Structural, biochemical, and clinical characterization of epidermal growth factor receptor (EGFR) exon 20 insertion mutations in lung cancer

Abstract

Epidermal growth factor receptor (EGFR) gene mutations (G719X, exon 19 deletions/insertions, L858R, and L861Q) predict favorable responses to EGFR tyrosine kinase inhibitors (TKIs) in advanced non-small cell lung cancer (NSCLC). However, EGFR exon 20 insertion mutations (~10% of all EGFR mutations) are generally associated with insensitivity to available TKIs (gefitinib, erlotinib, and afatinib). The basis of this primary resistance is poorly understood. We studied a broad subset of exon 20 insertion mutations, comparing in vitro TKI sensitivity with responses to gefitinib and erlotinib in NSCLC patients, and found that most are resistant to EGFR TKIs. The crystal structure of a representative TKI-insensitive mutant (D770_N771insNPG) reveals an unaltered adenosine triphosphate-binding pocket, and the inserted residues form a wedge at the end of the C helix that promotes the active kinase conformation. Unlike EGFR-L858R, D770_N771insNPG activates EGFR without increasing its affinity for EGFR TKIs. Unexpectedly, we find that EGFR-A763_Y764insFQEA is highly sensitive to EGFR TKIs in vitro, and patients whose NSCLCs harbor this mutation respond to erlotinib. Analysis of the A763_Y764insFQEA mutant indicates that the inserted residues shift the register of the C helix in the N-terminal direction, altering the structure in the region that is also affected by the TKI-sensitive EGFR-L858R. Our studies reveal intricate differences between EGFR mutations, their biology, and their response to EGFR TKIs.

Figure 1

EGFR exon 20 insertion mutations and their response to EGFR TKIs. A. Structure of the EGFR kinase in the inactive conformation, highlighting the locations of diverse EGFR mutations (drawn from PDB ID 1XKK). The schematic on the right depicts the site of EGFR exon 20 insertion mutations studied here. B. and C. Dose-dependent cell growth inhibition of Ba/F3 cells expressing EGFR-delL747_P753insS, delL747_P753insS+T790M, A763_Y764insFQEA, Y764_V765insHH, M766_A767insAI, A767_V769dupASV, D770_N771insNPG, D770_N771insSVD, H773_V774insH, L858R and L858R+T790M. Ba/F3 cells expressing aforementioned EGFR mutations were treated with the indicated doses of erlotinib for 72 hours. Cell survival was measured using a CellTiter Aqueous One Solution Cell Proliferation Assay. Error bars indicate standard deviation (n=3). Calculated average IC₅₀ values of eleven EGFR mutation types are shown (n=3). D. Inhibition of EGFR signaling by erlotinib. Ba/F3 cells expressing all generated EGFR mutations were treated with 1 µM erlotinib for 6 hours. Phosphorylation of EGFR, AKT, and ERK proteins were detected by immunoblotting. E. Dose-response of erlotinib in Ba/F3 cells expressing EGFR L858R, L858R–T790M, A763_Y764insFQEA, and V769_D770dupASV. The cells were treated with indicated doses of erlotinib for 24 hours. Immunoblotting was done against the indicated proteins (EGFR, AKT, and ERK, as well as full length [flPARP] or cleaved PARP [clPARP] and isoforms of BIM [extra long, BIM_EL; long, BIM_L; and short, BIM_S]).

Figure 2

Response to EGFR TKIs of NSCLCs harboring EGFR exon 20 insertion mutations. A. Waterfall plot of best responses of target tumor lesions after exposure to gefitinib or erlotinib in relation to baseline measurements for each patient. The plot highlights that all A763_Y764insFQEA bearing tumors decreased in size after exposure to erlotinib, while other mutations had either minimal changes or increase in target lesions. Yellow bars indicate partial response (PR), blue bars stable disease (SD) and red bars progressive disease (PD). * indicates patients who displayed non-measurable PD. B. Detailed response of each individual mutation type analyzed.

Figure 3

BID007, a cell line expressing EGFR-A763_Y764insFQEA. A. Sequence of BID007’s DNA confirms EGFR-A763_Y764insFQEA. B. siRNA knockdown of EGFR in NSCLC cell lines A549, BID007, and H3255 cells for 72 hours (n=3). EGFR protein was detected by immunoblotting. Compared to A549 cells, siRNA1 and siRNA2 inhibited proliferation in BID007 (p=0.005 and p=0.001, respectively) and H3255 (p=0.003 and p=0.003, respectively). C. Dose-dependent cell growth inhibition of H3255, BID007, HCC827, H1975 and PC9 cells. The cells were treated with the indicated doses of erlotinib for 72 hours. Error bars indicate standard deviation (n=3). Calculated average IC₅₀ values of H3255, BID007, HCC827, H1975 and PC9 cells are shown (n=3). D. Inhibition of EGFR signaling by erlotinib in NSCLC cell lines. PC9, HCC827, H3255, BID007, and H1975 cells were treated with or without 1 µM erlotinib for 6 hours. Phosphorylation of EGFR, AKT, and ERK proteins was detected by immunoblotting. E. Dose-response of erlotinib in BID007, H3255, and H1975 cells. The cells were treated with indicated doses of erlotinib for 24 hours. Immunoblotting was done against the indicated proteins (EGFR, AKT, ERK, clPARP and BIM).

Figure 4

Implications of the crystal structure of the EGFR exon 20 insertion D770_N771insNPG (insNPG). A. Crystal structure of the insNPG mutant. The inhibitor PD168393, covalently bound to Cys797 (C797), is shown in stick form with carbon atoms colored green. The inserted NPG sequence is highlighted in magenta. B. Detailed view of the NPG insertion. The insNPG structure is shown in yellow with the inserted residues in magenta in stick form and is superimposed on the L858R structure (blue ribbon). C. Superposition of the active site region of the insNPG and L858R mutants bound to inhibitor PD168393. The compound binds in an essentially identical manner in both structures, forming a covalent bond with C797.

Figure 5

Homology modeling of the TKI-sensitive EGFR-A763_Y764insFQEA mutation (insFQEA). A. Two plausible alignments of the wild-type (WT) and insFQEA sequences in the region of the C-helix. The inserted FQEA residues (bold) could shift the register of the C-helix in the C-terminal direction (upper panel) or in the N-terminal direction (lower panel) and maintain a glutamic acid in the position of Glu762, a key active site residue. A C-terminal shift would be expected to lengthen the loop following the C-helix (αC-β4), while an N-terminal shift would lengthen the loop preceding it (β3-αC). B. EGFR mutant constructs introduced into Cos-7 cells. EGFR phosphorylation and presence of transfected constructs (as measured by HA tag levels) are depicted. Four different constructs, insFQEA, insFQQA (E->Q mutation in the inserted sequence), E762Q_insFQEA (E->Q mutation in the endogenous E762) and L858R + T790M, were transiently transfected into COS-7 cells. Phosphorylation of EGFR and HA tag were detected by immunoblotting. Mutagenesis of the respective glutamic acid residues indicates a shift in the N-terminal direction; and the region that would be altered relative to WT, is boxed in orange (lower panel A). C. Homology model of the insFQEA mutant (yellow) superimposed on the WT EGFR structure (blue ribbon) in the active conformation. The inserted residues are labeled in bold, and the shifted sequence is colored orange (corresponding to the boxed region in panel A); the glutamic acid residue in the FQEA insertion assumes the position of E762, and is positioned to form a salt bridge with K745. D. Homology model of the insFQEA mutant in the inactive conformation (yellow, with the structurally altered region in orange) superimposed on the WT EGFR structure in the inactive conformation (drawn from PDB ID: 1XKK). A cluster of hydrophobic residues (yellow side chains) is important for the stability of the inactive state; L858 and L861 are part of this cluster. The insFQEA insertion will shift an alanine residue into the position of I759 in this cluster (I759>A). The insertion is also expected to alter the length and conformation of the β3-αC loop, which is the site of exon 19 deletion mutations.