To explore the possibility that deregulated HOX gene expression might commonly occur during leukemic hematopoiesis, we compared the relative levels of expression of these and related genes in phenotypically and functionally defined subpopulations of AML blasts and normal hematopoietic cells. Initially, a semi-quantitative RT-PCR technique was used to amplify total cDNA from total leukemic blast cell populations from 20 AML patients and light density cells from four normal bone marrows. Expression of HOX genes (A9, A10, B3 and B4), MEIS1 and MLL was easily detected in the majority of AML samples with the exception of two samples from patients with AML subtype M3 (which expressed only MLL). Low levels of HOXA9 and A10 but not B3 or B4 were seen in normal marrow while MLL was easily detected. PBX1a was difficult to detect in any AML sample but was seen in three of four normal marrows. Cells from nine AML patients and five normal bone marrows were FACS-sorted into CD34 ؉ CD38 ؊ , CD34 ؉ CD38 ؉ and CD34 ؊ subpopulations, analyzed for their functional properties in long-term culture (LTC) and colony assays, and for gene expression using RT-PCR. 93 ؎ 14% of AML LTC-initiating cells, 92 ± 14% AML colony-forming cells, and Ͼ99% of normal LTC-IC and CFC were CD34 ؉ . The relative level of expression of the four HOX genes in amplified cDNA from CD34 ؊ as compared to CD34 ؉ CD38 ؊ normal cells was reduced Ͼ10-fold. However, in AML samples this down-regulation in HOX expression in CD34 ؊ as compared to CD34 ؉ CD38 ؊ cells was not seen (P Ͻ 0.05 for comparison between AML and normal). A similar difference between normal and AML subpopulations was seen when the relative levels of expression of MEIS1, and to a lesser extent MLL, were compared in CD34 ؉ and CD34 ؊ cells (P Ͻ 0.05). In contrast, while some evidence of down-regulation of PBX1a was found in comparing CD34 ؊ to CD34 ؉ normal cells it was difficult to detect expression of this gene in any subpopulation from most AML samples. Thus, the down-regulation of HOX, MEIS1 and to some extent MLL which occurs with normal hematopoietic differentiation is not seen in AML cells with similar functional and phenotypic properties.

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N-MYC encodes a basic helix-loop-helix/leucine zipper (bHLH/ LZ) transcription factor that is frequently overexpressed in human neuroblastoma. N-MYC overexpression has also been reported in human acute myeloid leukemias (AML), which we show here is a frequent event. Myeloid cells in N-Mycoverexpressing mouse bone marrow hyperproliferate but those in c-MYC-overexpressing bone marrow do not. The NH 2 -terminal transactivation domain, nuclear localization signal, and bHLH/LZ domain of N-Myc are essential for this effect. Microarray analysis revealed 969 differentially expressed genes between N-Myc-and c-MYC-overexpressing myeloid cells. N-Myc-overexpressing cells showed decreased transforming growth factor B signaling and increased c-Jun-NH 2 -kinase signaling, both of which are associated with proliferation and leukemic transformation of myeloid cells. Mice transplanted with bone marrow expressing wild-type N-Myc developed clonal and transplantable AML after f1 month; those transplanted with bone marrow expressing mutant N-Myc did not. Twist, a known suppressor of the p19Arf/p53 pathway, was up-regulated in all tumors. These results show that N-Myc overexpression is highly oncogenic in mouse myeloid cells and suggest that N-MYC up-regulation contributes to human myeloid leukemogenesis. [Cancer Res 2007;67(22):10677-85]

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