IntroductionSeveral decades ago, variability of leukemias among patients was recognized by morphologic differences and recurrent chromosomal aberrations. In the last decade, a multitude of somatic mutations has been identified in acute myeloid leukemia (AML) patients without cytogenetic aberrations, such as mutations in NPM1, 1 FLT3, 2,3 C/EBP␣, 4 or MLL. 5 Many of these mutations are associated with a distinct prognosis, thus underscoring the heterogeneity of individual leukemias. Heterogeneity has been found not only between but also within individual leukemias. Moreover, in human AML and murine models only a subset of leukemic cells has shown the ability to engraft recipient mice and confer leukemia. 6-9 These findings have provided support for the cancer stem cell (CSC) model, in which tumor growth relies on a subset of cancer cells at the apex of a cellular hierarchy, and in which heterogeneity within the tumor is ascribed to differentiation from the cancer stem cell. 10 Enhanced self-renewal is the hallmark of CSCs and novel treatment approaches aim to specifically target the aberrant self-renewal machinery in CSCs. 11 Whether the heterogeneity among leukemias is represented at the stem cell level has not been systematically investigated. However, understanding the mechanisms underlying aberrant self-renewal in individual leukemias will be of paramount importance to developing leukemia stem cell-specific therapies. To functionally measure leukemia stem cell potential, the operational assay of leukemia-initiating cells (LICs) has been developed. Cells that can initiate the leukemia upon transplantation are denoted LICs, and can be quantified by limiting dilution transplantation. As an approach to modeling leukemias with differing leukemia stem cell properties, we analyzed leukemias engineered by the potent oncogene MN1 alone or in combination with the leukemogenic oncogenes HOXA9 or NUP98-HOXD13. These combinations were chosen based on evidence that these oncogenes may impact distinct pathways in AML patients: we hypothesized that coexpression of MN1 and a HOX gene may recapitulate characteristics of poor risk AML, which presumably is driven by the most aggressive leukemia stem cells, and in which multiple pathways are activated. 12 NPM1-mutated AML, accounting for 50% of patients with cytogenetically normal AML, is characterized by up-regulation of a HOX gene signature and the HOX cofactor MEIS1, whereas NPM1-wild-type AML is characterized by up-regulation of genes such as CD34, MN1, or BAALC that are also expressed in normal hematopoietic stem and progenitor cells. [13][14][15][16] Several HOX genes and MN1 induce myeloid malignancies in mice that represent these NPM1-mutated AML and NPM1-wild-type AML, respectively.The MN1 gene was first identified as a fusion partner of TEL, an ETS transcription factor, in patients with myeloid leukemia or myelodysplastic syndrome containing the translocation t(12; 22)(p13;q11). 17 More recent studies show that MN1 is a potent oncogene producing myeloid malignancy alone...