High-throughput retroviral tagging to identify components of specific signaling pathways in cancer

Genetic screens carried out in lower organisms such as yeast(1), Drosophila melanogaster(2) and Caenorhabditis elegans 3 have revealed many signaling pathways. For example, components of the RAS signaling cascade were identified using a mutant eye phenotype in D. melanogaster as a readout(2). Screening is usually based on enhancing or suppressing a phenotype by way of a known mutation in a particular signaling pathway. Such in vivo screens have been difficult to carry out in mammals, however, owing to their relatively long generation times and the limited number of animals that can be screened. Here we describe an in vivo mammalian genetic screen used to identify components of pathways contributing to oncogenic transformation. We applied retroviral insertional mutagenesis in Myc transgenic (EmuMyc) mice lacking expression of Pim1 and Pim2 to search for genes that can substitute for Pim1 and Pim2 in lymphomagenesis. We determined the chromosomal positions of 477 retroviral insertion sites (RISs) derived from 38 tumors from EmuMyc Pim1(-/-) Pim2(-/-) mice and 27 tumors from EmuMyc control mice using the Ensembl and Celera annotated mouse genome databases. There were 52 sites occupied by proviruses in more than one tumor. These common insertion sites (CISs) are likely to contain genes contributing to tumorigenesis. Comparison of the RISs in tumors of Pim-null mice with the RISs in tumors of EmuMyc control mice indicated that 10 of the 52 CISs belong to the Pim complementation group. In addition, we found that Pim3 is selectively activated in Pim-null tumor cells, which supports the validity of our approach.