Computational identification of insertional mutagenesis targets for cancer gene discovery

Insertional mutagenesis is a potent forward genetic screening technique used to identify candidate cancer genes in mouse model systems. An important, yet unresolved issue in the analysis of these screens, is the identification of the genes affected by the insertions. To address this, we developed Kernel Convolved Rule Based Mapping (KC-RBM). KC-RBM exploits distance, orientation and insertion density across tumors to automatically map integration sites to target genes. We perform the first genome-wide evaluation of the association of insertion occurrences with aberrant gene expression of the predicted targets in both retroviral and transposon data sets. We demonstrate the efficiency of KC-RBM by showing its superior performance over existing approaches in recovering true positives from a list of independently, manually curated cancer genes. The results of this work will significantly enhance the accuracy and speed of cancer gene discovery in forward genetic screens. KC-RBM is available as R-package.     

Retroviral insertions in the VISION database identify molecular pathways in mouse lymphoid leukemia and lymphoma

AKXD recombinant inbred (RI) strains develop a variety of leukemias and lymphomas due to somatically acquired insertions of retroviral DNA into the genome of hematopoetic cells that can mutate cellular proto-oncogenes and tumor suppressor genes. We generated a new set of tumors from nine AKXD RI strains selected for their propensity to develop B-cell tumors, the most common type of human hematopoietic cancers. We employed a PCR technique called viral insertion site amplification (VISA) to rapidly isolate genomic sequence at the site of provirus insertion. Here we describe 550 VISA sequence tags (VSTs) that identify 74 common insertion sites (CISs), of which 21 have not been identified previously. Several suspected proto-oncogenes and tumor suppressor genes lie near CISs, providing supportive evidence for their roles in cancer. Furthermore, numerous previously uncharacterized genes lie near CISs, providing a pool of candidate disease genes for future research. Pathway analysis of candidate genes identified several signaling pathways as common and powerful routes to blood cancer, including Notch, E-protein, NFκB, and Ras signaling. Misregulation of several Notch signaling genes was confirmed by quantitative RT-PCR. Our data suggest that analyses of insertional mutagenesis on a single genetic background are biased toward the identification of cooperating mutations. This tumor collection represents the most comprehensive study of the genetics of B-cell leukemia and lymphoma development in mice. We have deposited the VST sequences, CISs in a genome viewer, histopathology, and molecular tumor typing data in a public web database called VISION (Viral Insertion Sites Identifying Oncogenes), which is located at . Keith C. Weiser and Bin Liu are authors that contributed equally to this work.     

Clonal Expansion Analysis of Transposon Insertions by High-Throughput Sequencing Identifies Candidate Cancer Genes in a PiggyBac Mutagenesis Screen

Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10⁶ reads corresponding to >10⁴ insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors.     

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

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.     

Tnfrsf13c (Baffr) is mis-expressed in tumors with murine leukemia virus insertions at Lvis22

In susceptible strains of mice, leukemia is caused by the somatic integration of murine leukemia retroviruses into the host genome. Integration sites that are common to several tumors are likely to affect genes that are important in oncogenesis. Here we present the analysis of a common site of retroviral integration on mouse chromosome 15, which includes the genomic structure of three genes near the integration site. One of the genes misexpressed at the insertion site has recently been characterized as a B-cell receptor, Tnfrsf13c (formerly Baffr), indicating that this approach is useful in defining genes that function in lymphocyte development and tumor progression. Current genome databases provide powerful resources for the rapid identification of genes at common proviral insertion sites. The characterization of these genes in tumor samples will allow a function to be assigned to many novel loci identified by the genome sequencing projects.     

Common sites of retroviral integration in mouse hematopoietic tumors identified by high-throughput,single nucleotide polymorphism-based mapping and bacterial artificial chromosome hybridization

Retroviral insertional mutagenesis in mouse hematopoietic tumors provides a powerful cancer gene discovery tool. Here, we describe a high-throughput, single nucleotide polymorphism (SNP)-based method, for mapping retroviral integration sites cloned from mouse tumors, and a bacterial artificial chromosome (BAC) hybridization method, for localizing these retroviral integration sites to common sites of retroviral integration (CISs). Several new CISs were identified, including one CIS that mapped near Notch1, a gene that has been causally associated with human T-cell tumors. This mapping method is applicable to many different species, including ones where few genetic markers and little genomic sequence information are available. It can also be used to map endogenous proviruses.     

Retroviral insertions in Evi12, a novel common virus integration site upstream of Tra1/Grp94, frequently coincide with insertions in the gene encoding the peripheral cannabinoid receptor Cnr2

The common virus integration site (VIS) Evi11 was recently identified within the gene encoding the hematopoietic G-protein-coupled peripheral cannabinoid receptor Cnr2 (also referred to as Cb2). Here we show that Cnr2 is a frequent target (12%) for insertion of Cas-Br-M murine leukemia virus (MuLV) in primary tumors in NIH/Swiss mice. Multiple provirus insertions in Evi11 were cloned and shown to be located within the 3' untranslated region of the candidate proto-oncogene Cnr2. These results suggest that proviral insertion in the Cnr2 gene is an important step in Cas-Br-M MuLV-induced leukemogenesis in NIH/Swiss mice. To isolate Evi11/Cnr2 collaborating proto-oncogenes, we searched for novel common VISs in the Cas-Br-M MuLV-induced primary tumors and identified a novel frequent common VIS, Evi12 (14%). Interestingly, 54% of the Evi11/Cnr2-rearranged primary tumors contained insertions in Evi12 as well, which suggests cooperative action of the target genes in these two common VISs in leukemogenesis. By interspecific backcross analysis it was shown that Evi12 resides on mouse chromosome 10 in a region that shares homology with human chromosomes 12q and 19p. Sequence analysis demonstrated that Evi12 is located upstream of the gene encoding the molecular chaperone Tra1/Grp94, which was previously mapped to mouse chromosome 10 and human chromosome 12q22-24. Thus, Tra1/Grp94 is a candidate target gene for retroviral activation or inactivation in Evi12. However, Northern and Western blot analyses did not provide evidence that proviral insertion had altered the expression of Tra1/Grp94. Additional studies are required to determine whether Tra1/Grp94 or another candidate proto-oncogene in Evi12 is involved in leukemogenesis.     

Orthologous microRNA genes are located in cancer-associated genomic regions in human and mouse

Background

MicroRNAs (miRNAs) are short non-coding RNAs that regulate differentiation and development in many organisms and play an important role in cancer.

Methodology/Principal Findings

Using a public database of mapped retroviral insertion sites from various mouse models of cancer we demonstrate that MLV-derived retroviral inserts are enriched in close proximity to mouse miRNA loci. Clustered inserts from cancer-associated regions (Common Integration Sites, CIS) have a higher association with miRNAs than non-clustered inserts. Ten CIS-associated miRNA loci containing 22 miRNAs are located within 10 kb of known CIS insertions. Only one CIS-associated miRNA locus overlaps a RefSeq protein-coding gene and six loci are located more than 10 kb from any RefSeq gene. CIS-associated miRNAs on average are more conserved in vertebrates than miRNAs associated with non-CIS inserts and their human homologs are also located in regions perturbed in cancer. In addition we show that miRNA genes are enriched around promoter and/or terminator regions of RefSeq genes in both mouse and human.

Conclusions/Significance

We provide a list of ten miRNA loci potentially involved in the development of blood cancer or brain tumors. There is independent experimental support from other studies for the involvement of miRNAs from at least three CIS-associated miRNA loci in cancer development.     

Approaches towards directed DNA integration by the use of retroviral integrases and transposases

The ability of retroviruses and transposases to insert own genome into a host-cell allow us to consider them as a preferable object for constructing gene therapy vectors. However, enzymes that perform the insertion of the genetic material do not display a selectivity towards target nucleotide sequences that results in an almost random DNA introduction into the recipient cell genome. Random insertion leads to mutations which might cause a number of undesirable consequences including neoplastic transformation in the cell. Thereby, in order to achieve a successful functioning of retroviral and trasposonal genetic therapy systems, it is essential to modify them in such a way that directed integration of the vector in a target sequence in the human genome could be achieved. In the review approaches that have been developed for a high specific modification of genome, including the construction of hybrid proteins on the basis of retroviral integrases, transposases, as well as cellular factors interacting with these enzymes, are presented.