Large-scale mutagenesis in p19(ARF)- and p53-deficient mice identifies cancer genes and their collaborative networks

Abstract

p53 and p19(ARF) are tumor suppressors frequently mutated in human tumors. In a high-throughput screen in mice for mutations collaborating with either p53 or p19(ARF) deficiency, we identified 10,806 retroviral insertion sites, implicating over 300 loci in tumorigenesis. This dataset reveals 20 genes that are specifically mutated in either p19(ARF)-deficient, p53-deficient or wild-type mice (including Flt3, mmu-mir-106a-363, Smg6, and Ccnd3), as well as networks of significant collaborative and mutually exclusive interactions between cancer genes. Furthermore, we found candidate tumor suppressor genes, as well as distinct clusters of insertions within genes like Flt3 and Notch1 that induce mutants with different spectra of genetic interactions. Cross species comparative analysis with aCGH data of human cancer cell lines revealed known and candidate oncogenes (Mmp13, Slamf6, and Rreb1) and tumor suppressors (Wwox and Arfrp2). This dataset should prove to be a rich resource for the study of genetic interactions that underlie tumorigenesis.

Figure 1

Accelerated Tumor Formation in MuLV-Infected Mice and Identification of Common Insertion Sites Using the GKC Framework (A) Cumulative survival of p19^ARF−/−, p53^−/−, and wild-type mice infected with MuLV and noninfected p19^ARF−/− and p53^−/− mice is plotted against lifespan in days (p19^ARF−/− versus wild-type, p value < 0.0001; p53^−/− versus wild-type, p value < 0.0001, log-rank test). (B) Flow chart for retrieving retroviral insertions. (C) Number of CISs varies with kernel size and significance. (D) Identification of CISs near Myc with different kernel sizes. Red line represents insertion density for 300 kb kernel, green line for 30 kb, and the blue line for 5 kb. Blue and red denote sense and antisense insertions, respectively. (E) Insertions from p53^−/−, p19^ARF−/−, and wild-type tumors were analyzed together with a 30 kb kernel to determine insertion density over the genome (left panel). The cut-off (p value < 0.05) for significant insertion density is indicated (red line). CISs (p value < 0.05) are indicated by green vertical bars. A list of the insertion density of the 15 most significant CISs is included (right panel).

Figure 2

Genotype-Specific Distribution of Insertions (A) Analysis of all panels combined will detect CISs that occur relatively frequently (peaks B and C) but may not identify CISs in regions that have a relatively small number of insertions in tumors from the same genotype (peaks A). Conversely, CISs constituted of a number of insertions from different panels, may not reach significance when panels are analyzed separately (peak B). (B) Schematic overview of genotype-specific CISs found in p19^ARF−/−, p53^−/−, and wild-type mice. CISs that have a significant bias (p value < 0.05) toward a particular genotype are depicted. CISs more frequently found in one genotype versus another (e.g., Pim2 in p53^−/− versus p19^ARF−/−) are depicted at the green end of the wedge pointing toward the other genotype (p19^ARF−/−). (C) Venn-diagram representing the overlap in numbers of “individual panel” CISs found between genotypes. The number of CISs per genotype is indicated outside the diagram.

Figure 3

Mapping Interaction Networks between Common Insertion Sites (A) Co-occurrence between the top 25 300 kb CISs. CIS names and CIS rank are indicated on vertical axis, numbers on horizontal axis are CIS rank. The horizontal axis represents CISs that are assumed to be the predisposing, more clonal event and the vertical axis represents CISs that are presumed to be subsequent, subclonal events. (B) Mutual exclusivity between the top 25 300 kb CISs. Set up of the figure as described in (A). (C) CIS interaction network representing the co-occurrence or mutual exclusivity of the 20 most significant CISs. Co-occurring CISs are connected by green lines (thin line, 0.001 < p value < 0.05; heavy line, p value < 0.001), mutual exclusive CISs are connected with red lines (thin line, 0.001 < p value < 0.05; heavy line, p value < 0.001).

Figure 4

Clusters of Insertions within Flt3 and Notch1 Induce Oncogenic Proteins with Distinct Properties (A) Analysis of Flt3, Notch1, Ikaros and Jundm2 with a 5 kb kernel size reveals multiple CISs. Blue bars represent sense insertions, red bars antisense insertions, green bars introns and black bars exons. (B) Chimeric transcripts of MuLV and Flt3 sequences are formed by splicing of MuLV splice donor (SD) to splice acceptor of Flt3 exon10 producing a 1682nt mutant transcript (upper panel), and expression of truncated Flt3 proteins in tumors containing fusion transcripts. Translation starts from an in-frame start codon in exon10 (lower panel). Protein lysates of tumors with (+) or without (−) Flt3 insertion were separated with SDS/Page followed by immunoblotting with the indicated antibodies. Tumors with insertions upstream of Flt3 (US). (C) 300 kb CISs co-occurring or mutually exclusive with the separate 5 kb CISs found in Notch1 and Flt3. 5 kb CIS names are indicated on vertical axis, 300 kb CIS names on horizontal axis.

Figure 5

Insertions Identify Tumor Suppressors and Oncogenes in Human Cancers (A–C) Rreb1 (A), Slamf6 (B), and Mmp13 (C) are mutated by multiple insertions and also frequently found amplified in human cancers. (D and E) Mapping of retroviral insertions in the murine Wwox (D) and Arfrp2 (E) genes to deletions found in human cancer cell lines. Upper part, copy number of chromosomal regions in the human cell lines is depicted in color. Names of human cell lines and tissue of origin are provided. Lower part, insertions in murine tumors. Blue bars represent insertions in sense orientation, red bars antisense insertions, green bars introns and black bars exons. Positions on the murine and human chromosomes are indicated on the black horizontal bars in kb and Mb, respectively.