Insertional Mutagenesis Identifies Genes That Promote the Immortalization/Self-Renewal of Primary Bone Marrow Progenitor Cells.

Retroviruses can induce hematopoietic disease via insertional mutagenesis of cancer genes and provide valuable molecular tags for cancer gene discovery. Here we show that insertional mutagenesis can also identify genes that promote the immortalization of primary mouse bone marrow progenitor cells, which normally have only limited self-renewal. Transduction of bone marrow cells with replication-incompetent murine stem cell virus (MSCV) expressing only a neo marker gene, followed by serial passage in liquid culture containing SCF and IL-3, produced immortalized immature myeloid cell lines with neutrophil and macrophage differentiation potential in about 50% of the infected cultures. More than half of the lines have MSCV insertions at Evi1 or Prdm16. These loci encode transcription factor homologues and are validated human myeloid leukemia genes.

Integrations are located in intron 1 or 2 where they promote expression of truncated proteins lacking the PR domain, similar to what is observed in human leukemias with EVI1 or PRDM16 mutations. Evi1 overexpression alone appears sufficient to immortalize immature myeloid cells and does not seem to require any other cooperating mutations. Similarly, immortalized cells that are blocked at a very early hematopoietic progenitor stage can be produced by serially passaging the infected cells in SCF and FLT3L. In this case, viral integration deregulates the expression of Setbp1, a gene that is thought to regulate the activity of SET, which is mutated in acute human undifferentiated leukemia.

Interestingly, recurrent retroviral vector integrations at the Evi1 locus have also been recently identified in blood cells of rhesus monkeys transplanted up to 7 years earlier with retrovirally infected CD34+ bone marrow cells (Calmels et al, Blood. in press), and similar results have also been reported for mice (Kustikova et al, Science 2005 308:1171-4). Our results suggest that these cells were selected in transplant recipients due to their increased self-renewal or engraftment potential, which resulted from insertional mutations at Evi1. These studies highlight a new potential problem for human gene therapy, the selection of immortalized cells in transplant recipients that carry activating mutations in human leukemia oncogenes and a possible concomitant increased propensity for developing hematopoietic cancer as the patients age. Based on these studies, it will be important to assay the safety of other retroviral vectors, such as SIN vectors, that may be less apt to activate the expression of an oncogene following viral integration. The immortalization assay we describe here provides a means for assessing this safety.