The pathways by which oncogenes, such as MLL-AF9, initiate transformation and leukemia in humans and mice are incompletely defined. In a study of target cells and oncogene dosage, we found that Mll-AF9, when under endogenous regulatory control, efficiently transformed LSK (Lin(-)Sca1(+)c-kit(+)) stem cells, while committed granulocyte-monocyte progenitors (GMPs) were transformation resistant and did not cause leukemia. Mll-AF9 was expressed at higher levels in hematopoietic stem (HSC) than GMP cells. Mll-AF9 gene dosage effects were directly shown in experiments where GMPs were efficiently transformed by the high dosage of Mll-AF9 resulting from retroviral transduction. Mll-AF9 upregulated expression of 192 genes in both LSK and progenitor cells, but to higher levels in LSKs than in committed myeloid progenitors.

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Identification of the targets of mixed lineage leukemia (MLL) fusion genes will assist in understanding the biology of MLL fusion gene leukemias and in development of better therapies. Numerous studies have implicated HOXA9 as one of the possible targets of MLL fusion proteins. To determine if HOXA9 was required for leukemia development by MLL fusion genes, we compared the effects of the Mll-AF9 knock-in mutation in mice in the presence or absence of Hoxa9. Both groups of mice showed myeloid expansion at 8 weeks and then developed myeloid leukemia with a similar incidence and time course. The leukemia in the mice lacking Hoxa9 generally displayed a more immature myeloid phenotype than that in the mice that were wild-type for Hoxa9. Gene expression profiling revealed that expression of Mll-AF9 led to overexpression of Hoxa5, Hoxa6, Hoxa7, Hoxa9, and Hoxa10. Thus, genes of the Hox-a cluster are important in defining the phenotype but not the incidence of Mll-AF9 leukemia. These results demonstrate that the Mll-AF9 fusion gene disrupts the expression of several Hox genes, none of which as a single gene is likely to be necessary for development of leukemia. Instead, we propose that the "Hox code" minimally defined by the Hoxa5-a9 cluster is central to MLL leukemogenesis. ( IntroductionTranslocations involving the mixed lineage leukemia (MLL, ALL-1, HRX) gene are encountered in both myeloid and lymphoid leukemias. These MLL leukemias are often found in infants and also in adults previously treated with chemotherapy for other cancers. 1 The mechanisms by which the translocations cause leukemia remain unknown. Gene expression profile studies demonstrate that lymphoid leukemias with MLL rearrangements exhibit an increase in expression of certain homeobox (HOX) genes compared with phenotype-matched leukemias without MLL rearrangements. [2][3][4][5] The HOXA9 gene may hold an important key to the MLL leukemias because it is the one homeobox gene most frequently overexpressed in these leukemias. 5 Recent evidence also indicates that MLL is part of a multiprotein complex that regulates the transcription of HOXA9 by directly binding to promoter sequences. 6,7 Overexpression of Hoxa9 is also known to transform primary myeloid bone marrow cells. [8][9][10] HOXA9 is directly involved in human leukemia caused by the NUP98-HOXA9 fusion gene 11 and in the BXH-2 mouse model of leukemia. 12 This encouraged us to study the relationship between Hoxa9 and Mll fusion genes and to ask whether Hoxa9 is necessary for the development of Mll leukemia. We were able to test this hypothesis in Mll-AF9 ϩ/Ϫ /Hoxa9 Ϫ/Ϫ mice. Mice expressing Mll-AF9 as a heterozygous knock-in mutation develop myeloid leukemia. 13 The leukemia in these mice occurs with a latency period of about 6 months and is preceded by a preleukemic phase characterized by expansion of myeloid precursors. 14, 15 We compared the Mll-AF9-mediated myeloid expansion and leukemia development in the presence and absence of Hoxa9.To identify other Hox genes that may be involved in leuke...

Complete and robust human genome duplication requires loading minichromosome maintenance (MCM) helicase complexes at many DNA replication origins, an essential process termed origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single-cell analyses, we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation toward all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.