Novel Cre-Lox tools

Despite the many advantages offered by the Cre-Lox-based GEMMs described earlier, the newly discovered breadth of mutations in this disease and the need to rapidly evaluate different combinations of mutations warrant the development of more efficient modeling strategies. To address this need, there have been a number of recent efforts to generate non-germline Cre-Lox models. One approach involves the culture of embryonic hepatoblasts from Alb-Cre KrasLSL-G12D Tp53LSL-R172H mice, which can then be orthotopically transplanted into a syngeneic host to generate iCCA within several months.51These mutant hepatoblasts can be further manipulated to validate other oncogenic drivers and evaluate their potential as a therapeutic target. For example, to determine the impact of a newly identified fusion gene, FIG-ROS1, on iCCA growth, a Tet-inducible FIG-ROS1 gene was introduced into the hepatoblast-allograft model, dramatically accelerating tumor development. Furthermore, turning off FIG-ROS1 expression via doxycycline withdrawal resulted in dramatically slower tumor progression. A similar strategy could express shRNAs to determine the impact of knocking down putative tumor suppressors or putative tumor-promoting genes. In addition to the improved flexibility and ease of generation in comparison to traditional GEMMs, this approach has the additional advantage of not generating mutations throughout the entire organ, thus maintaining an environment that consists of cells not genetically predisposed to cancer, more akin to human tumors.


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Another promising strategy has been developed to study pancreatic cancer and represents a system that could be adapted to study liver malignancies as well.52 Embryonic stem cells (ESCs) were established that contain a pancreas-specific driver (Pdx-Cre or p48-Cre) and the KrasLSL-G12D allele. In addition to these features, the cells harbor a “recombinase-mediated cassette exchange” strategy for rapid exchange of Tet-inducible shRNA- or cDNA-expressing constructs accompanied by a fluorescent reporter and a Cre-dependent Tet-transactivator accompanied by a different fluorescent reporter. The ESCs can be manipulated in vitro to introduce the shRNA/cDNAs of interest and subsequently used to generate chimeric mice for study. This flexible technology enables a variety of questions to be asked, illustrated by the following examples. First, by introducing a doxycycline-inducible shRNA-targeting Pten, they demonstrated that knockdown of a tumor suppressor can accelerate Kras-induced tumorigenesis. Furthermore, doxycycline withdrawal, resulting in reexpression of Pten, causes a dramatic decrease in tumor burden and an increase in survival. To determine the role of Myc in pancreatic tumor development, they employed tandem shRNA technology to introduce an shRNA targeting Tp53, which similarly accelerates tumorigenesis, and an shRNA targeting either Myc or a control shRNA. Compared to the control shRNA, tumor development in the Myc knockdown cohort was significantly impaired, confirming a role for Myc in promoting tumor development.

Thus, these technologies maintain many of the advantages of traditional germline GEMMs: the ability to make temporal- and tissue-specific genetic perturbations in an immune-competent environment, thereby generating cancers that closely follow the histopathologic progression of human disease. However, they offer flexible strategies to bypass the extensive breeding required to address similar questions with germline GEMMs.

SB MODELS

Given the limitations associated with Cre-Lox GEMMs, a number of alternative approaches have been developed. The mouse liver is readily accessible via hydrodynamic tail vein injection (HTVI) for delivery of plasmids, but due to the transient nature of traditional expression plasmids, sustained expression of transcripts for oncogenic studies is a challenge.53 Thus, the SB transposon system has found particular relevance in enabling stable integration of transgenes in the mouse liver (Table 3). The SB transposon is a cut and paste element, which was created based on the inactive Tc1/mariner superfamily of transposons in fish. The transposase recognizes inverted repeat (IR) sequences that flank a DNA sequence, excise that DNA, and insert it at thymine and adenine sites elsewhere in the genome. The endogenous SB transposon is a single element, where the SB transposase gene is flanked by IR/direct repeat (DR) sequences. In order to use this system experimentally, the SB transposase and IR/DR sequences have been separated where the IR/DR sequences flank the gene of interest and are often introduced as unique vectors.

(To view a larger version of Table 3, click here.)

To drive iCCA development, a number of studies have employed an activated form of Notch signaling in combination with various other perturbations including activation of PI3K signaling, activation of Yap signaling, mutant IDH1 expression, and Tp53 knockdown.43,46,54,55 Notch-independent models have also been generated, including coactivation of Yap/PI3K signaling and mutant Kras/PI3K signaling.15,46,56–61 While tail vein injection is believed to largely target hepatocytes, targeting the biliary tract specifically was achieved by injecting plasmids directly into the gallbladder following bile-duct ligation, thereby preventing spillover into the gut. It was further demonstrated that Yap and Aktexpression in this setting cooperate to induce iCCA in a small proportion of mice, a phenotype that significantly increased when IL-33 is systemically administered.

SB technology has also been adapted for forward genetic mutagenesis screens to identify genes that are critical in regulating liver cancer development.62,63 This is achieved using a Cre-Lox-regulated SB transposase and a T2/onc transposon which can lead to expression of an oncogene or inactivation of a tumor suppressor. In an SB mutagenesis screen using livers predisposed to tumor development via expression of dominant negative Tp53 (Tp53LSL-R270H), sequencing of the resulting tumors revealed genes that are known HCC drivers such as Egfr and Met, as well as a number of novel genes not previously implicated in cancer. Another experiment used Myc overexpression to predispose the liver to tumorigenesis and a similar SB mutagenesis strategy to identify Ncoa2 as a novel tumor suppressor. Currently, these screens in the liver have focused on HCC; thus, similar approaches remain to be employed in a setting that will lead to iCCA.

CRISPR/Cas9 models

Another especially promising gene-editing tool is the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system.64 Like SB, CRISPR/Cas9 offers an efficient method to introduce oncogene expression, but it has additional flexibility to inactivate genes such as tumor suppressors and repair genes with point mutations. In brief, the Cas9 nuclease is directed to a target sequence via a single-guide RNA (sgRNA), where double-strand breaks are repaired by error-prone nonhomologous end joining (NHEJ), often resulting in insertion/deletions (indels) that can disrupt a locus through introduction of reading frame shifts. Alternatively, investigators can use homology-directed repair (HDR) to create specific mutations through use of a DNA template to guide homologous recombination (HR).

To date, there are three hallmark studies that illustrate various ways in which CRISPR/Cas9 technology can be applied to the murine liver. One group used HTVI to introduce a single vector containing both Cas9 and sgRNAs to target Pten and Tp53.65 Phenocopying the comparable Cre-Lox model, loss of Ptenalone led to up-regulation of AKT, and loss of both Pten and Tp53 resulted in iCCA. Investigators also showed that gain-of-function mutations can be introduced through HDR, paving way for CRISPR/Cas9 modeling of oncogenic mutations commonly used in GEMMs, such as KrasG12D. Another group, which also sought to disrupt tumor suppressors, attempted to achieve long-term in vivo CRISPR/Cas9 activity by combining it with the SB transposon system to incorporate Cas9 and the sgRNAs into the genome.66Although very few tumors had stable integration of the CRISPR/Cas9 vectors, this study established an effective strategy to model mutations in large gene sets in the mouse liver, which is particularly advantageous for verifying tumor-causing mutations (ie, driver versus passenger mutations). Finally, CRISPR/Cas9 was used to repair a disease-causing point mutation in the Fah gene, that causes hereditary tyrosinemia type I in mice.67 Investigators were able to successfully mend the mutation and also demonstrate that the mice regained weight, signifying alleviation of the disease burden. An interesting therapeutic application related to this is the possibility of repairing point mutations important for tumorigenesis. Currently, however, it should be noted that this strategy corrects mutations in a very small proportion of cells, which may be best suited for instances where the repaired cells have a selective growth advantage. Thus, although promising, much improvement is needed regarding the delivery and repair efficiency of this system for it to be applied therapeutically to reverse cancer causing mutations.

Patient-derived models

The utility of studying human cancer cell lines in the culture dish and as xenografts is well established, and they will continue to be critical in understanding the biology of the disease and in developing therapeutic strategies. In this review, we highlight novel approaches that can offer some advantages over these traditional models.