HOX genes, notably members of the HOXA cluster, and HOX cofactors have increasingly been linked to human leukemia. Intriguingly, HOXD13, a member of the HOXD cluster not normally expressed in hematopoietic cells, was recently identified as a partner of NUP98 in a t(2;11) translocation associated with t-AML/MDS. We have now tested directly the leukemogenic potential of the NUP98-HOXD13 t (

Rehovot 76100, Israel chains have been identified: IFNAR1 (Uze et al., 1990) 1 Corresponding author and IFNAR2-2 (or IFNAR2-c, Domanski et al., 1995;Lutfalla et al., 1995), which is a long form of IFN-α/βR The intracytoplasmic domain (IC) of cytokine receptors (Novick et al., 1994). Transduction of the signal generated provides docking sites for proteins which mediate by IFN-α,β,ω involves protein tyrosine kinases of the signal transduction. Thus, in interferon-α,β receptors Janus kinases (Jak) family and transcription factors of the (IFNAR1 and 2), the IC region binds protein-tyrosine Stat family (Velazquez et al., 1992;Darnell et al., 1994). and -serine/threonine kinases which phosphorylate the Proteins of the Jak-Stat pathways have been shown to receptor and the associated Stat transcription factors.bind to the intracytoplasmic (IC) domains of the IFNAR1 A two-hybrid screening was carried out to identify and 2 receptor chains. Jak1 is constitutively associated additional proteins which could interact with the IC with IFNAR2 (Novick et al., 1994), whereas tyk2 is bound domain of the IFNAR1 chain of the IFN-α,β receptor.to the IC domain of IFNAR1 (Abramovich et al., 1994b; Several positive clones representing a protein sequence Colaminici et al., 1994). IFNAR1 can also bind Stat2 (Abramovich et al., 1994b), and the docking site for latent designated IR1B4 were recovered from a human cDNA Stat2 was identified as a peptide containing phosphotyrolibrary. IR1B4 was identified as the human homolog sine Y466, adjacent to the tyk2 binding site (amino acids of PRMT1, a protein-arginine methyltransferase from 479-511) in the 100 amino acid long IFNAR1-IC region rat cells. Flag-IR1B4 fusion proteins bind to the isolated (Yan et al., 1996). IC was observed . Protein-tyrosine antibodies from untreated human cells. IR1B4/PRMT1 phosphatases PTP1C and D reversibly associate with is involved in IFN action since U266 cells rendered IFNAR1 upon IFN addition (David et al., 1995a). In deficient in this methyltransferase by antisense oligonuaddition, two serine/threonine protein kinases, the 48 kDa cleotides become more resistant to growth inhibition ERK2 MAP kinase (David et al., 1995b) and the cAMPby IFN. Methylation of proteins by enzymes which can activated protein kinase A (PKA; David et al., 1996) attach to the IC domains of receptors may be a signaling bind to the isolated membrane-proximal 50 residues of mechanism complementing protein phosphorylation.IFNAR1-IC. Therefore, the IC domains of type I IFN Among substrates methylated by PRMT1 are RNAreceptors serve as docking sites for multiple proteins binding heterogeneous nuclear ribonucleoproteins involved in phosphorylation and dephosphorylation. Both (hnRNPs) which are involved in mRNA processing, IFNAR1 and IFNAR2 become tyrosine phosphorylated in splicing and transport into the cytoplasm.response to IFN treatment, and binding of a tyrosineKeywords: interferon/methyltransferase/protein phosphorylated protein (βPTyr) to IFNAR1 is seen spemethylation/receptor/signaling cif...

NUP98-Hox fusion genes are newly identified oncogenes isolated in myeloid leukemias. Intriguingly, onlyAbd-B Hox genes have been reported as fusion partners, indicating that they may have unique overlapping leukemogenic properties. To address this hypothesis, we engineered novel NUP98 fusions with Hox genes not previously identified as fusion partners: the Abd-B-like gene HOXA10 and two Antennepedia-like genes, HOXB3 and HOXB4. Notably, NUP98-HOXA10 and NUP98-HOXB3 but not NUP98-HOXB4 induced leukemia in a murine transplant model, which is consistent with the reported leukemogenic potential ability of HOXA10 and HOXB3 but not HOXB4. Thus, the ability of Hox genes to induce leukemia as NUP98 fusion partners, although apparently redundant for Abd-B-like activity, is not restricted to this group, but rather is determined by the intrinsic leukemogenic potential of the Hox partner. We also show that the potent leukemogenic activity of Abd-B-like Hox genes is correlated with their strong ability to block hematopoietic differentiation. Conversely, coexpression of the Hox cofactor Meis1 alleviated the requirement of a strong intrinsic Hox-transforming potential to induce leukemia. Our results support a model in which many if not all Hox genes can be leukemogenic and point to striking functional overlap not previously appreciated, presumably reflecting common regulated pathways.

The identification of Hox genes as HSC regulators has been exploited to develop strategies to efficiently expand HSCs ex vivo, a key step to the success of therapies based on HSC transplantation and the understanding of mechanisms underlying HSC regulation. As leukemia is the result of deregulation of normal HSC development, the elucidation of the role of Hox in the pathobiology of the disease is helping to understand how HSCs self-renew and differentiate, and moreover, should facilitate the development of strategies for the management of leukemia.

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The human interferon alpha‐receptor (IFNAR gene product) is a transmembranal protein of 557 amino acids with an intracytoplasmic domain of 100 amino acids containing four tyrosines. Antibodies to a C‐terminal peptide (residues 521‐536) were developed which efficiently immunoprecipitate the 105 kDa IFNAR protein from detergent extracts of human cells. We show that the IFNAR protein becomes tyrosine phosphorylated within 5 min after treatment of human myeloma U266 cells with IFN‐alpha 2, IFN‐alpha 8 or IFN‐beta. The IFNAR chain interacts with both IFN‐alpha 2 and IFN‐beta, as demonstrated by cross‐linking. Among elements involved in signal transduction by type I IFNs, the tyrosine kinase Tyk2 but not Jak1, and the ISGF3 transcription factor subunit Stat2 (p113) but not Stat1 (p91), are found associated with the IFNAR protein. After IFN‐beta treatment for 5 min, a tyrosine‐phosphorylated protein of approximately 95 kDa (beta‐PTyr) is found bound to IFNAR, but can be dissociated by denaturation. The beta‐PTyr protein is present on the cell surface, like IFNAR, as shown by extracellular biotin tagging. The ratio of beta‐PTyr to IFNAR tyrosine phosphorylation is much higher with IFN‐beta than with IFN‐alpha 2 or 8. Both are IFN dependent and abrogated by a monoclonal antibody which blocks IFNAR action. The beta‐PTyr component may represent an important difference in the action of IFN‐beta as compared with IFN‐alpha in their shared receptor system.

PBX1 is a homeodomain protein that functions in complexes with other homeodomain-containing proteins to regulate gene expression during developmental and/or differentiation processes. A yeast two-hybrid screen of a fetal liver-hematopoietic cDNA library using PBX1a as bait led to the discovery of a novel non-homeodomaincontaining protein that interacts with PBX1 as well as PBX2 and PBX3. RNA analysis revealed it to be expressed in CD34؉ hematopoietic cell populations enriched in primitive progenitors, as is PBX1; search of the expressed sequence tag data base indicated that it is also expressed in other early embryonic as well as adult tissues. The full-length cDNA encodes a 731-amino acid protein that has no significant homology to known proteins. This protein that we have termed hematopoietic PBX-interacting protein (HPIP) is mainly localized in the cytosol and in small amounts in the nucleus. The region of PBX that interacts with HPIP includes both the homeodomain and immediate N-terminal flanking sequences. Strikingly, electrophoretic mobility shift assays revealed that HPIP inhibits the ability of PBX-HOX heterodimers to bind to target sequences. Moreover, HPIP strongly inhibits the transactivation activity of E2A-PBX. Together these findings suggest that HPIP is a new regulator of PBX function.The PBC protein family (mammalian PBX, C. elegans CEH-20 and Drosophila extradenticle) make critical contributions to cell fate and segmental patterning during embryogenic development (1-3). PBX1, a member of the PBX family along with PBX2 and PBX3 (4), was initially identified as the chromosome 1 participant of the t(1;19) translocation, which occurs in 25% of pediatric pre-B cell acute lymphocytic leukemia and that creates a chimeric gene designated E2A-PBX1 (5, 6). The mechanism by which E2A-PBX1 causes leukemia is still unclear. However, the structure of the protein, in which the majority of PBX1, including the homeodomain, is fused to the transcriptional activation domain of E2A (6, 7), suggests that the oncogenic properties of E2A-PBX1 result from inappropriate regulation of target genes whose expression during hematopoiesis is normally regulated by wild type PBX proteins (8 -11).In vitro and in vivo data strongly suggest that PBX functions in combination with heterologous homeodomain proteins, including class I HOX proteins. As HOX cofactors, PBC proteins improve HOX specificity due to the increased size of the cooperative binding site and the strength of DNA binding, as well as by modulating recognition of cooperative binding sites by different groups of HOX proteins (12-14). In addition, cooperative DNA binding with PBC proteins may act to change the regulatory signal of HOX proteins, from repressors to activators (15). HOX-PBC interaction optimally requires the PBC homeodomain and a short carboxyl-terminal region, called the HOX cooperativity motif (16,17).Although PBC proteins contribute to HOX DNA binding specificity, PBC-HOX complexes exhibit little transcriptional activity and thus alone do not account ...

Hox genes are clearly implicated in leukemia; however, neither the specificity of the leukemogenic potential among Hox genes of different paralog groups nor the role of the homeodomain is clear. We tested the leukemogenic potential of various NUP98-Hox fusion genes alone and with MEIS1. All genes tested had a significant overlapping effect in bone marrow cells in vitro. However, not all formed strong leukemogenic NUP98 fusion genes; but together with overexpression of MEIS1, all induced myeloid leukemia. This phenomenon was also seen with NUP98 fusions containing only the homeodomain of the corresponding Hox protein. We then exploited the strong transforming potential of NUP98-HOXD13 and NUP98-HOXA10 to establish preleukemic myeloid lines composed of early myeloid progenitors with extensive in vitro self-renewal capacity, short-term myeloid repopulating activity, and low propensity for spontaneous leukemic conversion. We also showed that MEIS1 can efficiently induce their conversion to leukemic stem cells, thus providing a novel model for the study of leukemic progression. In contrast to the leukemogenic effect of most of the Hox genes tested, HOXB4 has the ability to increase the self-renewal of hematopoietic stem cells without disrupting normal differentiation. On the basis of the discovery that the leukemogenic gene HOXA9 can also expand hematopoietic stem cells, we compared the ability of NUP98-Hox fusions to that of HOXB4 to trigger HSC expansion in vitro. Our preliminary results indicate that the expanding potential of HOXB4 is retained and even augmented by fusion to NUP98. Moreover, even greater expansion may be possible using Abd-B-like Hox fusions genes.