Glioblastoma-derived epidermal growth factor receptor carboxyl-terminal deletion mutants are transforming and are sensitive to EGFR-directed therapies

Abstract

Genomic alterations of the epidermal growth factor receptor (EGFR) gene play a crucial role in pathogenesis of glioblastoma multiforme (GBM). By systematic analysis of GBM genomic data, we have identified and characterized a novel exon 27 deletion mutation occurring within the EGFR carboxyl-terminus domain (CTD), in addition to identifying additional examples of previously reported deletion mutations in this region. We show that the GBM-derived EGFR CTD deletion mutants are able to induce cellular transformation in vitro and in vivo in the absence of ligand and receptor autophosphorylation. Treatment with the EGFR-targeted monoclonal antibody, cetuximab, or the small molecule EGFR inhibitor, erlotinib, effectively impaired tumorigenicity of oncogenic EGFR CTD deletion mutants. Cetuximab in particular prolonged the survival of intracranially xenografted mice with oncogenic EGFR CTD deletion mutants, compared with untreated control mice. Therefore, we propose that erlotinib and, especially, cetuximab treatment may be a promising therapeutic strategy in GBM patients harboring EGFR CTD deletion mutants.

Figure 1. Identification and functional characterization of GBM-derived CT deletion mutants

(A) Quantitative PCR analysis using primer sets for exons 23 through 28 of EGFR, marked according to the color legend on the right, performed on skin DNA from TCGA-08-0359 as a normal control and five samples harboring candidate EGFR CTD deletions based on exon array analysis shown in Table S1. The relative copy number for each exon is normalized to EGFR exon 23. (B) Direct sequencing of EGFR PCR fragments from TCGA-02-0102 and TCGA-02-0043 DNA revealed the location of intragenic deletions of exon 27 and exon 25-27 in these samples, respectively. (C) Schematic showing the proposed splicing and resulting protein products of GBM-derived mutants as well as wild-type EGFR. Deletion of exon 27 generates a frame-shifted exon 28, with the addition of Asn (N) and Thr (T) after amino acid 1054 followed by early termination (red solid arrow). Deletion of exons 25-27 also resulted in the addition of N and T after amino acid 982 followed by a stop codon (red solid arrow). Given that the 3’ end of the exon 27-28 and exon 25-28 deletions were not determined, the detailed transcripts are not defined (red dashed arrows). The blue asterisks indicate the position of the stop codon of the wild-type EGFR transcript (blue arrow). Red asterisks indicate the predicted stop codons generated by frame-shift of the indicated aberrant RNA transcripts.

Figure 2. GBM-derived CT982NT and CT1054NT EGFR CTD deletion mutants are oncogenic in the absence of tyrosine phosphorylation

(A) CT982NT and CT1054NT mutants confer IL-3 independency to Ba/F3 cells. IL-3 independent cell proliferation ability of Ba/F3 cell lines stably expressing CT982NT, CT1054NT, vIII mutant EGFR as well as parental Ba/F3 cells, was assayed by counting cell numbers on 9, 12, 14, 15 and 16 days after IL-3 withdrawal. The results are indicated as means +/- SD of three independent counts. (B) Tyrosine phosphorylation of EGFR is dispensable for oncogenic activity of CT982NT and CT1054NT mutants. Whole cell lysates prepared from Ba/F3 cells analyzed in (A) were subjected to immunoblotting with antibodies against phospho-tyrosine (4G10), EGFR and actin. Schematic cartoon shows the location of tyrosine residues for phosphorylation within exons consisting of C-terminal domain of wild-type EGFR and CT deletion mutants.

Figure 3. CT982NT and CT1054NT oncogenic EGFR mutants are sensitive to erlotinib or cetuximab in vivo and in vitro

(A and B) Growth of Ba/F3 cells transformed with either CT982NT or CT1054NT EGFR mutant, but not the parental line with IL-3, was suppressed by either erlotinib (A) or cetuximab (B). Cells were treated with either erlotinib or cetuximab at the concentrations indicated for 72 hrs and assayed for cell viability. The results are indicated as mean +/- SD of three independent experiments. (C and D) Growth of mouse tumors driven by CT982NT and CT1054NT EGFR mutants are significantly suppressed by either erlotinib or cetuximab. LN443 cells expressing CT982NT (C) and CT1054NT (D) mutants were subcutaneously injected in the flanks of SCID mice (5 mice per group and 3 sites per each mouse). 20 days after cell injection, when tumors researched a size around 50-70 mm³, either erlotinib (50 mg/kg, gavage) or cetuximab (50 mg/kg, IP) was administered 3 times per week for 13 weeks. Tumor size was measured once a week, and volume was determined according to the formula (W2 × L)/2.

Figure 4. Anti-EGFR therapy is effective against brain tumors induced by oncogenic EGFR CTD deletion mutants

(A) Schematic to show the generation of the intracranial xenograft mouse model and the timing of anti-EGFR drug treatment. The indicated numbers of SCID mice were implanted intracranially with LN443 cells stably expressing either wild-type EGFR, EGFRvIII or CT Del1 mutants. After 1 week, each mutant group of xenografted mice was assigned to receive either no treatment or erlotinib or cetuximab three times per week with the dose indicated in the schematic. (B) Representative images of excised brains from the untreated group of mice xenografted with LN443 cells expressing wild-type EGFR or the EGFRvIII or CT Del1 mutants, respectively. (C) EGFRvIII and CT Del1 mutants are expressed and active in brain tumors originated from xenografted mice whereas wild-type EGFR is neither detected nor active. Brain tumors sections prepared from no treatment group of xenograft mice of wild-type EGFR or EGFRvIII or CT Del1 mutants were stained with H&E (top) or antibodies to total EGFR (middle) or phospho-EGFR (Tyr1197)(bottom). (D-F) Cetuximab treatment prolongs survival of xenografted mice with oncogenic EGFR CT Del1 mutant as well as EGFRvIII mutant. The survival of treated or non-treated groups of xenografted mice described in (A) were monitored and depicted as a Kaplan-Meier curve (M.S = median survival). P values were calculated between non-treated and drug-treated mouse groups.