times, each time in triplicate. A representative triplicate experiment is shown.

Fig. 2.

Comparison of biochemical potency of EGFR inhibitors against different EGFR variants. (A–F) Correlation plots showing pairwise comparison of inhibitor IC₅₀ values across WT and selected EGFR variants, as indicated, for the 20 inhibitors studied here. Each compound is plotted with its x-axis value corresponding to its IC₅₀ against the first EGFR variant and its y-axis value corresponding to its IC₅₀ against the second EGFR variant. Red dots indicate compounds with one or both IC₅₀ values beyond the range of the assay (>1 μM). The diagonal black line indicates equipotency (x = y) and is shown for easier identification of mutant-selective compounds.

Fig. 3.

Cellular potency of exon 20 inhibitors. (A–C) Dose-dependent cell growth inhibition of Ba/F3 cells expressing EGFR D770_N771insNPG, D770_N771insSVD, or WT EGFR in the presence of 50 ng/mL EGF. Ba/F3 cells expressing aforementioned EGFR mutations were treated with the indicated doses of exon 20 inhibitors for 72 h. Cell survival was measured using a CellTiter Aqueous One Solution Cell Proliferation Assay. Error bars indicate SD (n = 3). (D–F) Western blot analysis of the effects of exon 20 inhibitors on Ba/F3 cells expressing EGFR insNPG, insSVD, or WT EGFR in the presence of 50 ng/mL EGF. The cells were treated with indicated doses of inhibitors for 6 h. Phosphorylation of EGFR, AKT, and ERK proteins was detected by immunoblotting. (G) Table showing cellular IC₅₀ values for these exon 20 inhibitors against Ba/F3 cells expressing WT, insASV, or insSVD EGFR.

Fig. 4.

Crystal structures of EGFR kinase in complex with exon 20 inhibitors. In (A–F) EGFR is shown in a yellow ribbon representation with the C-helix colored orange and the activation loop red. Dashed lines indicate hydrogen bonds. (A) View of active site for WT EGFR in complex with BAY-33 showing interactions with the hinge region, C-helix, and F723 (PDB 8F1Z) (46). (B) Alternate view of BAY-33 (PDB 8F1Z). (C) Interactions of poziotinib in the active site of WT EGFR (PDB 8F1Y) (47). (D) Interactions of poziotinib in the active site of EGFR T790M/V948R (PDB 8F1W) (48). Note that the kinase adopts an inactive conformation in this structure, with the C-helix displaced in an outward position. (E) Interactions of TAK-788 in WT EGFR (PDB 8F1X) (49). (F) View of the interactions of TAS6417 in the active site of WT EGFR (PDB 8F1H) (50).