Genetically engineered mouse models for hereditary cancer syndromes

Abstract

Advances in molecular diagnostics have led to improved diagnosis and molecular understanding of hereditary cancers in the clinic. Improving the management, treatment, and potential prevention of cancers in carriers of predisposing mutations requires preclinical experimental models that reflect the key pathogenic features of the specific syndrome associated with the mutations. Numerous genetically engineered mouse (GEM) models of hereditary cancer have been developed. In this review, we describe the models of Lynch syndrome and hereditary breast and ovarian cancer syndrome, the two most common hereditary cancer predisposition syndromes. We focus on Lynch syndrome models as illustrative of the potential for using mouse models to devise improved approaches to prevention of cancer in a high-risk population. GEM models are an invaluable tool for hereditary cancer models. Here, we review GEM models for some hereditary cancers and their potential use in cancer prevention studies.

Keywords: cancer genetics; disease model; genetically engineered mice; hereditary cancers; mouse models.

FIGURE 1

Different strategies to manipulate the mouse genome. A, Targeting construct containing an antibiotic marker gene (NeoR) and homology to the gene to be targeted is used to knock out a gene. Homologous recombination between targeting construct and wild‐type (WT) allele replaces one or more exons with an antibiotic marker gene that leads to generation of nonfunctional allele. B, For the activation of gene, a construct containing a promoter, ≥1 exons and introns, and the rest of the cDNA with polyA tail is inserted into the mouse genome. This generates the transgenic allele. C, Conditional transgenic allele is generated by insertion of a construct that contains a promoter, ≥1 exons and introns, and the rest of the cDNA of the gene along with polyA tail and LoxP‐STOP‐LoxP sequences between the promoter and the first exon of the gene. The STOP sequence prevents the generation of protein unless it is removed by Cre‐mediated recombination. D, Conditional knockout allele of a gene is generated by targeting a construct in which ≥1 exon are flanked by LoxP sequences. For selection of the correctly targeted allele, an antibiotic marker (NeoR) is placed in an intron. The marker is flanked by FRT sequences and FLP‐mediated recombination is used to remove the marker gene after successful targeting. This generates an allele in which ≥1 exon of a gene is flanked by LoxP sequences placed in intron sequences. E, Cre‐mediated recombination to remove the sequences that are flanked by LoxP sequences. The 34‐bp‐long LoxP sequence is shown in the right. An 8 bp asymmetric spacer sequence separates two 13 bp palindromic sequences. Arrows indicate Cre‐mediated cleavage sites during recombination. Recombination between two LoxP sequences leads to the loss of the regions that are placed in between those sequences. F, In the doxycycline inducible gene activation system, the promoter driving the gene of interest is fused with tetO (tet‐operator) sequence, and a transgene driving the expression of transactivator rtTA (reverse tetracycline controlled transactivator) is inserted into the genome. Only in the presence of doxycycline, rtTA binds to tetO, leading to the transcription of the gene. G, CRISPR‐Cas9 mediated genetic engineering. A large deletion in a gene can be achieved by using two guide RNAs (sgRNA) to induce double‐strand breaks (DSB) at preferred locations of a gene. Repair of induced DSBs by nonhomologous end joining results in generation of a knockout allele. In the presence of the homologous DNA repair template, the repair of induced DSBs generates knock‐in allele. Colored boxes denote exons, and thick lines represent introns of a gene. Numbers in the boxes represent exon numbers, the promoter is marked by “P,” and arrows indicate gene transcription. Dotted crossed lines are used to represent homologous recombination.

FIGURE 2

Generation of different genetically engineered mouse models and their applications. A, Breeding of mice harboring knockout allele in tumor suppressor gene to generate homozygous, heterozygous, and wild‐type cohorts for tumor studies. B, Crossing mice homozygous for conditional knockout of any gene (gene X) with Cre‐expressing transgenic mice leads to generation of compound heterozygotes that are interbred to generate experimental cohorts. Transgenic mice expressing Cre from a tissue‐specific promoter develop tissue‐specific knockout of a gene. C, Breeding of mice to generate inducible conditional knockouts. Transgenic mice with rtTA (reverse tetracycline‐controlled transactivator) transgene and transgenic mice with tetO‐regulated promoter–driven Cre expression are crossed to generate double‐transgenic mice expressing rtTA and Cre. This double‐transgenic mouse is further crossed with homozygous conditional knockouts for any gene (geneX) to generate compound heterozygotes that are interbred to generate cohorts. Addition of doxycycline induces Cre expression that further promotes deletion of the conditional allele. Solid boxes and lines represent exons and introns, respectively. “X” is used to mark deletion and “P” is used to mark promoter. Solid triangles represent loxP site. The probability of obtaining a genotype from a cross is indicated in front of the genotype. D, Schematic summary of applications of genetically engineered mouse (GEM) models to understand basic biology (top half of figure) or in translational oncology (bottom half of figure). The scheme was created using Biorender (http://Biorender.com).

FIGURE 3

DNA repair pathways involved in Lynch syndrome (LS) and hereditary breast and ovarian cancer (HBOC). A, Mismatch repair pathway (MMR) in which mismatched bases are recognized by the MSH2‐MSH6 heterodimer and daughter strand is cleaved by MLH1‐PMS2–mediated endonuclease activity using assistance from the PCNA‐RFC complex. A segment of newly synthesized DNA is removed by exonuclease, and polymerase δ resynthesizes the DNA resulting in the repair of the mismatched DNA. B, Homologous recombination–mediated DNA repair. MRN complex (MRE11‐RAD50‐NBS1) recognizes the double‐stranded breaks (DSBs). BRCA1‐CtIP–mediated nuclease activity resects the DNA end from 5′ to 3′ and leads to the formation of single‐strand DNA (ssDNA) that gets coated with DNA replication protein A (RPA). Then, RAD51 nucleoprotein filament assembled by the multiprotein complex that involves BRCA2, PALB2, BRCA1 along with many other proteins replaces the RPA‐coated ssDNA, performs homology search, and mediates strand invasion. New synthesis of DNA using sister chromatid DNA as template followed by ligation and resolution of Holliday junction repairs the DSBs. Biorender (http://Biorender.com) was used to create the illustration.

FIGURE 4

Preclinical use of genetically engineered mouse (GEM) models for Lynch syndrome (LS). A, Vaccination of Msh2 ^loxp/loxp ; Vill‐Cre mice with recurrent frameshift peptide (FSP) antigens prolong survival. Survival curve of Msh2 ^loxp/loxp ; Vill‐Cre mice after treatment with aspirin (ASA), naproxen (NAP), FSP antigens, ASA + FSP antigen, or NAP+FSP antigens. Untreated mice were used as control. Six‐ to eight‐week‐old mice were enrolled in the study. A total of 50 μg of each FSP combined with 20 μg of the adjuvant were introduced subcutaneously to either the right or left flank four times biweekly, followed by four times monthly. At the age of weaning, 400 ppm ASA or 166 ppm NAP was incorporated into the diet of the mice assigned to those arms of the study. B, Survival curve of Mlh1 ^−/− mice with and without vaccination using cell line lysates. Two Mlh1 ^−/− cell lines (328 and A7450 T1 M1) lysates were injected subcutaneously. Application was initiated in 8‐10‐week‐old mice at the dose of 10 mg/kg body weight by 4‐weekly administration and then monthly administration for up to 12 vaccinations (n = 9/group). Control mice (n = 15) were left untreated. *** < 0.001 A7450 T1 M1 vs. control; Log‐rank (Mantel‐Cox) test.