NUP98-HOXA9, the chimeric protein resulting from the t(7;11)(p15;p15) chromosomal translocation, is a prototype of several NUP98 fusions that occur in myelodysplastic syndromes and acute myeloid leukemia. We examined its effect on differentiation, proliferation, and gene expression in primary human CD34 + hematopoietic cells. Colony-forming cell (CFC) assays in semisolid medium combined with morphologic examination and flow cytometric immunophenotyping revealed that NUP98-HOXA9 increased the numbers of erythroid precursors and impaired both myeloid and erythroid differentiation. In continuous liquid culture, cells transduced with NUP98-HOXA9 exhibited a biphasic growth curve with initial growth inhibition followed by enhanced long-term proliferation, suggesting an increase in the numbers of primitive self-renewing cells. This was confirmed by a dramatic increase in the numbers of long-term cultureinitiating cells, the most primitive hematopoietic cells detectable in vitro. To understand the molecular mechanisms underlying the effects of NUP98-HOXA9 on hematopoietic cell proliferation and differentiation, oligonucleotide microarray analysis was done at several time points over 16 days, starting at 6 hours posttransduction. The early growth suppression was preceded by up-regulation of IFNB1 and accompanied by marked up-regulation of IFN-induced genes, peaking at 3 days posttransduction. In contrast, oncogenes such as homeobox transcription factors, FLT3, KIT, and WT1 peaked at 8 days or beyond, coinciding with increased proliferation. In addition, several putative tumor suppressors and genes associated with hematopoietic differentiation were repressed at later time points. These findings provide a comprehensive picture of the changes in proliferation, differentiation, and global gene expression that underlie the leukemic transformation of human hematopoietic cells by NUP98-HOXA9. (Cancer Res 2006; 66(13): 6628-37)

ALK-positive histiocytosis is a rare subtype of histiocytic neoplasm first described in 2008 in three infants with multisystemic disease involving the liver and hematopoietic system. This entity has subsequently been documented in case reports and series to occupy a wider clinicopathologic spectrum with recurrent KIF5B-ALK fusions. The full clinicopathologic and molecular spectra of ALK-positive histiocytosis remain, however, poorly characterized. Here, we describe the largest study of ALK-positive histiocytosis to date, with detailed clinicopathologic data of 39 cases, including 37 cases with confirmed ALK rearrangements. The clinical spectrum comprised distinct clinical phenotypic groups: infants with multisystemic disease with liver and hematopoietic involvement, as originally described (Group 1A: 6/39), other patients with multisystemic disease (Group 1B: 10/39), and patients with single-system disease (Group 2: 23/39). Nineteen patients of the entire cohort (49%) had neurologic involvement (seven and twelve from Groups 1B and 2, respectively). Histology included classic xanthogranuloma features in almost one third of cases, whereas the majority displayed a more densely cellular, monomorphic appearance without lipidized histiocytes but sometimes more spindled or epithelioid morphology. Neoplastic histiocytes were positive for macrophage markers and often conferred strong expression of phosphorylated-ERK, confirming MAPK pathway activation. KIF5B-ALK fusions were detected in 27 patients, while CLTC-ALK, TPM3-ALK, TFG-ALK, EML4-ALK and DCTN1-ALK fusions were identified in single cases. Robust and durable responses were observed in 11/11 patients treated with ALK inhibition, ten with neurologic involvement. This study presents the existing clinicopathologic and molecular landscape of ALK-positive histiocytosis, and provides guidance for the clinical management of this emerging histiocytic entity.

Nuclear import of classical nuclear localization sequence-bearing proteins is mediated by karyopherin ␣͞␤1 heterodimers. A second nuclear import pathway, mediated by karyopherin ␤2 (transportin), recently was described for mRNA-binding proteins. Here we report the cloning and characterization of human karyopherin ␤3, which may be involved in a third pathway for nuclear import. Karyopherin ␤3 was localized mainly to the cytosol and the nucleus, particularly the nuclear rim. It bound to several of the repeat-containing nucleoporins (Nup358, Nup214, Nup153, Nup98, and p62) in overlay and solution-binding assays and was competed away by karyopherin ␤1. For Nup98, we localized this binding to the peptide repeat-containing region. Karyopherin ␤3 contains two putative Ran-binding homology regions and bound to Ran-GTP in a solution-binding assay with much higher affinity than to Ran-GDP. Furthermore, it interacted with two ribosomal proteins in an overlay assay. We suggest that karyopherin ␤3 is a nuclear transport factor that may mediate the import of some ribosomal proteins into the nucleus.The nuclear import of different subsets of proteins appears to proceed via several distinct import pathways. Proteins bearing a traditional nuclear localization sequence are bound by a heterodimer consisting of karyopherin ␣ and karyopherin ␤1, which then docks at the nuclear pore complex (1-11). Karyopherin ␣ binds to the nuclear localization sequence of the import substrate, whereas docking at the nuclear pore complex is mediated by interaction of karyopherin ␤1 with nucleoporins. Several nucleoporins contain characteristic peptide repeat regions (12). Karyopherin ␤1 docks at the nuclear pore complex by interacting with the repeat regions of these nucleoporins (3, 13-15). Docking is followed by an energy-dependent translocation step that requires the GTPase Ran and p10 (16-19). Ran-GTP binds to karyopherin ␤1 leading to the dissociation of the karyopherin ␣͞␤ heterodimer and translocation of karyopherin ␣ and the import substrate into the nucleus (14,(20)(21)(22).A separate nuclear import pathway recently was described for certain mRNA-binding proteins in yeast (23) and mammalian cells (24,25). In this pathway, the substrate-binding and docking͞import functions are performed by a single molecule of the karyopherin ␤ family. The yeast homologue, Kap104p (karyopherin ␤2), binds to the mRNA-binding proteins Nab2p and Nab4p and to repeat-containing nucleoporins, and is required for the nuclear localization of Nab2p. Two other proteins with a high degree of similarity to karyopherin ␤1, Kap121p (also called Pse1p or karyopherin ␤3) and Kap123p (karyopherin ␤4), have been identified in yeast and are involved in the import of ribosomal and other proteins (M. Rout, G.B., and J. Aitchison, unpublished data). Pse1p originally was described as an enhancer of protein secretion (26).Here we describe the cloning and characterization of the human homologue of Kap121p (Pse1p). We show by immunofluorescence that it localizes to the cytoso...