Identification of Genes That Collaborate with CALM-AF10 to Induce Leukemia by Retroviral Insertional Mutagenesis and Candidate Gene Sequencing

The t(10;11) translocation results in a CALM-AF10 fusion gene in a subset of patients with ALL or AML. Expression of a CALM-AF10 fusion gene in transgenic mice is leukemogenic, however, the prolonged latency period, and incomplete penetrance suggests that additional events are necessary to complement CALM-AF10 mediated leukemic transformation. We used retroviral insertional mutagenesis (RIM) to identify complementary genetic events that might collaborate with CALM-AF10 during leukemic transformation. Newborn CALM-AF10 mice were infected with a modified replication competent retrovirus (Mol4070LTR); by 7 months of age, 90% of the transgenic mice developed acute leukemia. Acute leukemia developed more rapidly in CALM-AF10 infected mice than either wild-type mice infected with retrovirus (p=0.004) or CALM-AF10 mice not infected with retrovirus (p=0.037). Ligation-mediated PCR of DNA isolated from leukemic spleens identified potential collaborating genes near the retroviral insertion sites. 262 unique integrations were identified in 40 CALM-AF10 mice. Of these, 55 integrations occurred at 20 common insertion sites, including Zeb2, Mn1, Evi1, Nf1, Ift57, Mpl, Kras, Vav1, and Gata1. Of the candidate genes identified, Zeb2 and Nf1 are of particular interest. Zeb2 encodes the transcriptional co-repressor Smad-interacting protein 1, which is a member of the TGF beta signaling pathway. Previous studies using RIM identified Zeb2 insertions only sporadically, never more than once in a single study, and in association with B-cell, T-cell, and myeloid leukemia. In this study retroviral integrations near Zeb2 were identified either by PCR or Southern blot analysis in 26% (11/42) of the CALM-AF10 mice. RIM studies often generate oligoclonal leukemias, with two or more independent leukemias arising in the same mouse. Southern blot analysis demonstrated that the Zeb2 integration was present in the dominant leukemic clone in most of these mice, and the Zeb2 transcript was overexpressed compared to wild-type spleen and bone marrow. Most leukemias that arise in CALM-AF10 mice without retroviral insertions are myeloid (17/20). However, almost all (10/11) of the CALM-AF10 mice with Zeb2 insertions developed B-lineage ALL suggesting that expression of Zeb2 strongly influences the phenotype of CALM-AF10 leukemias. The tumor suppressor gene Nf1 (Neurofibromatosis type 1) functions as a GTPase activating protein that regulates the activity of Ras proteins involved in cellular proliferation, and acts as a tumor suppressor gene in immature myeloid cells. Retroviral integrations into the Nf1 locus were identified by PCR or Southern blot in 26% (11/42) of the mice in this study. In at least two cases, Southern blot showed loss of the germline allele. The high frequency of Nf1 integrations identified in the RIM assay suggested that Ras pathway activation complemented the CALM-AF10 transgene. Interestingly, 4 other retroviral integrations were identified near Ras genes; 2 near Kras (one with a Kras activating mutation), and 2 near Rras2. Given the high frequency of Nf1 or Ras insertions in the RIM study, we sequenced Nras and Kras genes from CALM-AF10 mice not infected with retrovirus. Thus far, we have identified 3 activating mutations in 15 mice, demonstrating that spontaneous Ras mutations develop as complementary events. We have shown that retroviral infection accelerates the onset of acute leukemia, and identified genes that potentially collaborate with the CALM-AF10 fusion gene in the leukemic transformation process. Furthermore, by sequencing candidate genes, we have demonstrated that pathways (ie, Ras) identified by RIM can also be activated by spontaneous mutations and potentially collaborate with CALM-AF10 to induce leukemia.