Mutational landscape of EGFR-, MYC-, and Kras-driven genetically engineered mouse models of lung adenocarcinoma

Abstract

Genetically engineered mouse models (GEMMs) of cancer are increasingly being used to assess putative driver mutations identified by large-scale sequencing of human cancer genomes. To accurately interpret experiments that introduce additional mutations, an understanding of the somatic genetic profile and evolution of GEMM tumors is necessary. Here, we performed whole-exome sequencing of tumors from three GEMMs of lung adenocarcinoma driven by mutant epidermal growth factor receptor (EGFR), mutant Kirsten rat sarcoma viral oncogene homolog (Kras), or overexpression of MYC proto-oncogene. Tumors from EGFR- and Kras-driven models exhibited, respectively, 0.02 and 0.07 nonsynonymous mutations per megabase, a dramatically lower average mutational frequency than observed in human lung adenocarcinomas. Tumors from models driven by strong cancer drivers (mutant EGFR and Kras) harbored few mutations in known cancer genes, whereas tumors driven by MYC, a weaker initiating oncogene in the murine lung, acquired recurrent clonal oncogenic Kras mutations. In addition, although EGFR- and Kras-driven models both exhibited recurrent whole-chromosome DNA copy number alterations, the specific chromosomes altered by gain or loss were different in each model. These data demonstrate that GEMM tumors exhibit relatively simple somatic genotypes compared with human cancers of a similar type, making these autochthonous model systems useful for additive engineering approaches to assess the potential of novel mutations on tumorigenesis, cancer progression, and drug sensitivity.

Keywords: EGFR; GEMM; KRAS; MYC; exome.

Fig. 1.

Diagrams illustrating the mouse models of mutant Kras-, mutant EGFR-, and MYC-induced lung adenocarcinoma used in whole-exome sequencing. (A and B) Kras models. (A) Mice carrying conditional Kras^LSL-G12D and p53^flox/flox alleles develop lung adenocarcinomas upon administration of lenti-cre. Cell lines were generated from primary and metastatic lung tumors. Tumors and cell lines were collected for exome sequencing. (B) Mice carrying Kras^LA2-G12D;p53^−/− form lung adenocarcinomas spontaneously. Primary tumors were collected for exome sequencing. (C) EGFR model: Bitransgenic CCSP-rtTA;TetO-EGFR^L858R mice were treated with doxycycline at weaning to induce transgene expression (10). Tumors were collected from untreated tumor-bearing mice, or mice were treated with erlotinib as described until the appearance of resistant tumors (12). Untreated and erlotinib-resistant lung tumors were collected and used for exome sequencing. (D) MYC model: Bitransgenic CCSP-rtTA;TetO-MYC mice were treated with doxycycline at weaning to induce transgene expression. Overexpression of MYC in type II pneumocyte leads to the development of lung adenocarcinomas that were collected for whole-exome sequencing (26).

Fig. S1.

Inbred mouse strains to simulate somatic mutation. (A) Diagram of the simulation performed using serial dilution of exon capture libraries generated from inbred mouse strains. (B) Observed allelic fraction of germ-line variants in the diluted libraries, histograms using R’s density function; y-axis scale is in default arbitrary units. (C) Receiver–operator characteristic curve of sensitivity vs. simulated tumor purity of muTect vs. HaJaVa, vs. intersection of both callers. (D) Receiver–operator characteristic curve of sensitivity vs. false-positive rate per Mb of muTect vs. HaJaVa, vs. intersection of both callers.

Fig. 2.

Low mutational burden in GEMM models of lung cancer. (A) Dot plots showing the nonsynonymous mutation frequency observed from whole-exome sequencing datasets in murine LUADs induced by oncogenic Kras (tumors from either the LA2 or LSL models; tumor-derived cell lines are excluded; see below), EGFR, or overexpression of MYC. (B) Trp53 null vs. wild-type tumors. (C) Kras^LA2-G12D;p53^−/− vs. Kras^LSL-G12D;Trp53^fl/fl tumors. (D) Kras^LSL-G12D;Trp53^fl/fl tumors vs. tumor-derived cell lines. (E) Comparison of Kras^G12D-induced tumors to human lung adenocarcinomas (ref. 4) from smoking and nonsmoking patients, shown in log scale. (F) Untreated vs. drug-resistant EGFR^L858R-induced LUADs. Mean and SEM are shown.

Fig. S2.

Distinct mutation signatures in GEMM models of lung adenocarcinoma. Sum of parts graph for observed somatic single-nucleotide variants in each model. Note the similar pattern between Kras tumors and cell lines vs. the distinct pattern between EGFR- and Kras-driven models.

Fig. 3.

Mutational landscape of oncogene-induced mouse lung adenocarcinomas. Schematic diagram of genes mutated in the mouse lung adenocarcinomas and cell lines is shown. (Top) Kras-, EGFR-, and MYC-induced tumors are indicated and shaded in red, blue, and purple, respectively. The Trp53 status is indicated (black, null; gray, heterozygous). (Middle) Genes mutated in two or more samples are indicated. (Bottom) Genes mutated in the murine tumors that are also mutated in >15% of lung adenocarcinomas analyzed in the TCGA. Note that Csmd1, Hmcn1, and Kml2c were not recurrently mutated in murine tumor, whereas asterisk-indicated genes were recurrently mutated in human and murine tumors. Erlotinib-resistant tumors are indicated. Stars are used to highlight tumors harboring an EGFR T790M mutation (by conventional Sanger sequencing).

Fig. S3.

Heat map of sequencing coverage of the Trp53 locus. Integrative Genome Viewer snapshot of the coverage depth of the Trp53 locus in all samples is shown. Notably, exons 2–10 were demarcated by very low coverage in tumor-derived cell lines from the Kras^LSL-G12D;p53^fl/fl model (dark blue regions). Trp53 locus map is shown at the bottom of the figure.

Fig. 4.

Distinct patterns of DNA copy number alterations in Kras- and EGFR-driven GEMMs. (A) Heat map of DNA copy number alterations across all samples. Red, DNA copy number amplification; blue, DNA copy number loss. Models are grouped by initiating driver allele. Also shown is p53 status and whether the sample was a tumor or tumor-derived cell line. Point mutation frequency is shown in gray–black box, with the darker shade representing a higher mutation frequency. (B) Recurrent whole-chromosome DNA copy number gains in Kras^G12D-driven GEMM tumors and cell lines. (C) Recurrent whole-chromosome DNA copy number gains in EGFR-driven GEMM tumors.

Fig. S4.

Selective amplification of mutant Kras-bearing chromosome 6. Graph of the observed allelic fraction of the Kras^G12D mutation in the tail DNA and tumor cell line DNA of individual samples is shown. The increased allelic fraction in the tumor-derived cell lines suggests that the observed amplification of Chr6 is primarily due to increased copy number of the mutant allele.