A Screen for Mll-AF9 Cooperating Mutations in Leukemogenesis Using MLV-Based Mutagenesis.

Human translocations involving the MLL gene are associated with a variety of myeloid and lymphoid leukemia. The MLL-AF9 translocation is common in acute myeloid leukemia (AML). C57BL/6 mice with a Mll-AF9 knock-in mimic the phenotype observed in human patients with this translocation: 75% develop AML but only after a preleukemic phase and prolonged latency. A minority of these mice develop acute lymphoblastic leukemia (ALL) (Dobson, et al. EMBO J. 1999, 18(13): 3564). This model provides a system for understanding the evolution of AML initiated by a MLL fusion oncoprotein, including the identification of cooperating genetic events required for AML induction. The recombinant retrovirus MOL4070LTR (M4070) induces AML in some strains of mice (Wolff, et al. J Virol. 2003, 77(8): 4965). We hypothesized that this virus could cooperate with the MLL-AF9 oncoprotein to accelerate AML by acting as an insertional mutagen, providing second “hits” in leukemia progression. We bred Mll-AF9 heterozygous C57BL/6 males to 129/SvJ wild type (WT) females, and injected the 280 offspring at 3 days of age with either M4070 virus (212) or a control supernatant (68). All mice were genotyped and observed for disease progression. Infected Mll-AF9 /+ mice succumb to disease with a significantly reduced latency period when compared to virus treated WT mice (p < .0001) and uninfected Mll-AF9 /+ mice (p < .0001), indicating that M4070 infection causes significant leukemia acceleration in these mice. The gross pathology and preliminary analysis of the surface immunophentoype by flow cytometry and Southern blot indicate infected Mll-AF9 /+ animals present with primarily myeloid leukemia while infected WT animals present with lymphoid leukemia. To determine what genes, when mutated by M4070 insertion, can cooperate with Mll-AF9 , retroviral insertion sites have been cloned from over 140 diseased tissues from our accelerated leukemia animals using a shotgun-based, linker-mediated, cloning protocol. More than 1,000 independent insertions have been isolated and are being analyzed to identify common insertion sites (CIS). So far, we have found several CIS enriched in Mll-AF9 /+ AML accelerated by M4070 insertion. We plan to verify nearby genes for cooperation with MLL-AF9 in AML development using human microarray data and mouse modeling.