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Tyrosine kinase inhibitor (TKI) therapies induce clinical remission with remarkable effects on chronic myeloid leukemia (CML). However, very few TKIs completely eradicate the leukemic clone and persistence of leukemic stem cells (LSCs) remains challenging, warranting new, distinct targets for improved treatments. We demonstrated that the scaffold protein AHI-1 is highly deregulated in LSCs and interacts with multiple proteins, including Dynamin-2 (DNM2), to mediate TKI-resistance of LSCs. We have now demonstrated that the SH3 domain of AHI-1 and the proline rich domain of DNM2 are mainly responsible for this interaction. DNM2 expression was significantly increased in CML stem/progenitor cells; knockdown of DNM2 greatly impaired their survival and sensitized them to TKI treatments. Importantly, a new AHI-1-BCR-ABL-DNM2 protein complex was uncovered, which regulates leukemic properties of these cells through a unique mechanism of cellular endocytosis and ROS-mediated autophagy. Thus, targeting this complex may facilitate eradication of LSCs for curative therapies.

Meningioma 1 (MN1) is an independent prognostic marker for normal karyotype acute myeloid leukemia (AML), with high expression linked to all-trans retinoic acid resistance and poor survival. MN1 is also a potent and sufficient oncogene in murine leukemia models, strongly dependent on the MEIS1/AbdB-like HOX protein complex to transform common myeloid progenitors, block myeloid differentiation, and promote leukemic stem cell self-renewal. To identify key genes and pathways underlying leukemic activity, we functionally assessed MN1 cell phenotypic heterogeneity, revealing leukemic and non-leukemic subsets. Using gene expression profiling of these subsets combined with previously published comparisons of full-length MN1 and mutants with varying leukemogenic activity, we identified candidate genes critical to leukemia. Functional analysis identified Hlf and Hoxa9 as critical to MN1 in vitro proliferation, self-renewal and impaired myeloid differentiation. Although critical to transformation, Meis1 knockdown had little impact on these properties in vitro. However, we identified Meis2 as critical to MN1-induced leukemia, with essential roles in proliferation, self-renewal, impairment of differentiation and disease progression in vitro and in vivo. Here, we provide evidence of phenotypic and functional hierarchy in MN1-induced leukemic cells, characterise contributions of Hlf, Hoxa9 and Meis1 to in vitro leukemic properties, and reveal Meis2 as a novel player in MN1-induced leukemogenesis.

Myeloid differentiation is blocked in acute myeloid leukemia (AML), but the molecular mechanisms are not well characterized. Meningioma 1 (MN1) is overexpressed in AML patients and confers resistance to all-trans retinoic acid-induced differentiation. To understand the role of MN1 as a transcriptional regulator in myeloid differentiation, we fused transcriptional activation (VP16) or repression (M33) domains with MN1 and characterized these cells in vivo. Transcriptional activation of MN1 target genes induced myeloproliferative disease with long latency and differentiation potential to mature neutrophils. A large proportion of differentially expressed genes between leukemic MN1 and differentiation-permissive MN1VP16 cells belonged to the immune response pathway like interferon-response factor (Irf) 8 and Ccl9. As MN1 is a cofactor of MEIS1 and retinoic acid receptor alpha (RARA), we compared chromatin occupancy between these genes. Immune response genes that were upregulated in MN1VP16 cells were co-targeted by MN1 and MEIS1, but not RARA, suggesting that myeloid differentiation is blocked through transcriptional repression of shared target genes of MN1 and MEIS1. Constitutive expression of Irf8 or its target gene Ccl9 identified these genes as potent inhibitors of murine and human leukemias in vivo. Our data show that MN1 prevents activation of the immune response pathway, and suggest restoration of IRF8 signaling as therapeutic target in AML.

Summary The transcription factor Meis1 drives myeloid leukemogenesis in the context of Hox gene overexpression but is currently considered undruggable. We therefore investigated whether myeloid progenitor cells transformed by Hoxa9 and Meis1 become addicted to targetable signaling pathways. A comprehensive (phospho)proteomic analysis revealed that Meis1 increased Syk protein expression and activity. Syk upregulation occurs through a Meis1-dependent feedback loop. By dissecting this loop, we show that Syk is a direct target of miR-146a, whose expression is indirectly regulated by Meis1 through the transcription factor PU.1. In the context of Hoxa9 overexpression, Syk signaling induces Meis1, recapitulating several leukemogenic features of Hoxa9/Meis1-driven leukemia. Finally, Syk inhibition disrupts the identified regulatory loop, prolonging survival of mice with Hoxa9/Meis1-driven leukemia., Graphical Abstract, Highlights • Meis1 regulates expression and activation of Syk in Hoxa9-driven leukemia • Syk upregulation in Hoxa9/Meis1-driven leukemia is mediated by miR-146a • Increased Syk activity induces a Meis1 transcriptional program • In vivo, Meis1 sensitizes Hoxa9-driven leukemia to Syk inhibition, Mohr et al. identify a Meis1-dependent regulatory loop involving PU.1, miR-146a, and Syk, resulting in the addiction to Syk activity in Hoxa9/Meis1-transformed myeloid progenitor cells. Syk inhibition disrupts the regulatory loop and prolongs survival of mice with Hoxa9/Meis1-driven acute myeloid leukemia.

HOX genes have been implicated as regulators of normal and leukemic stem cell functionality, but the extent to which these activities are linked is poorly understood. Previous studies revealed that transduction of primitive mouse hematopoietic cells with a NUP98HOXA10homeodomain (NA10HD) fusion gene enables a subsequent rapid and marked expansion in vitro of hematopoietic stem cell numbers without causing their transformation or deregulated expansion in vivo. To determine whether forced expression of NA10HD in primitive human cells would have a similar effect, we compared the number of long-term culture-initiating cells (LTC-ICs) present in cultures of lenti-NA10HD versus control virus-transduced CD34(+) cells originally isolated from human cord blood and chronic phase (CP) chronic myeloid leukemia (CML) patients. We found that NA10HD greatly increases outputs of both normal and Ph(+)/BCR-ABL(+) LTC-ICs, and this effect is particularly pronounced in cultures containing growth factor-producing feeders. Interestingly, NA10HD did not affect the initial cell cycle kinetics of the transduced cells nor their subsequent differentiation. Moreover, immunodeficient mice repopulated with NA10HD-transduced CP-CML cells for more than 8 months showed no evidence of altered behavior. Thus, NA10HD provides a novel tool to enhance both normal and CP-CML stem cell expansion in vitro, without apparently altering other properties.

Abstract 4194 Cytogenetically normal acute myeloid leukemia (CN-AML) patients with high BAALC or MN1 expression have a poor prognosis. Whereas the oncogenic function of MN1 is well established, the functional role of BAALC in hematopoiesis is not known. We therefore compared the expression of BAALC and MN1 in 140 CN-AML patients by quantitative PCR. To further assess the impact of BAALC on leukemogenesis we used retroviral gene transfer into primary murine bone marrow cells and cells immortalized with NUP98-HOXD13 (ND13) and HOXA9. Transduced cells were assessed in vitro by colony forming assays and for their sensitivity to treatment with all-trans retinoic acid (ATRA). They were also evaluated by in vivo transplantation into lethally-irradiated mice. In the 140 CN-AML patients analyzed, the expression of BAALC and MN1 was highly correlated (R=0.71). Retroviral overexpression of MN1 or BAALC in the Hox gene-immortalized bone marrow cells did not cause upregulation of the other gene, suggesting that these genes do not regulate each other. In murine bone marrow cells BAALC did not immortalize the cells in vitro as assessed by serial replating of transduced cells in methylcellulose assays. Transplantation of transduced cells resulted in negligible engraftment of approximately 1 percent at 4 weeks after transplantation. However, co-transduction of BAALC into NUP98-HOXD13 cells (which are very sensitive to the treatment with all-trans retinoic acid) increased the 50 percent inhibitory concentration (IC50) of ATRA by 4.3-fold, suggesting a negative impact of BAALC on myeloid differentiation. We next evaluated whether the differentiation inhibiting effects of BAALC may cooperate with the self renewal-promoting effects of HOXA9 to induce leukemia in mice. Mice receiving transplants of murine bone marrow cells transduced with BAALC and HOXA9 developed myeloid leukemias with a median latency of 139.5 days that were characterized by leukocytosis, massively enlarged spleens (up to 1.02 g), anemia and thrombocytopenia. Infiltrations of myeloid cells were also found in liver, spleen, and kidney. The disease was transplantable into secondary animals. By Southern blot analysis we found one to two BAALC viral integrations per mouse, suggesting that clonal disease had developed from BAALC-transduced cells. We demonstrate for the first time that BAALC blocks myeloid differentiation and promotes leukemogenesis when combined with the self-renewal promoting oncogene HOXA9. Due to its prognostic and functional effects BAALC may become a valuable therapeutic target in leukemia patients. Disclosures: No relevant conflicts of interest to declare.

A chimaera of stem cell factor (SCF) and a cellulose-binding domain from the xylanase Cex (CBDCex) effectively immobilizes SCF on a cellulose surface. The fusion protein retains both the cytokine properties of SCF and the cellulose-binding characteristics of CBDCex. When adsorbed on cellulose, SCF–CBDCex is up to 7-fold more potent than soluble SCF–CBDCex and than native SCF at stimulating the proliferation of factor-dependent cell lines. When cells are incubated with cellulose-bound SCF–CBDCex, activated receptors and SCF–CBDCex co-localize on the cellulose matrix. The strong binding of SCF–CBDCex to the cellulose surface permits the effective and localized stimulation of target cells; this is potentially significant for long-term perfusion culturing of factor-dependent cells. It also permits the direct analysis of the effects of surface-bound cytokines on target cells.

Huntington disease (HD) is a neurodegenerative disorder associated with polyglutamine expansion in a recently identified protein, huntingtin. Huntingtin is widely expressed and plays a crucial role in development, because gene-targeted HD-/- mouse embryos die early in embryogenesis. To analyze the function of normal huntingtin, we have generated HD-/- embryonic stem (ES) cells and used an in vitro model of ES cell differentiation to analyze their ability to develop into neuronal cells. Expression analysis of wild-type ES cells revealed that huntingtin is expressed at all stages during ES cell differentiation with high expression in neurons. Expression levels increased with the maturation of differentiating neurons, demonstrating that expression of huntingtin is developmentally regulated in cell culture and resembles the pattern of expression observed in differentiating neurons in the mouse brain. It is interesting that HD-/- ES cells could differentiate into mature postmitotic neurons that expressed functional voltage- and neurotransmittergated ion channels. Moreover, both excitatory and inhibitory spontaneous postsynaptic currents were observed, indicating the establishment of functional synapses in the absence of huntingtin. These results demonstrate that huntingtin is not required for the generation of functional neurons with features characteristic of postmitotic neurons in the developing mouse brain.

Introduction of erythropoietin receptors (EpoRs) into the interleukin-3 (IL-3)-dependent murine hemopoietic cell line, Ba/F3, enables these cells to not only proliferate, after an initial lag in G1, but also to increase beta-globin mRNA levels in response to erythropoietin (Epo). With IL-3 and Epo costimulation, IL-3-induced signaling appears to be dominant since no increase in beta-globin mRNA occurs. Differentiation and proliferation signals may be uncoupled since EpoRs lacking all eight intracellular tyrosines were compromised in proliferative signaling but retained erythroid differentiation ability. Intriguingly, a chimeric receptor of the extracellular domain of the EpoR and the transmembrane and intracellular domains of IL-3RbetaIL-3 chain (EpoR/IL-3RbetaIL-3) was capable of Epo-induced proliferative and differentiating signaling, suggesting either the existence of a second EpoR subunit responsible for differentiation or that the alpha subunit of the IL-3 receptor (IL-3R) prevents it. Arguing against the former, a truncated EpoR lacking an intracellular domain was incapable of promoting proliferation or differentiation. An EpoR/IL-3Ralpha chimera, in contrast, was capable of transmitting a weak Epo-induced proliferative signal but failed to stimulate accumulation of beta-globin mRNA. Most significantly, coexpression of the EpoR/IL-3Ralpha chimera with either EpoR/IL-3Rbeta or wild-type EpoRs suppressed Epo-induced beta-globin mRNA accumulation. Taken together, these results suggest an active role for the IL-3Ralpha subunit in inhibiting EpoR-specific differentiating signals.

The murine heat-stable antigen (HSA) is a glycosyl-phosphatidylinositol-linked cell surface protein which has been implicated in cellular adhesion processes, the co-stimulation of CD4+ T cells, and B cell memory. We have recently demonstrated a significant reduction in pro-B and pre-B lymphocytes in transgenic mice that overexpress HSA. We now report that cross-linking HSA with the M1/69 monoclonal antibody induces the apoptosis of cultured B cell precursors in a stomal cell and cytokine-independent manner and that sensitivity to HSA-mediated cell death increases with developmental maturity. The cross-linking of HSA does not induce apoptosis in mature splenic B cells, but instead inhibits their ability to proliferate in response to anti-CD40 + IL-4. Taken together, these data implicate HSA as a potent negative regulator of B cell development and activation.

We recently cloned and sequenced a cDNA encoding a 145-kD protein from the murine hematopoietic cell line B6SUtA, that becomes tyrosine phosphorylated and associated with Shc after cytokine stimulation. Based on its domains and enzymatic activity, we named this protein SHIP for SH2-containing inositol phosphatase (Damen et al, Proc Natl Acad Sci USA 93:1689, 1996). We describe here the cloning of the human homologue of murine SHIP (mSHIP) from a human megakaryocytic cell line (MO7e) lambda gt11 cDNA library using two nonoverlapping mSHIP cDNA fragments as probes. Northern blot analysis suggests that human SHIP (hSHIP) is expressed as a 5.3-kb mRNA in human bone marrow and a wide variety of other tissues. Sequence analysis of this cDNA predicts a protein of 1188 amino acids exhibiting 87.2% overall sequence identity with mSHIP. Contained within the defined open reading frame is an N-terminal, group l src homology 2 (SH2) domain; three NXXY motifs that, if phosphorylated, could be bound by phosphotyrosine binding (PTB) domains; a C-terminal proline-rich region; and two centrally located inositol polyphosphate 5-phosphatase motifs. Fluorescence in situ hybridization, using the full-length hSHIP cDNA as a probe, mapped hSHIP to the long arm of chromosome 2 at the border between 2q36 and 2q37.

Corrective gene transfer for therapeutic intervention in metabolic and hematopoietic disorders has been hampered by the relatively inefficient transduction of human hematopoietic stem cells. To overcome this, a bicistronic recombinant retrovirus has been generated that delivers both a therapeutic glucocerebrosidase (GC) cDNA for the treatment of Gaucher disease, and a small murine cell surface antigen (heat-stable antigen [HSA]) as a selectable marker. An amphotropic retroviral- producing cell clone was created, and filtered supernatant was used to transduce NIH 3T3 cells. Sorting of transduced cells by flow cytometry enabled separation into populations based on cell surface fluorescence intensity derived from the expressed HSA. Significant increases in GC enzyme activity were seen for the transduced and especially the transduced and sorted cells. Similarly, increases in GC specific activity were seen in transduced and sorted skin fibroblasts from a patient with Gaucher disease. To streamline future transfer and sorting protocols for hematopoietic cells, transformed B-cell lines from Gaucher patients were created. Type I B cells were transduced and sorted, and large increases in GC specific activity occurred with concomitant increases in integrated retroviral copy numbers. In addition, toward the goal of using this selectable approach for corrective gene transfer to bone marrow stem cells, CD34+ cells were isolated from normal BM donors, transduced, and sorted based on cell surface expression of HSA. Proviral DNA was detected in approximately 40% of clonogenic progenitor colonies derived from unsorted, transduced CD34+ cells, demonstrating the high titer of the vector. However, after sorting, 100% of the colonies had the corrective GC cDNA, demonstrating the efficiency of this selective system for human hematopoietic progenitors. It is expected that strategies based on this approach will allow sorting of transduced cells of many types before implantation of transduced cells to animals or patients. This vector system may also be used to simplify manipulations and studies on retroviral-mediated gene delivery in vitro and for in vivo models.

A 145-kDa tyrosine-phosphorylated protein that becomes associated with Shc in response to multiple cytokines has been purified from the murine hemopoietic cell line B6SUtA1. Amino acid sequence data were used to clone the cDNA encoding this protein from a B6SUtA1 library. The predicted amino acid sequence encodes a unique protein containing an N-terminal src homology 2 domain, two consensus sequences that are targets for phosphotyrosine binding domains, a proline-rich region, and two motifs highly conserved among inositol polyphosphate 5-phosphatases. Cell lysates immunoprecipitated with antiserum to this protein exhibited both phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate polyphosphate 5-phosphatase activity. This novel signal transduction intermediate may serve to modulate both Ras and inositol signaling pathways. Based on its properties, we suggest the 145-kDa protein be called SHIP for SH2-containing inositol phosphatase.

Recombinant retroviruses offer many advantages for the genetic modification of human hematopoietic cells, although their use in clinical protocols has thus far given disappointing results. There is therefore an important need to develop new strategies that will allow effectively transduced primitive hematopoietic target populations to be both rapidly characterized and isolated free of residual nontransduced but biologically equivalent cells. To address this need, we constructed a murine stem cell virus (MSCV)-based retroviral vector containing the 228-bp coding sequence of the murine heat-stable antigen (HSA) and generated helper virus-free amphotropic MSCV-HSA producer cells by transfection of GP-env AM12 packaging cells. Light density and, in some cases, lineage marker-negative (lin-) normal human marrow or mobilized peripheral blood cells preactivated by exposure to interleukin-3 (IL- 3), IL-6, and Steel factor in vitro for 48 hours were then infected by cocultivation with these MSCV-HSA producer cells for a further 48 hours in the presence of the same cytokines. Fluorescence-activated cell sorting (FACS) analysis of the cells 24 hours later showed 21% to 41% (mean, 27%) of those that were still CD34+ to have acquired the ability to express HSA. The extent of gene transfer to erythroid and granulopoietic progenitors (burst-forming unit-erythroid and colony- forming unit-granulocyte-macrophage), as assessed by the ability of these cells to form colonies of mature progeny in the presence of normally toxic concentrations of G418, averaged 11% and 12%, respectively, in 6 experiments. These values could be increased to 100% and 77%, respectively, by prior isolation of the CD34+HSA+ cell fraction and were correspondingly decreased to an average of 2% and 5%, respectively, in the CD34+HSA- cells. In addition, the extent of gene transfer to long-term culture-initiating cells (LTC-IC) was assessed by G418 resistance. The average gene transfer to LTC-IC-derived colony- forming cells in the unsorted population was < or = 7% in 4 experiments. FACS selection of the initially CD34+HSA+ cells increased this value to 86% and decreased it to 3% for the LTC-IC plated from the CD34+HSA- cells. Transfer of HSA gene expression to a phenotypically defined more primitive subpopulation of CD34+ cells, ie, those expressing little or no CD38, could also be shown by FACS analysis of infected populations 24 hours after infection. These findings underscore the potential use of retroviral vectors encoding HSA for the specific identification and non-toxic selection immediately after infection of retrovirally transduced populations of primitive human hematopoietic cells. In addition, such vectors should facilitate the subsequent tracking of their marked progeny using multiparameter flow cytometry.

The gene transfer efficiency of human hematopoietic stem cells is still inadequate for efficient gene therapy of most disorders. To overcome this problem, a selectable retroviral vector system for gene therapy has been developed for gene therapy of Gaucher disease. We constructed a bicistronic retroviral vector containing the human glucocerebrosidase (GC) cDNA and the human small cell surface antigen CD24 (243 bp). Expression of both cDNAs was controlled by the long terminal repeat enhancer/promoter of the Molony murine leukemia virus. The CD24 selectable marker was placed downstream of the GC cDNA and its translation was enhanced by inclusion of the long 5' untranslated region of encephalomyocarditis virus internal ribosomal entry site. Virus-producing GP+envAM12 cells were created by multiple supernatant transductions to create vector producer cells. The vector LGEC has a high titer and can drive expression of GC and the cell surface antigen CD24 simultaneously in transduced NIH 3T3 cells and Gaucher skin fibroblasts. These transduced cells have been successfully separated from untransduced cells by fluorescence-activated cell sorting, based on cell surface expression of CD24. Transduced and sorted NIH 3T3 cells showed higher GC enzyme activity than the unsorted population, demonstrating coordinated expression of both genes. Fibroblasts from Gaucher patients were transduced and sorted for CD24 expression, and GC enzyme activity was measured. The transduced sorted Gaucher fibroblasts had a marked increase in enzyme activity (149%) compared with virgin Gaucher fibroblasts (17% of normal GC enzyme activity). Efficient transduction of CD34+ hematopoietic progenitors (20-40%) was accomplished and fluorescence-activated cell sorted CD24(+)-expressing progenitors generated colonies, all of which (100%) were vector positive. The sorted, CD24-expressing progenitors generated erythroid burst-forming units, colony-forming units (CFU)-granulocyte, CFU-macrophage, CFU-granulocyte/macrophage, and CFU-mix hematopoietic colonies, demonstrating their ability to differentiate into these myeloid lineages in vitro. The transduced, sorted progenitors raised the GC enzyme levels in their progeny cells manyfold compared with untransduced CD34+ progenitors. Collectively, this demonstrates the development of high titer, selectable bicistronic vectors that allow isolation of transduced hematopoietic progenitors and cells that have been metabolically corrected.

Hox genes were first recognized for their role in embryonic development and may also play important lineage-specific functions in a variety of somatic tissues including the hematopoietic system. We have recently shown that certain members of the Hox A and B clusters, such as HOXB3 and HOXB4, are preferentially expressed in subpopulations of human bone marrow that are highly enriched for the most primitive hematopoietic cell types. To assess the role these genes may play in regulating the proliferation and/or differentiation of such cells, we engineered the overexpression of HOXB4 in murine bone marrow cells by retroviral gene transfer and analyzed subsequent effects on the behavior of various hematopoietic stem and progenitor cell populations both in vitro and in vivo. Serial transplantation studies revealed a greatly enhanced ability of HOXB4-transduced bone marrow cells to regenerate the most primitive hematopoietic stem cell compartment resulting in 50-fold higher numbers of transplantable totipotent hematopoietic stem cells in primary and secondary recipients, compared with serially passaged neo-infected control cells. This heightened expansion in vivo of HOXB4-transduced hematopoietic stem cells was not accompanied by identifiable anomalies in the peripheral blood of these mice. Enhanced proliferation in vitro of day-12 CFU-S and clonogenic progenitors was also documented. These results indicate HOXB4 to be an important regulator of very early but not late hematopoietic cell proliferation and suggest a new approach to the controlled amplification of genetically modified hematopoietic stem cell populations.

Incubation of chronic myeloid leukemia (CML) marrow for 10 days in vitro causes a marked and selective loss of very primitive Philadelphia chromosome (Ph)+ as compared with Ph- progenitors. We have autografted 22 patients with CML (16 in first chronic phase [group 1] and 6 with more advanced disease [group 2]) with marrow treated in this way to facilitate restoration of Ph- hematopoiesis after intensive therapy. Hematologic recovery to greater than 0.5 x 10(9)/L neutrophils occurred in 16 patients, and to greater than 20 x 10(9)/L platelets in 15 of 21 evaluable patients at a median of 29 and 48 days postautograft, respectively. Regenerating marrow cells were 100% Ph- in 13 patients and 75% to 94% Ph- in 3. Between 4 and 36 months (median 12) postautograft, Ph+ cells became detectable in all but 1 (who died in remission) of the 13 patients who achieved complete cytogenetic remission. Four of 7 evaluable patients treated with low-dose interferon alpha were returned to complete cytogenetic remission. Thirteen group 1 patients (81%) are alive 1.0 to 5.7 years (median 2.6) after autografting: 4 in complete cytogenetic remission, 2 in hematologic remission, 6 in chronic phase, and 1 in myeloid blast phase. Three group 2 patients (50%) are alive at 2.6, 3.8, and 4.3 years after autografting: 1 in partial cytogenetic remission, 1 in chronic phase, and 1 in accelerated phase. Thus, autografts of cultured marrow can result in prolonged restoration of Ph- hematopoiesis for some patients with CML.

Retrovirus-mediated gene transfer of the human beta-globin gene into hematopoietic stem cells is an attractive approach to the therapy of human beta-globin gene disorders. However, expression of the transduced beta-globin gene linked to its proximal cis-acting sequences (-0.8 to +0.3 kb from the cap site) is considerably below the level required for a significant therapeutic effect. The discovery of the beta-locus control region (beta-LCR), organized in four major DNase I hypersensitive sites far upstream of the human beta-like globin gene cluster, provided a potential means to achieve a high level of expression of a linked human beta-globin gene, but initial attempts to incorporate beta-LCR derivatives in retroviral vectors resulted in the production of low-titer viruses with multiple rearrangements of the transmitted proviral structures. We now describe how extensive mutagenesis of the transduced beta-globin gene, eliminating a 372 bp intronic segment and multiple reverse polyadenylation and splicing signals, increases viral titer significantly and restores stability of proviral transmission upon infection of cell lines and bone marrow-repopulating cells. These optimized vectors have enabled us to analyze the expression properties of various retrovirally transduced beta-LCR derivatives in dimethylsulfoxide-induced murine erythroleukemia cells and to achieve ratios of human beta-globin/murine beta maj-globin mRNA, on a per gene basis, as high as 80%.

The human cell surface antigen, CD24, is a glycosyl phosphatidylinositol (GPI)-linked glycoprotein that has been implicated in the differentiation and activation of granulocytes and B lymphocytes. Changes in expression of the antigen occur at critical times during B lineage development. CD24 was cloned by its homology to mouse heat-stable antigen. Southern blot analysis suggested the presence of multiple CD24 related sequences in the human genome. We have now mapped CD24 homologous sequences to chromosomes 6q21, 15q21-q22, and Yq11 by screening a panel of somatic cell hybrid DNAs and by in situ hybridization. At least two additional homologues, one located on chromosome 1 at band p36 and one tentatively mapped to chromosome 20, that are distantly related to CD24 were identified. Southern analysis of male and female DNA samples confirmed the presence of CD24 homologous sequences on the human Y chromosome. Sequencing of DNA fragments amplified from monochromosomal somatic cell hybrids showed that the CD24 cDNA was derived from a transcript originating from a gene on chromosome 6. The CD24 gene on the Y chromosome had many base changes compared to the cDNA but had retained an open reading frame, leaving open the question of whether this gene is functional. Both ATGs in the translation initiation region of CD24 homologous sequences on chromosome 15 were converted to GTGs, making it unlikely that this gene, if functional, encodes a protein similar to CD24. CD24, therefore, is a member of a multigene family, but it remains to be determined whether any of the related genes are functional.

Heat-stable antigen (HSA) is a small, glycosyl phosphatidylinositol-anchored protein that can act as a costimulatory molecule for antigen-dependent activation of helper T cells. In addition to being expressed on antigen-presenting B cells, HSA is also expressed during the initial stages of T cell development in the thymus. HSA levels are very high on immature CD4-, CD8- double negative thymocytes, but are reduced on CD4+, CD8+ double positive cells undergoing selection in the thymus, and are entirely eliminated when these cells differentiate into immunologically competent CD4+ or CD8+ single positive T cells. To examine the potential roles of this molecule in T cell development and selection, we generated transgenic mice in which HSA was highly expressed on all classes of thymocytes. The consequence of deregulated HSA expression was a pronounced reduction in the numbers of double positive and single positive thymocytes, whereas the numbers of their double negative precursors were largely unaffected. These results demonstrate that downregulation of HSA expression at the double positive stage is a critical event in thymocyte development. The depletion of thymocytes resulting from HSA overexpression begins at the same time as the onset of negative selection, suggesting that HSA may provide signals that contribute to determining the efficiency of this process.

Gene therapy as well as methods capable of returning cells to a pluripotent state (iPS) have enabled the correction of genetic deficiencies in syngenic adult progenitors, reducing the need for immunosuppression in cell therapy approaches. However, in diseases involving mutations that lead to the complete lack of a protein, such as Duchenne muscular dystrophy, the main immunogens leading to rejection of transplanted cells are the therapeutic proteins themselves. In these cases even iPS cells would not circumvent the need for immunosuppression, and alternative strategies must be developed. One such potential strategy seeks to induce immune tolerance using hematopoietic stem cells originated from the same donor or iPS line from which the therapeutic progenitors are derived. However, donor hematopoietic stem cells (HSCs) are available in limiting numbers and embryonic stem (ES) cell-derived HSCs engraft poorly in adults. While these limitations have been circumvented by ectopic expression of HOXB4, overexpression of this protein is associated with inefficient lymphoid reconstitution. Here we show that adult HSCs expanded with a NUP98-HOXA10hd fusion protein sustain long-term engraftment in immunologically mismatched recipients and generate normal numbers of lymphoid cells. In addition, NUP98-HOXA10hd-expanded cells induce functional immune tolerance to a subsequent transplant of myogenic progenitors immunologically matched with the transplanted HSCs.

Genomic imprinting is an epigenetic phenomenon that ensures the parental-specific expression of a subset of genes and is required for the normal mammalian development. Loss of genomic imprinting represents one of the most common genetic abnormalities in growth disorders and cancer. DNA methylation is a key component of genomic imprinting regulation in eutherian mammals, and most imprinted genes are associated with differentially methylated CpG-rich regions.

In this report we describe the purification of the murine interleukin 3 receptor (mIL-3R) to apparent homogeneity using a two-step procedure involving biotinylated mIL-3 (B-mIL-3) and affinity binding to immobilized antiphosphotyrosine and streptavidin agarose (SA). Purification was monitored using an assay for detergent solubilized-mIL-3Rs that utilized unglycosylated 125I-mIL-3 and concanavalin A (ConA)-Sepharose beads. The final material consisted of a 140-kDa tyrosine and serine phosphorylated protein that was greater than 98% pure as assessed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of either [35S]methionine-labeled, silver-stained, or radioiodinated preparations. Characterization of the purified receptor revealed that it migrated identically under reducing and nonreducing conditions in SDS gels, possessed 10 kDa of N-linked carbohydrate, and was cleaved upon storage at 4 degrees C to a 70-kDa form. These properties suggested that the purified mIL-3R was identical to that identified by cross-linking studies. The KD of the purified receptor was 1-5 nM, similar to estimates obtained using intact normal mouse bone marrow cells and mIL-3-dependent cell lines. The two-step purification procedure also isolated a 120-kDa serine phosphorylated but nontyrosine phosphorylated mIL-3R species. Apart from phosphorylation differences, the 140- and 120-kDa species were apparently identical, yielding, after alkaline phosphatase treatment, the same molecular mass on SDS gels and similar chymotryptic peptide maps. Amino acid sequences and composition data obtained from the more abundant and more stable serine phosphorylated 120-kDa mIL-3R, further purified by SDS-polyacrylamide gel electrophoresis, suggested that the purified mIL-3R may be identical to the predicted sequence of the recently isolated cDNA clone AIC2A. This was further suggested by comparing chymotryptic maps of the 120-kDa mIL-3R with the Aic2A protein and using antibodies corresponding to the amino and carboxyl termini of the AIC2A cDNA product. However, the Aic2A protein, when expressed on the surface of COS or 3T3 cells or following detergent solubilization and partial purification with biotinylated mIL-3 and SA, displayed a substantially lower affinity for mIL-3.

Clinical uses of gene transfer to bone marrow transplants require the establishment of a reproducible method for infecting large numbers of very primitive hematopoietic cells at high efficiency using cell-free retrovirus-containing media. In this study we report the results of experiments with preparations of a high-titer (2-5 x 10(7)/ml) helper-free recombinant neo(r) retrovirus that indicate this goal can now be achieved based on measurements of gene transfer efficiencies to cells referred to as long-term culture initiating cells (LTC-IC) because they give rise to clonogenic cells after greater than or equal to 5 wk in long-term culture (LTC). Intermittent, repeated exposure of normal human marrow mononuclear cells to virus-containing supernatant over a 3-d period of cell maintenance on an IL-3/granulocyte colony-stimulating factor (G-CSF) producing stromal layer resulted in gene transfer efficiencies to LTC-IC of 41%; a level previously obtainable only using co-cultivation infection techniques. Marrow cells enriched greater than or equal to 500-fold for LTC-IC (1-2% pure) by flow cytometry showed gene transfer efficiencies of 27% when infected in a similar fashion over a shorter period (24 h), but in the presence of added soluble IL-3 and G-CSF without stromal feeders, and this increased to 61% when Steel factor was also present during the infection period. By using a less highly enriched population of LTC-IC obtained by a bulk immunoselection technique applicable to large-scale clinical marrow harvests, gene transfer efficiencies to LTC-IC of 40% were achieved and this was increased to 60% by short-term preselection in G418. Southern analysis of DNA from the nonadherent cells produced by these LTC over a 6-wk period provided evidence of clonal evolution of LTC-IC in vitro. Leukemic chronic myelogenous leukemia LTC-IC were also infected at high efficiency using the same supernatant infection strategy with growth factor supplementation. These data demonstrate the feasibility of using cell-free virus preparations for infecting clinical marrow samples suitable for transplantation, as well as for further analysis of human marrow stem cell dynamics in vitro.

Numerous factors that can influence the proliferation and differentiation in vitro of cells at various stages of hematopoiesis have been identified, but the mechanisms used by stromal cells to regulate the cycling status of the most primitive human hematopoietic cells are still poorly understood. Previous studies of long-term cultures (LTC) of human marrow have suggested that cytokine-induced variations in stromal cell production of one or more stimulators and inhibitors of hematopoiesis may be important. To identify the specific regulators involved, we performed Northern analyses on RNA extracted from human marrow LTC adherent layers, or stromal cell types derived from or related to those present in the adherent layer. These analyses showed marked increases in interleukin-1 beta (IL-1 beta), IL-6, and granulocyte colony-stimulating factor (G-CSF) mRNA levels within 8 hours after treatments that lead to the activation within 2 days of primitive hematopoietic progenitors in such cultures. Increases in granulocyte-macrophage (GM)-CSF and M-CSF mRNA were also sometimes seen. Bioassays using cell lines responsive to G-CSF, GM-CSF, and IL-6 showed significant elevation in growth factor levels 24 hours after IL- 1 beta stimulation. Neither IL-3 nor IL-4 mRNA was detectable at any time. In contrast, transforming growth factor-beta (TGF-beta) mRNA and nanogram levels of TGF-beta bioactivity in the medium were detected at all times in established LTC, and these levels were not consistently altered by any of the manipulations that stimulated hematopoietic growth factor production and primitive progenitor cycling. We also found that addition of anti-TGF-beta antibody could prolong or reactivate primitive progenitor proliferation when added to previously stimulated or quiescent cultures, respectively. Together, these results indicate a dominant negative regulatory role of endogenously produced TGF-beta in unperturbed LTC, with activation of primitive hematopoietic cells being achieved by mechanisms that stimulate stromal cells to produce G-CSF, GM-CSF, and IL-6. Given the similarities between the LTC system and the marrow microenvironment, it seems likely that the control of human stem cell activation in vivo may involve similar variations in the production of these factors by stromal cells.

In addition to the 85-95 kD CD44 species found on most hemopoietic cell types, the human myelomonocytic cell line KG1a expresses proteins of approximately 115 kD and 130 kD that react with monoclonal antibodies belonging to CD44. The possibility that these higher molecular weight species may represent novel CD44 isoforms containing additional protein sequence was investigated. CD44 cDNA clones were isolated from a plasmid-based expression library prepared from KG1a mRNA. One of the three clones obtained (clone 2.3) was found to encode a CD44 molecule of approximately 130 kD in transfected COS cells. Sequences analysis indicated that the molecule encoded by this cDNA clone, designated CD44R1, was essentially identical to CD44 except for the presence of an additional 132 amino acids inserted into the extracellular domain. This inserted region is rich in serine and threonine residues that may serve as sites of O-linked glycosylation, and contains a potential site of N-linked glycosylation and a potential site of chondroitin sulphate attachment. PCR analysis using primers that flank the inserted region present within CD44R1 identified an additional CD44 isoform, designated CD44R2, that contains only the last 69 amino acids present within the unique region of CD44R1. Peripheral blood mononuclear cells and granulocytes from normal individuals and patients with chronic myelogenous leukemia, polycythemia vera, or acute myelomonocytic leukemia, express both CD44R1 and CD44R2. In contrast, CD44R1 and CD44R2 appear to be differentially expressed in various CD44-positive cell lines. Thus KG1a, and the Epstein-Barr Virus-transformed B cell lines WalkDR4 and Way-1 express both CD44 and the CD44 isoforms CD44R1 and CD44R2, while the myeloid cell lines HL60 and U937 express high levels of CD44, but only very low levels of CD44R1 and CD44R2. The CD44-negative cell lines DHL-4, DHL-10, Jurkat, and K562 are also negative for CD44R1 and CD44R2.

The ability of granulocyte-macrophage colony-stimulating factor (GM- CSF) and G-CSF to influence hematopoiesis in long-term cultures (LTC) of human marrow was studied by cocultivating light density normal human marrow cells with human marrow fibroblast feeders engineered by retroviral infection to constitutively produce one of these growth factors. Feeders producing stable levels of 4 ng/mL GM-CSF or 20 ng/mL G-CSF doubled the output of mature nonadherent cells. The numbers of both colony forming unit-GM (CFU-GM) and erythroid burst forming unit (BFU-E) in the G-CSF LTC were also increased (twofold and fourfold, respectively, after 5 weeks in culture), but this effect was not seen with the GM-CSF feeders. At the time of the weekly half medium change 3H-thymidine suicide assays showed primitive adherent layer progenitors in LTC to be quiescent in both the control and GM-CSF cultures. In contrast, in the G-CSF cultures, a high proportion of primitive progenitors were in S-phase. A single addition of either recombinant GM- CSF or G-CSF to LTC in doses as high as 80 ng/mL and 150 ng/mL, respectively, failed to induce primitive progenitor cycling. However, three sequential daily additions of 150 ng/mL G-CSF did stimulate primitive progenitors to enter S-phase and a single addition of 5 or 12.5 ng/mL of G-CSF together with 10 ng/mL GM-CSF was able to elicit the same effect. Thus, selective elevation of G-CSF in human LTC stimulates proliferation of primitive clonogenic progenitors, which may then proceed through to the terminal stages of granulopoiesis. In contrast, the effects of GM-CSF in this system appear limited to terminally differentiating granulopoietic cells. However, when both GM- CSF and G-CSF are provided together, otherwise biologically inactive doses show strong stimulatory activity. These findings suggest that the production of both of these growth factors by normal stromal cells may contribute to the support and proliferation of hematopoietic cells, not only in LTC, but also in the microenvironment of the marrow in vivo.

PMX1 is a member of a non-clustered homeobox gene family, not normally expressed in hematopoietic cells, and first identified for its role in enhancing the binding of the serum response factor (SRF) to the serum responsive element (SRE). PMX1 has never been linked to leukemia on its own, raising the possibility of unique mechanisms underlying the oncogenicity of NUP98-PMX1. To elucidate the leukemogenic potential of NUP98-PMX1, we compared the effects of PMX1 and NUP98-PMX1 and, through strategic mutations, the involvement of the SRE in NUP98-PMX1-mediated leukemia. NUP98-PMX1, but not PMX1, had potent ability to impair differentiation, promote proliferation of myeloid progenitors, induce lethal myeloproliferative disease and to activate a number of genes previously linked to leukemic stem cells. Similar to NUP98-HOX fusions, the transforming potential of NUP98-PMX1 required the NUP98 portion and DNA-binding capability of the PMX1 homeodomain and collaborated with Meis1 to induce more rapid onset myeloproliferative-like myeloid leukemia. The transforming activity of NUP98-PMX1 was independent of its ability to interact with SRF. These findings provide novel evidence of the contributory role of the NUP98 sequence in conferring leukemogenic properties on a partner gene and point to common leukemogenic pathways for NUP98-PMX1 and NUP98-clustered HOX fusions.

Although hematopoiesis is known to originate in a population of very primitive cells with both lymphopoietic and myelopoietic potential, a procedure for enumerating such cells has to date not been available. We now describe a quantitative assay for long-term repopulating stem cells with the potential for reconstituting all hematopoietic lineages. This assay has two key features. The first is the use of competitive repopulation conditions that ensure not only the detection of a very primitive class of hematopoietic stem cells but also the survival of lethally irradiated mice transplanted with very low numbers of such cells. The second is the use of a limiting-dilution experimental design to allow stem cell quantitation. The assay involves transplanting limiting numbers of male "test" cells into lethally irradiated syngeneic female recipients together with 1-2 x 10(5) syngeneic female marrow cells whose long-term repopulating ability has been compromised by two previous cycles of marrow transplantation. The proportion of assay recipients whose regenerated hematopoietic tissues are determined to contain greater than or equal to 5% cells of test cell origin (male) greater than or equal to 5 weeks later is then used to calculate the frequency of competitive repopulating units (CRU) in the original male test cell suspension (based on Poisson statistics). Investigation of this assay system has shown that all three potential sources of stem cells (test cells, compromised cells, and the host) can under appropriate circumstances contribute to long-term hematopoietic regeneration, thus establishing both the competitive pressure of hematopoietic stem cells in the cotransplanted compromised population and in the host, and the need to use genetic markers to track the specific contribution of the injected test cells. Analysis of the frequency of CRU in test marrow suspensions that varied widely in their CRU content gave similar values when endpoints of either 5 or 10 weeks posttransplantation were used and when either recipient marrow or thymus was used to identify progeny populations. In addition, repopulation of marrow and thymus was found to be associated in most mice injected with limiting numbers of test cells. These findings are consistent with the conclusion that the assay is highly selective for a very primitive, totipotent, reconstituting hematopoietic stem cell and should therefore be particularly useful in future gene therapy-oriented research as well as for more basic studies of hematopoietic stem cell regulation and differentiation.

A large number of biologic, technological, and clinical studies await the development of procedures that will allow totipotent hematopoietic stem cells to be expanded in vitro. Previous work has suggested that hematopoiesis can be reconstituted using transplants of cells from long- term marrow cultures. We have used retrovirus mediated gene transfer to demonstrate that marked totipotent hematopoietic stem cells are both maintained and can be amplified in such cultures, and then subsequently regenerate and sustain lympho-myeloid hematopoiesis in irradiated recipients. Marrow cells from 5-fluorouracil-treated male mice were infected with a recombinant virus carrying the neomycin resistence gene and seeded onto irradiated adherent layers of pre-established, long- term marrow cultures of female origin. At 4 weeks, cells from individual cultures were transplanted into single or multiple female recipients. Southern blot analysis of hematopoietic tissue 45 days posttransplantation showed retrovirally marked clones common to lymphoid and myeloid tissues in 14 of 23 mice examined. Strikingly, for 3 of 4 long-term cultures, multiple recipients of cells from a single flask showed marrow and thymus repopulation with the same unique retrovirally marked clone. These results establish the feasibility of retroviral-marking techniques to demonstrate the maintenance of totipotent lympho-myeloid stem cells for at least 4 weeks in the long- term marrow culture system and provide the first evidence of their proliferation in vitro. Therefore, such cultures may serve as a starting point for identifying factors that stimulate totipotent hematopoietic stem cell expansion.

Procedures have been developed to label human erythropoietin (Ep) with biotin to detect and isolate the Ep-receptor. The labeling method used the abundant carbohydrate groups on Ep and resulted in biologically active biotin-Ep (b-Ep) containing 8 to 10 biotins per Ep molecule. Specific binding of b-Ep to cells from spleens of mice made anemic by phenylhydrazine injections was demonstrated using 125I-labeled streptavidin. B-Ep, together with fluorescently tagged streptavidin, was found to specifically detect Ep-receptor-bearing cells by flow cytometry. This was demonstrated in several ways. First, approximately 90% of nucleated spleen cells from phenylhydrazine-treated mice were clearly fluorescent after staining with b-Ep and streptavidin- phycoerythrin, whereas only background fluorescence was detected using spleen cells from untreated mice. In addition, Ep-receptors were detected on 5% to 10% of normal mouse bone marrow cells, and these cells could be identified as erythroid in nature by separating the cells into subpopulations based on light-scatter properties. Third, Ep- receptor expression was found to correlate positively with expression of transferrin receptors, confirming the erythroid nature of these cells. B-Ep was also used to isolate Ep-receptors from monkey COS cells transfected with the murine Ep-receptor cDNA. In these experiments a cell-surface-bound protein of approximately 65 Kd and an intracellular protein of approximately 60 Kd were isolated from these cells. The procedures described in this report for detecting Ep-receptor expressing cells and for isolating the Ep-receptor should be valuable for purifying erythroid cells from heterogeneous cell populations, for elucidating the structure of the Ep-receptor, and for studying the biological activities of Ep at the cellular and molecular level.

Procedures have been developed to label human erythropoietin (Ep) with biotin to detect and isolate the Ep-receptor. The labeling method used the abundant carbohydrate groups on Ep and resulted in biologically active biotin-Ep (b-Ep) containing 8 to 10 biotins per Ep molecule. Specific binding of b-Ep to cells from spleens of mice made anemic by phenylhydrazine injections was demonstrated using 125I-labeled streptavidin. B-Ep, together with fluorescently tagged streptavidin, was found to specifically detect Ep-receptor-bearing cells by flow cytometry. This was demonstrated in several ways. First, approximately 90% of nucleated spleen cells from phenylhydrazine-treated mice were clearly fluorescent after staining with b-Ep and streptavidin- phycoerythrin, whereas only background fluorescence was detected using spleen cells from untreated mice. In addition, Ep-receptors were detected on 5% to 10% of normal mouse bone marrow cells, and these cells could be identified as erythroid in nature by separating the cells into subpopulations based on light-scatter properties. Third, Ep- receptor expression was found to correlate positively with expression of transferrin receptors, confirming the erythroid nature of these cells. B-Ep was also used to isolate Ep-receptors from monkey COS cells transfected with the murine Ep-receptor cDNA. In these experiments a cell-surface-bound protein of approximately 65 Kd and an intracellular protein of approximately 60 Kd were isolated from these cells. The procedures described in this report for detecting Ep-receptor expressing cells and for isolating the Ep-receptor should be valuable for purifying erythroid cells from heterogeneous cell populations, for elucidating the structure of the Ep-receptor, and for studying the biological activities of Ep at the cellular and molecular level.

Gene expression analyses, gene targeting experiments and retroviral overexpression studies in the murine bone marrow transplantation model have provided strong correlative evidence for the involvement of clustered Hox genes in normal hematopoiesis. The data strongly support the hypothesis that the role of Hox genes in normal hematopoiesis is primarily at the level of hematopoietic stem cell function. A large body of evidence now links Hox genes to leukemic transformation including dysregulated HOX expression in leukemic patient samples, their involvement as oncogenic fusion proteins with NUP98 and their requirement for the oncogenicity of Mll fusions. In recent years, much attention has been devoted to the identification and characterization of leukemic stem cells. Given the documented role of Hox genes in hematopoiesis and leukemogenesis, we propose that Hox-dependent pathways are closely linked to the self-renewal program crucial to the origin and function of leukemic stem cells.

Transplantation of immunodeficient mice with human hematopoietic cells has greatly facilitated studies of the earliest stages of human hematopoiesis. These include demonstration of the ability of injected 'human-specific' hematopoietic growth factors to enhance the production of human cells at multiple levels of differentiation. In contrast, the effects of continuous exposure to such molecules have not been well investigated. Here, we show that nonobese diabetic severe combined immunodeficiency mice genetically engineered to produce ng/ml serum levels of human interleukin-3 (IL-3), granulocyte/macrophage-stimulating factor (GM-CSF) and Steel factor (SF) display a complex phenotype when transplanted with primitive human bone marrow (BM) or fetal liver cells. This phenotype is characterized by an enhancement of terminal human myelopoiesis and a matched suppression of terminal human erythropoiesis, with a slight reduction in human B-lymphopoiesis in the BM of the engrafted mice. Human clonogenic progenitors are more prevalent in the blood of the transplanted growth factor-producing mice and this is accompanied by a very marked reduction of more primitive human cells in the BM. Our findings suggest that long-term exposure of primitive human hematopoietic cells to elevated levels of human IL-3, GM-CSF and SF in vivo may deleteriously affect the stem cell compartment, while expanding terminal myelopoiesis.

CD24 (heat-stable antigen) is expressed in a developmentally regulated fashion by B cell precursors in mouse bone marrow (BM), but its role in B lymphopoiesis remains obscure. A slight overexpression of CD24 in transgenic (Tg) mice leads to depletion of B lymphoid cells in BM. The present study examines whether CD24 is involved in apoptotic selection of B lineage cells under normal microenvironmental conditions in vivo. Double immunofluorescence labeling and flow cytometry have been used to quantitate the apoptotic rates of phenotypically defined B cell populations in BM of CD24-Tg mice. Apoptosis of pre-B cells expressing cytoplasmic mu heavy chains of IgM but lacking surface (s)IgM was increased both ex vivo and in short-term culture, while the number of pre-B cells was halved compared to BM of normal mice. In contrast, B220+mu- pro-B cells and sIgM+ B lymphocytes showed no significant change in either apoptosis or number. The findings provide evidence that CD24 can play a role in vivo in modulating pre-B cell apoptosis, a quality control checkpoint in B cell development.

Considerable progress has been made in unraveling key pathogenetic steps in human leukemogenesis. In recent years, cytogenetic analyses and the molecular characterization of chromosomal breakpoints in patients with acute leukemia have proven that homeobox (HOX) genes, an evolutionarily highly conserved family of transcription factors, are critically involved in human leukemogenesis. HOX genes themselves, as well as their upstream regulators and cofactors, are implicated in the pathogenesis of leukemia, and experimental models using knock-in strategies or retrovirally induced overexpression of candidate genes have shown the leukemogenicity of homeobox genes. This review summarizes the recent advances in this field.

After bone marrow transplantation (BMT), there is a rapid regeneration to normal pretransplantation levels in the number of hematopoietic progenitors and mature end cells, whereas hematopoietic stem cell (HSC) numbers recover to only 5% to 10% of normal levels. This suggests that HSC are significantly restricted in their self-renewal behavior and hence in their ability to repopulate the host stem cell compartment. Previously, we have reported that HSC engineered to overexpress the homeobox transcription factor HOXB4 have a large repopulation advantage over untransduced cells as assessed at 4 months in a murine transplantation model (Sauvageau et al, Genes Dev 9:1753, 1995). This phenomenon has now been examined in detail for periods extending to 12 months in cohorts of mice transplanted with various numbers of HOXB4-transduced HSC. In all mice analyzed, HOXB4-transduced HSC were capable of fully reconstituting the HSC compartment, resulting, on average, in some 14-fold greater numbers of HSC than observed when transplanting control, non-HOXB4-transduced bone marrow cells. These data indicate that HOXB4 is a limiting factor in the regeneration of HSC to normal levels after BMT. Furthermore, we show that HOXB4-transduced HSC did not expand above levels normally observed in unmanipulated mice, indicating that its overexpression does not override the regulatory mechanisms that maintain the HSC pool size within normal limits.

A chimaera of stem cell factor (SCF) and a cellulose-binding domain from the xylanase Cex (CBDCex) effectively immobilizes SCF on a cellulose surface. The fusion protein retains both the cytokine properties of SCF and the cellulose-binding characteristics of CBDCex. When adsorbed on cellulose, SCF-CBDCex is up to 7-fold more potent than soluble SCF-CBDCex and than native SCF at stimulating the proliferation of factor-dependent cell lines. When cells are incubated with cellulose-bound SCF-CBDCex, activated receptors and SCF-CBDCex co-localize on the cellulose matrix. The strong binding of SCF-CBDCex to the cellulose surface permits the effective and localized stimulation of target cells; this is potentially significant for long-term perfusion culturing of factor-dependent cells. It also permits the direct analysis of the effects of surface-bound cytokines on target cells.

In 1996, three groups independently cloned the hemopoietic specific src homology 2 (SH2)-containing inositol 5′-phosphatase, SHIP (Damen et al. 1996; Lioubin et al. 1996; Kavanaugh et al. 1996). Although this intracellular enzyme, which is capable in vitro of hydrolyzing the 5′-phosphate from phosphatidylinositol-3,4,5-trisphosphate (PIP3) and inositol-1,3,4,5-tetrakisphosphate (IP4) (Damen et al. 1996), was originally identified as a 145-kDa protein that became both tyrosine phosphorylated and associated with the adaptor protein Shc in response to multiple cytokines and to B- and T-cell receptor engagement (reviewed in Liu et al. 1997), it is currently in the limelight because of its interaction with the negative co-receptor FcγRIIB. Specifically, SHIP is now known to inhibit immune receptor-mediated activation in both mast cells and B cells by binding to the tyrosine phosphorylated immunoreceptor tyrosine-based inhibitory motif (ITIM) of the inhibitory core-ceptor FcγRIIB (Vely et al. 1997; Tridandapani et al. 1997) and inhibiting FcɛRl- and B cell receptor-induced degranulation and proliferation, respectively (Ono et al. 1996; Ono et al. 1997). However, while activation of the B cell receptor in the absence of FcγRIIB co-clustering does not lead to significant tyrosine phosphorylation of SHIP (Chacko et al. 1996), activation of the FcɛRl alone does (Huber et al. 1998). We therefore investigated whether SHIP plays any role in modulating IgE-induced signaling via this immunoreceptor tyrosine based activation motif (ITAM)-containing receptor in the absence of FcγRIIB co-clustering. In this review we discuss our findings in this area.

Recent studies have shown efficient gene transfer to primitive progenitors in human cord blood (CB) when the cells are incubated in retrovirus-containing supernatants on fibronectin-coated dishes. We have now used this approach to achieve efficient gene transfer to human CB cells with the capacity to regenerate lymphoid and myeloid progeny in nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. CD34(+) cell-enriched populations were first cultured for 3 days in serum-free medium containing interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor, Flt3-ligand, and Steel factor followed by two 24-hour incubations with a MSCV-NEO virus-containing medium obtained under either serum-free or serum-replete conditions. The presence of serum during the latter 2 days made no consistent difference to the total number of cells, colony-forming cells (CFC), or long-term culture-initiating cells (LTC-IC) recovered at the end of the 5-day culture period, and the cells infected under either condition regenerated similar numbers of human CD34(+) (myeloid) CFC and human CD19(+) (B lymphoid) cells for up to 20 weeks in NOD/SCID recipients. However, the presence of serum increased the viral titer in the producer cell-conditioned medium and this was correlated with a twofold to threefold higher efficiency of gene transfer to all progenitor types. With the higher titer viral supernatant, 17% +/- 3% and 17% +/- 8%, G418-resistant in vivo repopulating cells and LTC-IC were obtained. As expected, the proportion of NEO + repopulating cells determined by polymerase chain reaction analysis of in vivo generated CFC was even higher (32% +/- 10%). There was no correlation between the frequency of gene transfer to LTC-IC and colony-forming unit-granulocyte-macrophage (CFU-GM), or to NOD/SCID repopulating cells and CFU-GM (r2 = 0.16 and 0.17, respectively), whereas values for LTC-IC and NOD/SCID repopulating cells were highly and significantly correlated (r2 = 0.85). These findings provide further evidence of a close relationship between human LTC-IC and NOD/SCID repopulating cells (assessed using a/= 6-week CFC output endpoint) and indicate the predictive value of gene transfer measurements to such LTC-IC for the design of clinical gene therapy protocols.

The use of gene transfer procedures has greatly facilitated the investigation of cell lineage relationships and other developmental processes in a variety of primary tissues. In this report we described the infection and selection of primary human breast epithelial cells using retroviral vectors (Jzen-HSA-NEO and MSCV-HSA.NEO) containing the complete 228 bp coding sequence of a murine cell surface marker (Heat Stable Antigen, HSA) as well as the neomycin resistance (neo(r)) gene. Expression of the transduced HSA gene was detectable using either flow cytometry or immunohistochemistry after staining cells with an anti-murine HSA-specific antibody (M1/69). Expression of the transduced neo(r) gene conferred resistance to G418. In initial experiments with the MCF-7 breast cancer cell line, continued expression of both markers was demonstrated in a high proportion of cells for at least 4 weeks after their infection by positive M1/69 staining of cells that had been selected by prior incubation in G418. Evidence of gene transfer to early stage (9 days old) primary cultures of normal human breast epithelial cells (15 experiments with cells from 12 normal individuals) was also obtained using an infection protocol in which these calls were exposed to helper-free viral supernatants (2 incubations, 4 to 6 hr each) after being subcultured for 12 to 18 hr to increase their rate of proliferation. The presence of 5-50% (mean = 26%) HSA+ cells was demonstrated in these experiments within 5 days after their infection and the HSA+ populations included both myoepithelial and luminal phenotypes. The transduced (HSA+) cells within both of these subpopulations could also be separately isolated by FACS and subcultured. These results should provide an important starting point for future studies of genetically modified or marked primary human breast epithelial cell populations.

Although studies with truncated erythropoietin receptors (EpoRs) have suggested the tyrosine phosphorylation (Yphos) of the EpoR may not play a significant role in Epo-induced proliferation, we found, using a full length EpoR mutant designed Null, in which all 8 of the intracellular tyrosines (Ys) were substituted with phenylalanines (Fs), that Null cells required 5-10 fold more Epo than wild type (WT) EpoR containing cells in order to proliferate as well. Moreover, a comparison of Epo-induced proliferation with Epo-induced Yphos patterns, using DA-3 cells expressing WT, Null and various Y to F EpoR point mutants revealed that Stat5 Yphos and activation correlated directly with proliferation and was mediated primarily throuhg the most membrane proximal Y, i.e., Y343, although other tyrosines (most likely Y401 and Y431) within the EpoR could activate Stat5 in its absence. We also found that EpoR Yphos was essential for the Yphos of Shc and for the Yphos and association of a 145 kDa protein with Shc. We purified and cloned this Shc-associated 145 kDa protein and found that it was a unique SH2 containing inositol polyphosphate-5-phosphatase. This novel enzyme, which we have called SHIP for SH2-containing inositol-phosphatase, may modulate both Ras and inositol signaling pathways.

Several homeobox genes of the HOXA and HOXB clusters are expressed in primitive blood cells, suggesting a role for HOX genes in normal hematopoiesis. The HOXA9 gene is expressed in CD34+ marrow cells and in developing lymphocytes. We examined blood-forming organs of mice homozygous for an interrupted HOXA9 allele to determine if loss of HOX gene function is deleterious to hematopoiesis. HOXA9-/- mice have approximately 30% to 40% reductions in total leukocytes and lymphocytes (P.001) and a blunted granulocytic response to granulocyte colony-stimulating factor (G-CSF). Homozygous mice have significantly smaller spleens and thymuses. Myeloid/erythroid and pre-B progenitors in the marrow are significantly reduced, but no significant decreases are noted in mixed colonies, day 12 colony-forming units-spleen (CFU-S), or long-term culture-initiating cells (LTC-IC), suggesting little or no perturbation in earlier progenitors. Heterozygous animals display no hematopoietic defects. The abnormalities in leukocyte production are transplantable, indicating that the defect resides in the hematopoietic cells. These studies demonstrate a physiologic role for a HOX gene in blood cell differentiation, with the greatest apparent influence of HOXA9 at the level of the committed progenitor.

Recent assessment of the long-term repopulating activity of defined subsets of hematopoietic cells has offered new insights into the characteristics of the transplantable stem cells of this system; however, as yet, there is very little known about mechanisms that regulate their self-renewal in vivo. We have now exploited the ability to quantitate these cells using the competitive repopulating unit (CRU) assay to identify the role of both intrinsic (ontological) and extrinsic (transplanted dose-related) variables that may contribute to the regulation of CRU recovery in vivo. Ly5.1 donor cells derived from day-14.5 fetal liver (FL) or the bone marrow (BM) of adult mice injected 4 days previously with 5-fluorouracil were transplanted at doses estimated to contain 10, 100, or 1,000 long-term CRU into irradiated congenic Ly5.2 adult recipient mice. Eight to 12 months after transplantation, there was a complete recovery of BM cellularity and in vitro clonogenic progenitor numbers and a nearly full recovery of day-12 colony-forming unit-spleen numbers irrespective of the number or origin of cells initially transplanted. In contrast, regeneration of Ly5.1+ donor-derived CRU was incomplete in all cases and was dependent on both the origin and dose of the transplant, with FL being markedly superior to that of adult BM. As a result, the final recovery of the adult marrow CRU compartment ranged from 15% to 62% and from 1% to 18% of the normal value in recipients of FL and adult BM transplantation, respectively, with an accompanying maximum CRU amplification of 150-fold for recipients of FL cells and 15-fold for recipients of adult BM cells. Interestingly, the extent of CRU expansion from either source was inversely related to the number of CRU transplanted. These data suggest that recovery of mature blood cell production in vivo may activate negative feedback regulatory mechanisms to prematurely limit stem cell self-renewal ability. Proviral integration analysis of mice receiving retrovirally transduced BM cells confirmed regeneration of totipotent lymphomyeloid repopulating cells and provided evidence for a greater than 300-fold clonal amplification of a single transduced stem cell. These results highlight the differential regenerative capacities of CRU from fetal and adult sources that likely reflect intrinsic, genetically defined determinants of CRU expansion but whose contribution to the magnitude of stem cell amplification ultimately obtained in vivo is also strongly influenced by the initial number of CRU transplanted. Such findings set the stage for attempts to enhance CRU regeneration by administration of agents that may enable full expression of regenerative potential or through the expression of intracellular gene products that may alter intrinsic regenerative capacity.

We recently cloned and sequenced a cDNA encoding a 145-kD protein from the murine hematopoietic cell line B6SUtA, that becomes tyrosine phosphorylated and associated with Shc after cytokine stimulation. Based on its domains and enzymatic activity, we named this protein SHIP for SH2-containing inositol phosphatase (Damen et al, Proc Natl Acad Sci USA 93:1689, 1996). We describe here the cloning of the human homologue of murine SHIP (mSHIP) from a human megakaryocytic cell line (MO7e) lambda gt11 cDNA library using two nonoverlapping mSHIP cDNA fragments as probes. Northern blot analysis suggests that human SHIP (hSHIP) is expressed as a 5.3-kb mRNA in human bone marrow and a wide variety of other tissues. Sequence analysis of this cDNA predicts a protein of 1188 amino acids exhibiting 87.2% overall sequence identity with mSHIP. Contained within the defined open reading frame is an N-terminal, group l src homology 2 (SH2) domain; three NXXY motifs that, if phosphorylated, could be bound by phosphotyrosine binding (PTB) domains; a C-terminal proline-rich region; and two centrally located inositol polyphosphate 5-phosphatase motifs. Fluorescence in situ hybridization, using the full-length hSHIP cDNA as a probe, mapped hSHIP to the long arm of chromosome 2 at the border between 2q36 and 2q37.

Recombinant retroviruses offer many advantages for the genetic modification of human hematopoietic cells, although their use in clinical protocols has thus far given disappointing results. There is therefore an important need to develop new strategies that will allow effectively transduced primitive hematopoietic target populations to be both rapidly characterized and isolated free of residual nontransduced but biologically equivalent cells. To address this need, we constructed a murine stem cell virus (MSCV)-based retroviral vector containing the 228-bp coding sequence of the murine heat-stable antigen (HSA) and generated helper virus-free amphotropic MSCV-HSA producer cells by transfection of GP-env AM12 packaging cells. Light density and, in some cases, lineage marker-negative (lin-) normal human marrow or mobilized peripheral blood cells preactivated by exposure to interleukin-3 (IL-3), IL-6, and Steel factor in vitro for 48 hours were then infected by cocultivation with these MSCV-HSA producer cells for a further 48 hours in the presence of the same cytokines. Fluorescence-activated cell sorting (FACS) analysis of the cells 24 hours later showed 21% to 41% (mean, 27%) of those that were still CD34+ to have acquired the ability to express HSA. The extent of gene transfer to erythroid and granulopoietic progenitors (burst-forming unit-erythroid and colony-forming unit-granulocyte-macrophage), as assessed by the ability of these cells to form colonies of mature progeny in the presence of normally toxic concentrations of G418, averaged 11% and 12%, respectively, in 6 experiments. These values could be increased to 100% and 77%, respectively, by prior isolation of the CD34+HSA+ cell fraction and were correspondingly decreased to an average of 2% and 5%, respectively, in the CD34+HSA- cells. In addition, the extent of gene transfer to long-term culture-initiating cells (LTC-IC) was assessed by G418 resistance. The average gene transfer to LTC-IC-derived colony-forming cells in the unsorted population wasor = 7% in 4 experiments. FACS selection of the initially CD34+HSA+ cells increased this value to 86% and decreased it to 3% for the LTC-IC plated from the CD34+HSA- cells. Transfer of HSA gene expression to a phenotypically defined more primitive subpopulation of CD34+ cells, ie, those expressing little or no CD38, could also be shown by FACS analysis of infected populations 24 hours after infection. These findings underscore the potential use of retroviral vectors encoding HSA for the specific identification and non-toxic selection immediately after infection of retrovirally transduced populations of primitive human hematopoietic cells. In addition, such vectors should facilitate the subsequent tracking of their marked progeny using multiparameter flow cytometry.

To study the role of the murine heat-stable Ag (HSA) in lymphocyte maturation, we generated transgenic mice in which the HSA cDNA was under the transcriptional control of the TCR V beta promoter and Ig mu enhancer. The HSA transgene was expressed during all stages of B lymphocyte maturation. Expression was first detected in the earliest lymphoid-committed progenitors, which normally do not express HSA, and subsequently reached the highest levels in pro- and pre-B cells. In bone marrow, the number of IL-7-responsive clonogenic progenitors was4% of normal, whereas the frequency of earlier B lymphocyte-restricted precursors, detectable as Whitlock-Witte culture-initiating cells, was normal. Pro- and pre-B cells detected by flow cytometry were reduced by approximately 50% relative to controls. Mature splenic B cells were also reduced but to a lesser extent than in marrow, and their response to LPS stimulation was impaired. Reconstitution of SCID and BALB/c-nu/nu mice with HSA transgenic marrow indicated that the perturbations in B lymphopoiesis were not caused by a defective marrow microenvironment or by abnormal T cells. Our previous studies showed elevated HSA expression throughout thymocyte development, which resulted in a profound depletion of CD4+CD8+ double-positive and single-positive thymocytes. Together, these results indicate that HSA levels can determine the capacity of early T and B lymphoid progenitors to proliferate and survive. Therefore, HSA could serve as an important regulator during the early stages of B and T lymphopoiesis.