Structural chromosomal rearrangements of the Nucleoporin 98 gene (NUP98), primarily balanced translocations and inversions, are associated with a wide array of hematopoietic malignancies. NUP98 is known to be fused to at least 28 different partner genes in patients with hematopoietic malignancies, including acute myeloid leukemia, chronic myeloid leukemia in blast crisis, myelodysplastic syndrome, acute lymphoblastic leukemia, and bilineage/biphenotypic leukemia. NUP98 gene fusions typically encode a fusion protein that retains the amino terminus of NUP98; in this context, it is important to note that several recent studies have demonstrated that the amino-terminal portion of NUP98 exhibits transcription activation potential. Approximately half of the NUP98 fusion partners encode homeodomain proteins, and at least 5 NUP98 fusions involve known histone-modifying genes. Several of the NUP98 fusions, including NUP98-homeobox (HOX)A9, NUP98-HOXD13, and NUP98-JARID1A, have been used to generate animal models of both lymphoid and myeloid malignancy; these models typically up-regulate HOXA cluster genes, including HOXA5, HOXA7, HOXA9, and HOXA10. In addition, several of the NUP98 fusion proteins have been shown to inhibit differentiation of hematopoietic precursors and to increase self-renewal of hematopoietic stem or progenitor cells, providing a potential mechanism for malignant transformation. (Blood. 2011;118(24): 6247-6257) IntroductionOne of the oldest, and most useful, whole genome screens for genes involved in malignant transformation is a simple karyotype of the malignant cell. 1 Analysis of recurrent, nonrandom chromosomal translocation breakpoints has identified numerous genes important for malignant transformation and provided critical insight into the biology, classification, and prognosis of hematopoietic malignancies. 2 The study of these genes (such as BCR-ABL and BCL2) has led to vastly improved therapy 3 and has opened an entire field of scientific inquiry. 4 The Nucleoporin 98 gene (NUP98) was originally identified as a structural component of the nuclear pore complex (NPC), 5 and was subsequently shown to be a fusion partner with homeobox (HOX)A9 in acute myeloid leukemia (AML) patients with a t(7;11) (p15;p15). 6,7 Twenty-eight distinct NUP98 gene fusions have been identified, caused primarily by balanced translocations and inversions, in the malignant cells of patients with a wide array of distinct hematopoietic malignancies, including AML, chronic myeloid leukemia in blast crisis (CML-bc), myelodysplastic syndrome (MDS), acute lymphoblastic leukemia (ALL), and bilineage/ biphenotypic leukemia. 8 In this overview, we present a summary of the known roles of NUP98 in normal cell physiology, the association of NUP98 fusion proteins with hematopoietic malignancies, the incidence and prognostic importance of these fusions, and the mechanisms by which NUP98 fusion oncoproteins contribute to the process of malignant transformation. Normal functions of NUP98NUP98 is a component of the NPC NUP98 is an ϳ 90...

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Chromosomal aberrations occur with great frequency and some specificity in leukemia and other hematologic malignancies. The most common outcome of these rearrangements is the formation of a fusion gene, comprising portions of 2 genes normally present in the cell. These fusion proteins are presumed to be oncogenic; in many cases, animal models have proven them to be oncogenic. One of the most promiscuous fusion partner genes is the newly identified NUP98 gene, located on chromosome 11p15.5, which to date has been observed fused to 15 different fusion partners. NUP98 encodes a 98 kD protein that is an important component of the nuclear pore complex, which mediates nucleo-cytoplasmic transport of protein and RNA. The fusion partners of NUP98 form 2 distinct groups: homeobox genes and non-homeobox genes. All NUP98 fusions join the N-terminal GLFG repeats of NUP98 to the C-terminal portion of the partner gene, which, in the case of the homeobox gene partners, includes the homeodomain. Clinical findings are reviewed here, along with the findings of several in vivo and in vitro models have been employed to investigate the mechanisms by which NUP98 fusion genes contribute to the pathogenesis of leukemia.

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The NUP98-HOXD13 (NHD13) fusion gene occurs in patients with myelodysplastic syndrome (MDS) and acute nonlymphocytic leukemia (ANLL). We reported that transgenic mice expressing NHD13 develop MDS, and that more than half of these mice eventually progress to acute leukemia. The latency period suggests a requirement for at least 1 complementary event before leukemic transformation. We conducted a candidate gene search for complementary events focused on genes that are frequently mutated in human myeloid leukemia. We investigated 22 ANLL samples and found a high frequency of Nras and Kras mutations, an absence of Npm1, p53, Runx1, Kit and Flt3 mutations, and a single Cbl mutation. Our findings support a working hypothesis that predicts that ANLL cases have one mutation which inhibits differentiation, and a complementary mutation which enhances proliferation or inhibit apoptosis. In addition, we provide the first evidence for spontaneous collaborating mutations in a IntroductionWe previously reported a transgenic mouse model for myelodysplastic syndrome (MDS), in which NUP98-HOXD13 (NHD13) mice develop MDS at an early age, and progress to acute leukemia between 4 and 14 months of age. 1 This latency period is likely due to a requirement for additional genetic events before leukemic transformation. Many studies have investigated the nature of such secondary events through experimental induction of complementary events, such as retroviral insertional mutagenesis 2-4 or ENUinduced mutagenesis. 5,6 To our knowledge, no study has investigated the nature of complementary mutations that occur spontaneously. Therefore, we evaluated NHD13 ANLL samples for the presence of mutations commonly seen in patients with ANLL. Methods DNA and RNA isolationAll animal experiments were conducted with the approval of the NIH Intramural Animal Care and Use Committee. Peripheral blood complete blood counts were obtained, bone marrow was harvested for cytospins, and paraffin-embedded spleen and liver were stained with hematoxylin and eosin. Routine immunohistochemical stains included F4/80, CD3, B220, and myeloperoxidase (MPO), and ANLL diagnosis was based on the Bethesda proposals for hematopoietic neoplasms in mice. 7,8 Effaced spleen tissue from NHD13 mice with acute leukemia was snap frozen on dry ice. DNA and RNA were prepared by standard techniques. RT-PCR and PCRReverse transcription (RT) was performed using Superscript II (Invitrogen, Carlsbad, CA). Genomic-and RT-polymerase chain reaction (PCR) were performed using either Supermix (Invitrogen) or Taq DNA Polymerase (Invitrogen). Primers, thermal cycling profiles, and regions amplified are listed in Tables S1 and S2 (available on the Blood website; see the Supplemental Materials link at the top of the online article). PCR products were purified using Qiagen (Valencia, CA) protocols, and were directly sequenced (Retrogen, San Diego, CA). Sequence chromatograms were manually inspected to detect mutations ( Figure S1).Reference sequences (NCBI accession numbers 9 ) used were as follows...

Constitutive activation of FLT3 by internal tandem duplication (ITD) is one of the most common molecular alterations in acute myeloid leukemia (AML

The t(2;11)(q31;p15) chromosomal translocation results in a fusion between the NUP98 and HOXD13 genes and has been observed in patients with myelodysplastic syndrome (MDS) or acute myelogenous leukemia. We previously showed that expression of the NUP98-HOXD13 (NHD13) fusion gene in transgenic mice results in an invariably fatal MDS; approximately one third of mice die due to complications of severe pancytopenia, and about two thirds progress to a fatal acute leukemia. In the present study, we used retroviral insertional mutagenesis to identify genes that might collaborate with NHD13 as the MDS transformed to an acute leukemia. Newborn NHD13 transgenic mice and littermate controls were infected with the MOL4070LTR retrovirus. The onset of leukemia was accelerated, suggesting a synergistic effect between the NHD13 transgene and the genes neighboring retroviral insertion events. We identified numerous common insertion sites located near protein-coding genes and confirmed dysregulation of a subset of these by expression analyses. Among these genes were Meis1, a known collaborator of HOX and NUP98-HOX fusion genes, and Mn1, a transcriptional coactivator involved in human leukemia through fusion with the TEL gene. Other putative collaborators included Gata2, Erg, and Epor. Of note, we identified a common insertion site that was >100 kb from the nearest coding gene, but within 20 kb of the miR29a/miR29b1 microRNA locus. Both of these miRNA were up-regulated, demonstrating that retroviral insertional mutagenesis can target miRNA loci as well as protein-coding loci. Our data provide new insights into NHD13-mediated leukemogenesis as well as retroviral insertional mutagenesis mechanisms.

Eph receptor tyrosine kinases control cell-cell interactions during normal and oncogenic development, and are implicated in a range of processes including angiogenesis, stem cell maintenance and metastasis. They are thus of great interest as targets for cancer therapy. EphA3, originally isolated from leukemic and melanoma cells, is presently one of the most promising therapeutic targets, with multiple tumor-promoting roles in a variety of cancer types. This review focuses on EphA3, its functions in controlling cellular behavior, both in normal and pathological development, and most particularly in cancer.