NUP98 gene rearrangements occur in acute myeloid leukemia and result in the expression of fusion proteins. One of the most frequent is NUP98-DDX10 that fuses a portion of NUP98 to a portion of DDX10, a putative DEAD-box RNA helicase. Here we show that NUP98-DDX10 dramatically increases proliferation and self-renewal of primary human CD34+ cells, and disrupts their erythroid and myeloid differentiation. It localizes to their nuclei and extensively deregulates gene expression. Comparison to another leukemogenic NUP98 fusion, NUP98-HOXA9, reveals a number of genes deregulated by both oncoproteins, including HOX genes, COX-2, MYCN, ANGPT1, REN, HEY1, SOX4, and others. These genes may account for the similar leukemogenic properties of NUP98 fusion oncogenes. The YIHRAGRTAR sequence in the DDX10 portion of NUP98-DDX10 represents a major motif shared by DEAD-box RNA helicases that is required for ATP binding, RNA-binding, and helicase functions. Mutating this motif diminished the in vitro transforming ability of NUP98-DDX10, indicating that it plays a role in leukemogenesis. These data demonstrate for the first time the in vitro transforming ability of NUP98-DDX10 and show that it is partially dependent on one of the consensus helicase motifs of DDX10. They also point to common pathways that may underlie leukemogenesis by different NUP98 fusions.

NUP98-HOXA9 is the prototype of a group of oncoproteins associated with acute myeloid leukemia. It consists of an N-terminal portion of NUP98 fused to the homeodomain of HOXA9 and is believed to act as an aberrant transcription factor that binds DNA through the homeodomain. Here we show that NUP98-HOXA9 can regulate transcription without binding to DNA. In order to determine the relative contributions of the NUP98 and HOXA9 portions to the transforming ability of NUP98-HOXA9, the effects of NUP98-HOXA9 on primary human CD34+ cells were dissected and compared to those of wild-type HOXA9. In contrast to previous findings in mouse cells, HOXA9 had only mild effects on the differentiation and proliferation of primary human hematopoietic cells. The ability of NUP98-HOXA9 to disrupt the differentiation of primary human CD34+ cells was found to depend primarily on the NUP98 portion, whereas induction of long-term proliferation required both the NUP98 moiety and an intact homeodomain. Using oligonucleotide microarrays in primary human CD34+ cells, a group of genes was identified whose dysregulation by NUP98-HOXA9 is attributable primarily to the NUP98 portion. These include RAP1A, HEY1, and PTGS2 (COX-2). Their functions may reflect the contribution of the NUP98 moiety of NUP98-HOXA9 to leukemic transformation. Taken together, these results suggest that the effects of NUP98-HOXA9 on gene transcription and cell transformation are mediated by at least two distinct mechanisms: one that involves promoter binding through the homeodomain with direct transcriptional activation, and another that depends predominantly on the NUP98 moiety and does not involve direct DNA binding.

Different fusion oncogenes in acute myeloid leukemia (AML) have distinct clinical and laboratory features suggesting different modes of malignant transformation. Here we compare the in vitro effects of representatives of 4 major groups of AML fusion oncogenes on primary human CD34+ cells. As expected from their clinical similarities, MLL-AF9 and NUP98-HOXA9 had very similar effects in vitro. They both caused erythroid hyperplasia and a clear block in erythroid and myeloid maturation. On the other hand, AML1-ETO and PML-RARA had only modest effects on myeloid and erythroid differentiation. All oncogenes except PML-RARA caused a dramatic increase in long-term proliferation and self-renewal. Gene expression profiling revealed two distinct temporal patterns of gene deregulation. Gene deregulation by MLL-AF9 and NUP98-HOXA9 peaked 3 days after transduction. In contrast, the vast majority of gene deregulation by AML1-ETO and PML-RARA occurred within 6 hours, followed by a dramatic drop in the numbers of deregulated genes. Interestingly, the p53 inhibitor MDM2 was upregulated by AML1-ETO at 6 hours. Nutlin-3, an inhibitor of the interaction between MDM2 and p53, specifically inhibited the proliferation and self-renewal of primary human CD34+ cells transduced with AML1-ETO, suggesting that MDM2 upregulation plays a role in cell transformation by AML1-ETO. These data show that differences among AML fusion oncogenes can be recapitulated in vitro using primary human CD34+ cells and that early gene expression profiling in these cells can reveal potential drug targets in AML.

Many cases of acute myeloid leukemia (AML) are associated with non-random chromosomal rearrangements and most of these result in fusions involving retinoic acid receptor α (RARα), CBF transcription factors, MLL, and nucleoporins. Here, we report the effects of key members of these four major groups of AML-associated chimeric fusion proteins on differentiation, proliferation, self-renewal and gene expression in primary human CD34+ hematopoietic cells. We expressed the PML-RARα, AML1-ETO, MLL-AF9 and NUP98-HOXA9 fusion genes in human peripheral blood CD34+ cells by retroviral transduction and compared them to cells transduced with empty vector. By colony forming assays, morphological examination and flow cytometric immunophenotyping we found that PML-RARα causes some degree of myeloid differentiation block. AML1-ETO had no obvious effect on differentiation, while MLL-AF9 and NUP98-HOXA9 caused a block in both myeloid and erythroid differentiation. All fusion oncoproteins, except PML-RARα, induced dramatic long-term proliferation in liquid culture and a marked increase in the numbers of primitive long-term culture-initiating cells (LTC-ICs). In order to understand the molecular basis of these effects, gene expression profiling was performed for each fusion gene by oligonucleotide microarray analysis at 3 different time points (6 h, 3 days, and 8 days post transduction). At the 6 h time point both AML1-ETO and PML-RARα caused extensive changes in gene expression with a predominance of downregulated genes. The number of genes dysregulated by AML1-ETO and PML-RARα decreased dramatically at the 3-day time point. These data are consistent with the fact that these two oncoproteins are DNA-binding transcription factors that are known to repress transcription. In contrast, MLL-AF9 and NUP98-HOXA9 altered the expression of fewer genes at the 6 h time point with a preponderance of upregulated genes; at the 3 day time point, the number of genes dysregulated by NUP98-HOXA9 and MLL-AF9 increased. These findings suggest that NUP98-HOXA9 and MLL-AF9 may have delayed effects that are not due to direct transcriptional regulation. Homeobox transcription factors were upregulated by both MLL-AF9 and NUP98-HOXA9, but not by either AML1-ETO or PML-RARα. The results of the biological assays and gene profiling show marked similarities between NUP98-HOXA9 and MLL-AF9, and suggest that they transform cells by similar pathways that are different from those used by AML1-ETO and PML-RARα.

2966 Poster Board II-942 NUP98 gene rearrangements occur in acute myeloid leukemia and other hematopoietic malignancies, and result in the expression of fusion proteins. One of the most frequent NUP98 fusions is NUP98-DDX10 that consists of an N-terminal portion of NUP98 and a C-terminal segment of DDX10, a putative DEAD-box RNA helicase. Here we express NUP98-DDX10 in primary human CD34+ hematopoietic cells and show that it localizes within the nucleus in a punctate distribution. It dramatically increases the proliferation and self-renewal of primary human CD34+ cells and disrupts their erythroid and myeloid differentiation. Expression gene profiling shows dysregulation of many genes by NUP98-DDX10 in primary human CD34+ cells starting within 6 h. Comparison of the dysregulome of NUP98-DDX10 to that of another leukemogenic NUP98 fusion, NUP98-HOXA9, reveals a number of genes dysregulated by both oncoproteins, including HOX genes, COX-2, MYCN, angiopoietin-1, renin, HEY1, SOX4, and others, that may account for the induction of AML by these and other NUP98 fusion oncogenes. The HRAGRTAR sequence in the DDX10 portion of NUP98-DDX10 corresponds to a major motif shared by DEAD-box RNA helicases that is required for their ATP binding/hydrolysis, RNA-binding, and helicase functions. Mutating this motif diminished the transforming ability of NUP98-DDX10, indicating that it plays a role in leukemogenesis. These data demonstrate for the first time the transforming ability of NUP98-DDX10 and show that it is partially dependent on one of the consensus helicase motifs of DDX10. They also point to common pathways that may underlie leukemogenesis by different NUP98 fusions. Disclosures: No relevant conflicts of interest to declare.