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...

SUMMARY:Previously we showed reduced protein and mRNA expression of the SHP1 gene in lymphoma/leukemia cell lines and patient specimens by Northern blot, RT-PCR, Western blot, and immunohistochemical analyses. In this study, aberrant methylation in the SHP1 gene promoter was detected in many B-cell leukemia/lymphoma cell lines as well as in patient specimens, including diffuse large B-cell lymphoma (methylation frequency 93%), MALT lymphoma (82%), mantle cell lymphoma (75%), plasmacytoma (100%) and follicular lymphoma (96%) by methylation-specific PCR, bisulfite sequencing, and restriction enzyme-mediated PCR analyses. The methylation frequency was significantly higher in high-grade MALT lymphoma cases (100%) than in low-grade MALT lymphoma cases (70%), which correlated well with the frequency of no expression of SHP1 protein in high-grade (80%) and low-grade MALT lymphoma (54%). It suggests that the SHP1 gene silencing with aberrant CpG methylation relates to the lymphoma progression. SHP1 protein expression was recovered in B-cell lines after the treatment of the demethylating reagent: 5-aza-2'-deoxycytidine. Transfection of the intact SHP1 gene to the hematopoietic cultured cells, which show no expression of the SHP1 gene, induced growth inhibition, indicating that gene silencing of the SHP1 gene by aberrant methylation plays an important role to get the growth advantage of the malignant lymphoma/leukemia cells. The extraordinarily high frequency (75 to 100%) of CpG methylation of the SHP1 gene in B-cell lymphoma/leukemia patient specimens indicates that the SHP1 gene silencing is one of the critical events to the onset of malignant lymphomas/leukemias as well as important implications for the diagnostic or prognostic markers and the target of gene therapy. These data support the possibility that the SHP1 gene is one of the tumor suppressor genes. (Lab Invest 2003, 83:1849 -1858.

17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of the molecular chaperone heat shock protein 90, results in cell type-specific inhibition of proliferation of leukemic cells. GTP14564 is a tyrosine kinase inhibitor actively against FLT3. The current study evaluated the single and combined effects of 17-AAG and GTP14564, and the role of FLT3 in their inhibitory effects. The importance of FLT3 mutations was demonstrated using small interfering RNA (siRNA) targeted to FLT3. Similar to FLT3 siRNA, GTP14564 inhibited FLT3 internal tandem duplication (ITD) cells (MV4;11) and FLT3 amplified wild-type cells (SEMK2-M1), but not wild-type FLT3 cells (RS4;11). However, when RS4;11 cells were stimulated with FLT3-ligand, phosphorylation of STAT5 and GTP14564 inhibition were observed. Responses to GTP14564 in all cell types were directly related to the level of STAT5 phosphorylation in the cells. We observed synergistic effects of combined 17-AAG and GTP14564 in cell lines with FLT3-ITD and amplified wild-type FLT3. Combined treatment with 17-AAG and GTP14564 reduced the levels of p-FLT3 and p-STAT5, enhanced G0/G1 arrest and apoptosis in FLT3-ITD and amplified wild-type FLT3. The combination of 17-AAG with FLT3 kinase inhibitors can enhance targeted therapy in leukemias with FLT3 mutations, such as MLL fusion gene leukemias.

This study suggests that late relapse lymphoma may present as a new clone. Sequencing of the PCR products is important in the evaluation of clonal heterogeneity.