Emergence of KRAS mutations and acquired resistance to anti-EGFR therapy in colorectal cancer

Abstract

A main limitation of therapies that selectively target kinase signalling pathways is the emergence of secondary drug resistance. Cetuximab, a monoclonal antibody that binds the extracellular domain of epidermal growth factor receptor (EGFR), is effective in a subset of KRAS wild-type metastatic colorectal cancers. After an initial response, secondary resistance invariably ensues, thereby limiting the clinical benefit of this drug. The molecular bases of secondary resistance to cetuximab in colorectal cancer are poorly understood. Here we show that molecular alterations (in most instances point mutations) of KRAS are causally associated with the onset of acquired resistance to anti-EGFR treatment in colorectal cancers. Expression of mutant KRAS under the control of its endogenous gene promoter was sufficient to confer cetuximab resistance, but resistant cells remained sensitive to combinatorial inhibition of EGFR and mitogen-activated protein-kinase kinase (MEK). Analysis of metastases from patients who developed resistance to cetuximab or panitumumab showed the emergence of KRAS amplification in one sample and acquisition of secondary KRAS mutations in 60% (6 out of 10) of the cases. KRAS mutant alleles were detectable in the blood of cetuximab-treated patients as early as 10 months before radiographic documentation of disease progression. In summary, the results identify KRAS mutations as frequent drivers of acquired resistance to cetuximab in colorectal cancers, indicate that the emergence of KRAS mutant clones can be detected non-invasively months before radiographic progression and suggest early initiation of a MEK inhibitor as a rational strategy for delaying or reversing drug resistance.

Figures

Figure 1. KRAS amplification mediates acquired resistance to cetuximab in DiFi cells

(a) Parental and cetuximab resistant DiFi cells were treated for one week with increasing concentrations of cetuximab. Cell viability was assayed by the ATP assay. Data points represent means ± SD of three independent experiments. (b) Whole exome gene copy number analysis of parental and cetuximab resistant DiFi cells. Individual chromosomes are indicated on the x axis. The lines indicate the sequencing depth (y axis) over exome windows of 100,000 bp. (c) FISH analysis confirming KRAS amplification in DiFi-R but not parental DiFi cells. KRAS locus BAC DNA (probe RP11-707G18; green) and chromosome 12 paint (red) were hybridized to the metaphase spreads of DiFi cells. (d) DiFi cells were treated with cetuximab 35 nM for 24 hours, after which whole-cell extracts were subjected to Western blot analysis and compared to untreated cells. DiFi R1 and R2 were plated in the absence of cetuximab for 7 days or maintained in their normal growth medium (with cetuximab 35 nM) before protein analysis. Active KRAS (GTP-KRAS) was assessed by GST-Raf1 pull-down. Whole-cell extracts were blotted with phosphor-EGFR (Tyr 1068), total EGFR, total KRAS, phosphor-AKT (Thr 308), phosphor-AKT (Ser473), total AKT, total MEK1/2 and phospho-MEK1/2, total ERK1/2 and phospho-ERK1/2 antibodies. Vinculin was included as a loading control. (e) Western blot analysis of KRAS protein in DiFi cells infected with a KRAS lentivirus. Actin is shown as a loading control (f) Ectopic expression of wild-type KRAS in parental DiFi cells confers resistance to cetuximab.

Figure 2. KRAS mutations mediate acquired resistance to cetuximab in Lim1215 cells

(a) Parental and cetuximab resistant Lim1215 cells were treated for one week with increasing concentrations of cetuximab. Cell viability was assayed by the ATP assay. Data points represent means ± SD of three independent experiments. (b) Sanger sequencing of KRAS exon 2 in parental and two representative cetuximab-resistant Lim1215 cells obtained in independent selection procedures. (c) Western blot analysis of the EGFR signaling pathway in parental and cetuximab resistant Lim1215 cells. (d) Schematic representation of the vectors used to knock-in the G12R and G13D mutations into the genome of Lim1215 parental cell lines by AAV mediated homologous recombination. Targeting was assessed by Sanger sequencing. (e) Parental and isogenic Lim1215 cells carrying the indicated mutations were treated for one week with increasing doses of cetuximab.

Figure 3. Mutational analysis of the KRAS gene in patients

(a) Mutational analysis of KRAS in chemorefractory patients. (b) Mutational analysis of the KRAS gene in patients who progressed on anti-EGFR antibodies. The results are based on assays performed by Deep sequencing technologies a: 454 pyrosequencing; b: BEAMing. (c) Dot plot of percentage of mutated KRAS alleles in chemorefractory and anti-EGFR resistant patients: p-value was calculated by two-tailed unpaired Mann-Whitney test.

Figure 4. Detection of circulating KRAS mutant DNA in a patient with acquired resistance to cetuximab therapy

(a) Size of liver metastasis (blue bars) and CEA levels in blood (blue line) at the indicated time points showing an initial response to cetuximab followed by progression (Patient 8). (b) Quantitative analysis of Q61H mutant DNA in plasma as assessed by BEAMing (green line). (c) Two dimensional dot plot showing quantitative analysis of the KRAS Q61H mutation in plasma using BEAMing at individual time points (d) Mutational analysis of KRAS on tumor samples collected pre-cetuximab treatment and at the time of disease progression.