Oncogenic activity of epidermal growth factor receptor kinase mutant alleles is enhanced by the T790M drug resistance mutation

Abstract

Activating mutations in the epidermal growth factor receptor (EGFR) characterize a subset of non-small cell lung cancers (NSCLC) with extraordinary sensitivity to targeted tyrosine kinase inhibitors (TKI). A single secondary EGFR mutation, T790M, arising in cis with the primary activating mutation, confers acquired resistance to these drugs. However, the T790M mutation is also detected in the absence of drug selection, suggesting that it may provide a growth advantage. We show here that although T790M alone has only a modest effect on EGFR function, when combined with the characteristic activating mutations L858R or del746-750, it results in a dramatic enhancement of EGFR activity. The double mutants show potent ligand-independent receptor autophosphorylation associated with altered cellular phenotypes, soft agar colony formation, and tumorigenesis in nude mice. The significant gain-of-function properties of these double mutants may explain their initial presence before drug selection and their rapid selection as the single drug resistance mutation during therapy with gefitinib/erlotinib, and suggests that they may contribute to the adverse clinical course of TKI-resistant NSCLC.

Figure 1

Morphologic transformation of NIH3T3 cells expressing double-mutant EGFR. A, immunoblot of NIH3T3 cells infected with retroviruses encoding the indicated EGFR constructs demonstrating comparable expression of all EGFR constructs (H-RasV12 and actin controls). B, cells expressing EGFR T790M/L858R and EGFR T790M/Del double mutants have striking morphologic differences from parental cells and those expressing EGFR wild-type (wt) or single mutants, as determined by phase-contrast microscopy. Cells expressing H-RasV12 also differ morphologically.

Figure 2

EGFR T790M/Del activity is required for morphologic transformation. A, cells stably expressing various EGFR constructs (shown above) were treated with 10 μmol/L gefitinib, 10 μmol/L HKI-272, or DMSO for 2 h, and were then stimulated with 100 ng/mL EGF for 5 min. Top row, phosphorylated (Phospho) Y992 EGFR; bottom row, total EGFR. B, cells expressing the constructs were grown in 5% serum and treated with 1 μmol/L HKI-272 or DMSO for 6 d to show drug-induced reversion of transformed morphology. C, cells expressing EGFR T790M/Del were infected with lentivirus encoding shRNA against EGFR or a control. D, lysates of infected cells were analyzed by immunoblot to confirm knockdown of EGFR expression. Actin was used as a loading control.

Figure 3

Increased EGFR kinase activity by T790M mutation in cis with classic activating mutations. A, immunoblot of NIH3T3 cells expressing EGFR wild-type, EGFR T790M, and EGFR L858R show increased autophosphorylation of mutant EGFR, as measured by phosphorylated Y845 EGFR (left). Total EGFR was used as a control. The intensity of the phosphorylated Y845 EGFR band in T790M extracts or L858R extracts relative to wild-type extracts was measured in five independent experiments, using total EGFR as an internal control (right). A t test was done to determine the statistical significance (P = 0.015 for T790M versus wild-type and P = 0.009 for L858R versus wild-type). Bars, SE. B, lysates derived from cells expressing EGFR wild-type or mutants (H-RasV12 and vector as controls) were subjected to immunoblot analysis with a panel of phosphorylated EGFR antibodies (Y845, Y992, Y1045, Y1068, Y1148) and total EGFR. C, cells expressing EGFR wild-type or mutants were starved of serum for 24 h. Lysates derived from these cells were subjected to immunoblot analysis against phosphorylated EGFR, Src, Akt, STAT3, and MAPK, and the appropriate total proteins as indicated. D, cells expressing EGFR wild-type or EGFR mutants were grown in 10% serum. Where indicated, cells were serum starved for 24 h, then stimulated with 100 ng/mL EGF for 5 min. Total cell lysate was analyzed by immunoblot against phosphorylated Y992 EGFR and total EGFR. Ligandindependent activity of EGFR double mutants is evident.

Figure 4

Synergistic transforming activity of T790M in cis with L858R and Del EGFR mutations. A, soft-agar colony formation of NIH3T3 cells stably expressing the indicated EGFR construct or H-RasV12. Colonies were counted after 5 wk. This experiment was done in duplicate and is representative of three independent experiments (left). Right, similar soft-agar assay in the presence of 100 ng/mL EGF added to the top agar. Colonies were counted after 2 wk. B, soft agar colony formation of immortalized hTBE cells infected with the indicated EGFR constructs (left). Colonies were counted after 1 mo. Immunoblot of hTBE cells stably expressing EGFR constructs showsimilar expression levels of EGFR (right). Tubulin is used as a loading control. C, tumor formation in nude mice injected s.c. with NIH3T3 cells stably expressing the indicated EGFR construct or H-RasV12. Tumor volume was measured every 2 to 3 d. D, number of tumors formed and the total number of inoculations for each construct. Tumor formation was assessed 25 d after inoculation.

Figure 5

Model for T790M selection in EGFR mutant-expressing lung cancer. A somatic activating mutation at EGFR (e.g., L858R) is sufficient to initiate the formation of NSCLC (40, 41). A small proportion of cells acquire ligand-independent oncogenic potential by the secondary mutation T790M in cis with the primary mutation. Treatment with the TKIs gefitinib or erlotinib results in killing of cells expressing EGFR L858R, but has no effect on those expressing EGFR T790M/L858R. Drug treatment thus enriches for preexisting NSCLC cell populations expressing the highly transforming EGFR double mutant, resulting in a more aggressive tumor.