Genomic and biological characterization of exon 4 KRAS mutations in human cancer

Abstract

Mutations in RAS proteins occur widely in human cancer. Prompted by the confirmation of KRAS mutation as a predictive biomarker of response to epidermal growth factor receptor (EGFR)-targeted therapies, limited clinical testing for RAS pathway mutations has recently been adopted. We performed a multiplatform genomic analysis to characterize, in a nonbiased manner, the biological, biochemical, and prognostic significance of Ras pathway alterations in colorectal tumors and other solid tumor malignancies. Mutations in exon 4 of KRAS were found to occur commonly and to predict for a more favorable clinical outcome in patients with colorectal cancer. Exon 4 KRAS mutations, all of which were identified at amino acid residues K117 and A146, were associated with lower levels of GTP-bound RAS in isogenic models. These same mutations were also often accompanied by conversion to homozygosity and increased gene copy number, in human tumors and tumor cell lines. Models harboring exon 4 KRAS mutations exhibited mitogen-activated protein/extracellular signal-regulated kinase kinase dependence and resistance to EGFR-targeted agents. Our findings suggest that RAS mutation is not a binary variable in tumors, and that the diversity in mutant alleles and variability in gene copy number may also contribute to the heterogeneity of clinical outcomes observed in cancer patients. These results also provide a rationale for broader KRAS testing beyond the most common hotspot alleles in exons 2 and 3.

Figure. 1. Prevalence of KRAS, NRAS and BRAF mutations in patients with colorectal cancer

A. 415 colorectal tumors were screened for mutations in RAS and BRAF. Exon 2 KRAS mutations are shown in light blue, exon 3 KRAS mutations in dark blue, exon 4 KRAS mutations (K117 and A146) in red, NRAS mutations in yellow and BRAF mutations in green. B. Representative mass spectrometry and Sanger sequencing traces are shown for tumors harboring a V600E BRAF, Q22K KRAS, and A146T KRAS mutation.

Figure. 2. Concordance of KRAS, BRAF, PIK3CA and TP53 mutations

A. Exon 4 KRAS mutations were non-overlapping in distribution with mutations in KRAS exons 2 and 3 and BRAF. B. A146 KRAS mutations were identified in samples derived from all clinical states including adenomas, invasive primary colorectal adenocarcinomas, and liver metastases. C. Kaplan-Meier plot of disease-specific survival of 186 patients with stages 1-3 colorectal cancer as a function of KRAS/NRAS mutational status (p-value as indicated, log-rank test). No patients with stage 1-3 colorectal cancer whose tumor expressed an exon 3 or 4 KRAS or NRAS mutation died of colorectal cancer. D. Sanger and mass spectrometry traces of three tumors resected from a 75 year old woman who presented with synchronous primaries in the rectosigmoid (primary 1), cecum (primary 2), and four liver metastases. She is without evidence of disease 20 months following surgical resection of all disease sites. Primary 1 and the liver metastasis harbored A146T KRAS and R306* TP53 mutations. Primary 2 was G12V KRAS mutant and TP53 wild-type.

Figure. 3. Cell lines harboring an A146T KRAS mutation demonstrate elevated RAS-GTP expression and KRAS-dependence

A. HEK-293FT cells were transfected with KRAS mutants and GTP-bound RAS was measured by immuno-precipitating active RAS with recombinant Ras binding domain (RBD) of RAF. B. Colorectal cancer cell lines expressing A146T KRAS demonstrate elevated RAS-GTP expression as compared to cell lines with V600E BRAF mutation or those wild-type for both KRAS and BRAF. C. Knockdown of KRAS expression by siRNA in LS1034 cells (homozygous A146T KRAS mutant) inhibited colony formation.

Figure. 4. Colorectal tumors and cell lines harboring A146 KRAS mutations demonstrate evidence of copy number gain at the KRAS gene locus

A. Statistically significant genomic aberrations in 128 colorectal adenocarcinomas or adenomas (red and blue for amplifications and deletions respectively; FDR ≤ 15%). Profile is shown for the 22 autosomes in genomic coordinates (centromeres in red). B. Copy number gain at the KRAS locus was more common in tumors expressing A146T mutation versus those with exon 2 KRAS mutation or KRAS wild-type tumors (p-value = 0.014, one-tailed Fisher exact test). C. FISH analysis of the LS1034 (KRAS A146T) cell line demonstrating a hyper-triploid karyotype with three copies of chromosome 12 (red arrow) and two copies of an isochromosome for 12p (yellow arrows).

Figure. 5. MEK-dependence of A146T KRAS mutant cell lines

A. IC50 values for the selective MEK1/2 inhibitor PD0325901 for thirty-five colorectal, lung and breast cell lines including all seven models harboring exon 4 KRAS mutation (4 A146T, 2 A146V and 1 K117N). B. Representative IC50 curves for the LS1034 (A146T KRAS), C80 (A146V KRAS), CCCL-18 (A146T KRAS, PIK3CA E542K), HCT-15 (KRAS G13D, PIK3CA E545K) and COLO-205 (V600E BRAF) models. C. Treatment of A146T KRAS expressing cell lines with PD0325901 was associated with a decrease in pERK and cyclin D1 expression, induction of p27 and hypo-phosphorylation of RB. (D). Cell lines expressing exon 4 KRAS mutations were resistant to the EGFR inhibitor gefitinib. The gefitinib-sensitive H3255 (L858R EGFR) model is included for comparison.

Figure. 6. The growth of A146T KRAS mutant xenografts was MEK-dependent

A. Treatment of mice bearing established LS1034 xenografts with a single oral dose of PD0325901 (25 mg/kg) resulted in downregulation of pERK and cyclin D1 expression at 6 hours and upregulation of p27 at 24 hours. B. Mice with established LS1034 (A146T KRAS) xenografts were treated with PD0325901 (25 mg/kg 5x/week × 3 weeks) or vehicle only as control. (C). Mice were treated with 10 mg/kg of cetuximab or vehicle only as control twice weekly for three weeks.