Mll partial tandem duplication and Flt3 internal tandem duplication in a double knock-in mouse recapitulates features of counterpart human acute myeloid leukemias

Abstract

The MLL-partial tandem duplication (PTD) associates with high-risk cytogenetically normal acute myeloid leukemia (AML). Concurrent presence of FLT3-internal tandem duplication (ITD) is observed in 25% of patients with MLL-PTD AML. However, mice expressing either Mll-PTD or Flt3-ITD do not develop AML, suggesting that 2 mutations are necessary for the AML phenotype. Thus, we generated a mouse expressing both Mll-PTD and IntroductionAcute myeloid leukemia (AML) is a genetically heterogeneous disease. Recurrent cytogenetic and molecular gene aberrations have been used to classify AML patients into distinct subsets that differ in biologic, clinical, and prognostic characteristics. The MLL gene, located at chromosome band 11q23, encodes for a protein involved in epigenetic regulation of gene expression. 1 In AML, this gene is frequently involved in chromosome translocations at 11q23 and, at the molecular level, is fused with one of more than 50 different partners. 2 We first reported an internal duplication of MLL, an in-frame repeat producing an elongated protein retaining all functional domains, in cytogenetically normal (CN)-AML. 3 Approximately 5% to 7% of CN-AML patients have a MLL-partial tandem duplication (PTD) mutation, which is associated with unfavorable prognosis, 3-5 but if and how it contributes to myeloid leukemogenesis remain to be elucidated. Approximately 25% of CN-AML patients with the MLL-PTD had constitutive activation of FLT3, a tyrosine kinase receptor regulating proliferation and survival of hematopoietic cells, via an internal tandem duplication (FLT3-ITD), and have a very poor prognosis. 6 This suggests that both MLL-PTD and FLT3-ITD mutations are necessary for an AML phenotype, as supported by the broadly accepted 2-hit model. 7 Indeed, a Mll-PTD mouse, created by knocking in exons 5 to 11 in-frame and driven off of the endogenous Mll promoter, did not develop AML. 8 8,9 This model, which develops AML, is the first that requires the MLL-PTD. In this model, the 2 mutated genes are regulated by their respective endogenous promoters to recapitulate the Mll PTD/WT :Flt3 ITD/WT AML found in humans. This differs from some other doublemutant mouse models of human AML that carry one mutation driven by the endogenous promoter and the other mutation driven by transgenes, 10 or introduced via viral transduction, 11 or 2 mutations driven off 2 endogenous promoters but requiring BM transplantation. 12 Methods Mouse strainsThe Mll PTD/WT , Flt3 ITD/WT , and Flt3 ITD/ITD mice were generated and genotyped as previously described. 8,9 Male Flt3 ITD/WT Balb/c mice were backcrossed onto the C57Bl/6J strain to purity and then bred with Mll PTD/WT mice to generate Mll PTD/WT :Flt3 ITD/WT double knock-in offspring. Genotyping was performed as previously described. 8,9 All animals studied were compared within litters and/or age-and sex-matched. All experiments were conducted under an approved The Ohio State University Institutional Submitted March 2, 2012; accepted May 17, 2012. Prepublished online as Blood Firs...

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