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.

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...

The 2 most frequent human MLL hematopoietic malignancies involve either AF4 or AF9 as fusion partners; each has distinct biology but the role of the fusion partner is not clear. We produced Mll-AF4 knock-in (KI) mice by homologous recombination in embryonic stem cells and compared them with Mll-AF9 KI mice. Young Mll-AF4 mice had lymphoid and myeloid deregulation manifest by increased lymphoid and myeloid cells in hematopoietic organs. In vitro, bone marrow cells from young mice formed unique mixed pro-B lymphoid (B220 ؉ CD19 ؉ CD43 ؉ sIgM ؊ , PAX5 ؉ , TdT ؉ , IgH rearranged)/myeloid (CD11b/Mac1 ؉ , c-fms ؉ , lysozyme ؉ ) colonies when grown in IL-7-and Flt3 ligand-containing media. Mixed lymphoid/myeloid hyperplasia and hematologic malignancies (most frequently B-cell lymphomas) developed in Mll-AF4 mice after prolonged latency; long latency to malignancy indicates that Mll-AF4-induced lymphoid/myeloid deregulation alone is insufficient to produce malignancy. In contrast, young Mll-AF9 mice had predominately myeloid deregulation in vivo and in vitro and developed myeloid malignancies. The early onset of distinct mixed lymphoid/myeloid lineage deregulation in Mll-AF4 mice shows evidence for both "instructive" and "noninstructive" roles for AF4 and AF9 as partners in MLL fusion genes. The molecular basis for "instruction" and secondary cooperating mutations can now be studied in our Mll-AF4 model. IntroductionThe myeloid/mixed lymphoid leukemia gene (MLL) on human chromosome 11 was first described from a cell line derived from a patient with a hematologic malignancy that resulted from a reciprocal translocation involving chromosome 4. 1 MLL was subsequently shown to partner with many other genes to result in hematologic malignancy. 2 The fusion of MLL to AF4 family members, LAF4 and AF5, results in malignancies that are the most common and unique among the MLL fusion gene malignancies in that they are generally lymphoid or lymphoid/myeloid in type but rarely purely myeloid. They are also unique because of the high frequency in infants, extensive spread beyond the hematopoietic compartment, and a poor outcome with treatment. [3][4][5][6][7][8][9][10] In contrast to MLL-AF4, the fusion of MLL with most other partners, including the second most common partner AF9, 8 results in myeloid malignancies.To date, no murine model of MLL-AF4 translocation has been reported and thus neither the premalignant early events nor the eventual malignancies have been defined. In this study, we produced Mll-AF4 knock-in (KI) mice and compare them with Mll-AF9 mice developed previously. 11 The KI models, which have the advantage of having a single copy of the fusion gene in all stem/progenitor cells, permit control of bias introduced by a variable number of gene copies in the various progenitor populations in other models.The mechanisms for the association between the MLL partner gene and type of malignancy have not been elucidated. The MLL partner may be "instructive" in directing the selective expansion and transformation of cells t...

IntroductionLeukemias with MLL gene rearrangements occur most frequently in infants and as secondary malignancies and are associated with poor outcomes. Several studies have demonstrated that both human and murine MLL-rearranged leukemias have high expression of HOXA9 and MEIS1. 1-5 Recently, we found that the expression of 5Ј Hox-a genes and Meis1 in murine hematopoietic cells is proportional to the level of transformation, suggesting that the MLL-fusion gene induced overexpression of these genes is central to the pathogenesis of leukemia. 6 Wong et al have recently shown that retrovirally expressed MLL-fusion genes are incapable of transforming Meis1 Ϫ/Ϫ murine fetal liver cells. 7 In the current study, we report on Meis1 inhibition in murine Mll-AF9 knockin leukemia. The knockin model closely mimics the human disease because each cell contains a single copy of the gene, expressed from the endogenous promoter and the mice develop myeloid leukemia. 8 A cell line, derived from a leukemic Mll-AF9 knockin mouse with high Meis1 expression, was used in the current study. 9 We found that Meis1 is required for the growth and survival of Mll-AF9 leukemia cells in vitro and for growth of leukemia in vivo. Gene profiling data suggested mechanisms by which Meis1 might mediate these growth-and survival-promoting effects in this cell line. Finally, we show that human MLL-fusion gene leukemia cell lines also require MEIS1 for growth. Methods Cell cultureThe 4166 cell line was established from a leukemic Mll-AF9 knockin mouse. The human cell lines were obtained from ATCC (Manassas, VA). 10 Lentivirus shRNAsLentivirus short hairpin RNA (shRNA) clones were obtained from OpenBiosystems (Huntsville, AL); details are provided in the "Lentivirus" section in Document S1 (available on the Blood website; see the Supplemental Materials link at the top of the online article). For ease of description, these clones were designated M23, M24, M25, M26, and M27. The manually designed construct was labeled M1456. Cell-cycle and apoptosis analysis by flow cytometryThe 4166 cells were transduced with Meis1 shRNA at multiplicities of infection (MOI) of 10 to 100, and the cells were harvested at days 2 through 5 after transduction. Nuclei were stained with propidium iodide (PI), and analysis of nuclear DNA content was performed using the CellQuest-Pro software (BD Biosciences, San Jose, CA). Apoptosis was detected using the CaspaTag caspase activity kit (Millipore, Billerica, MA) as described previously. 11 Western blotting and immunohistochemistryWestern blotting was performed using anti-Meis1 antibody (Upstate Biotechnology, Charlottesville, VA) with antiactin (Sigma-Aldrich, St Louis, MO) used as a loading control. Cell-surface marker expression was determined by immunohistochemistry as previously described. 9 Myeloid colony-forming assayCells were cultured in methylcellulose medium under myeloid conditions, and colonies were counted and scored at 7 days as previously described. 9 TransplantationsFor in vivo experiments, 4166 cells were transd...

The proto-oncogene EVI1 (ecotropic viral integration site-1), located on chromosome band 3q26, is aberrantly expressed in human acute myeloid leukemia (AML) with 3q26 rearrangements.

While it is known that mice with genetic immune defects are useful for establishing durable engraftment of human tumor xenografts, the relative role of components of host innate and adoptive immunity in engraftment has not been determined. We directly compared the ability of four strains of genetically immunodeficient mice (NOD/SCID, SCID, Nude and Rag-1-deficient) to successfully engraft and support the human cell lines Daudi, Raji, Namalwa and Molt-4 as subcutaneous tumors. We additionally examined the effect of further immunosuppression of the mice by whole body irradiation at a dose of 600 cGy for Nude and Rag-1 and 300 cGy for SCID mice and by administration of anti-natural killer (asialo-GM1) antibody on tumor growth. Mice with each of the defects supported xenografts to varying degrees. We found differences in growth characteristics in the cell lines tested, with Namalwa consistently producing the largest tumors. With all cell lines studied, optimal growth was achieved using NOD/SCID mice. Overall, tumor growth was somewhat enhanced by pretreatment with radiation with little additional benefit from the addition of anti-asialo-GM1 antibody. The importance of multiple components of the innate and adoptive immune system in xenotransplantation were best demonstrated when results in untreated NOD/SCID mice were compared to SCID, nude and RAG-1-deficient mice. The NOD/SCID mouse with or without additional immunosuppression provides the optimal model for the study of the biology and treatment of human leukemias and lymphomas.

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Hepatic steatosis is a strong risk factor for the development of hepatocellular carcinoma (HCC), yet little is known about the molecular pathology associated with this factor. In this study, we performed a forward genetic screen using Sleeping Beauty (SB) transposon insertional mutagenesis in mice treated to induce hepatic steatosis, and compared the results to human HCC data. In humans, we determined that steatosis increased the proportion of female HCC patients, a pattern also reflected in mice. Our genetic screen identified 203 candidate steatosis-associated HCC genes, many of which are altered in human HCC and are members of established HCC-driving signaling pathways. The protein kinase A/cyclic AMP signaling pathway was altered frequently in mouse and human steatosis-associated HCC. We found that activated PKA expression drove steatosis-specific liver tumorigenesis in a mouse model. Another candidate HCC driver, the N-acetyltransferase NAT10, which we found to be overexpressed in human steatosis-associated HCC and associated with decreased survival in human HCC, also drove liver tumorigenesis in a steatotic mouse model. This study identifies genes and pathways promoting HCC that may represent novel targets for prevention and treatment in the context of hepatic steatosis, an area of rapidly growing clinical significance.