MIM regulates the trafficking of bone marrow cells via modulating surface expression of CXCR4.

Missing in metastasis (MIM) is abundantly expressed in hematopoietic cells. Here we characterized the impact of MIM deficiency on murine bone marrow (BM) cells. Although MIM(-/-) cells proliferated similarly to wild type (WT), they exhibited stronger response to chemokine stromal-derived factor 1 (SDF-1), increase in surface expression of CXCR4, impaired CXCR4 internalization and constitutive activation of Rac, Cdc42 and p38. Transplantation of MIM(-/-) BM cells into lethally irradiated mice showed enhanced homing to BM, which was abolished when mice were pretreated with a p38 antagonist. Interestingly, MIM(-/-) BM cells, including hematopoietic stem and progenitor cells (HSPCs), showed two- to fivefold increase in mobilization into the peripheral blood upon treatment with AMD3100. In vitro, MIM(-/-) leukocytes were susceptible to AMD3100 and maintained increased response to AMD3100 for mobilization even after transfer into WT mice. MIM(-/-) mice had also a higher level of SDF-1 in the circulation. Our data highlighted an unprecedented role of MIM in the homeostasis of BM cells, including HSPCs, through modulation of the CXCR4/SDF-1 axis and interactions of BM leukocytes with their microenvironments.

Mice deficient in MIM expression are predisposed to lymphomagenesis.

Missing in metastasis (MIM) is a member of newly emerged inverse Bin-Amphiphysin-Rvs (BAR) domain protein family and a putative metastasis suppressor. Although reduced MIM expression has been associated with bladder, breast and gastric cancers, evidence for the role of MIM in tumor progression remains scarce and controversial. Herein we characterized a MIM knockout mouse strain and observed that MIM-deficient mice often developed enlarged spleens. Autopsy and histological analysis revealed that nearly 78% of MIM(-/-) mice developed tumors with features similar to diffuse large B lymphoma during a period from 1 to 2 years. MIM(-/-) mice also exhibited abnormal distribution of B cells in lymphoid organs with decrease in the spleen but increase in the bone marrow and the peripheral blood. Furthermore, the bone marrow of MIM(-/-) mice contained a higher percentage of pre-B2 cells but fewer immature B-cells than wild-type mice. In response to CXCL13, a B-cell chemokine released from splenic stromal cells, MIM-deficient B-cells did not undergo chemotaxis or morphological changes in response to the chemokine and also did not internalize CXCR5, the receptor of CXCL13. Microarray analyses demonstrated that MIM is the only member of the I-BAR domain family that was highly expressed in human B cells. However, low or absent MIM expression was common in either primary B-cell malignancies or established B-cell acute lymphocytic leukemia or lymphomas. Thus, our data demonstrate for the first time an important role for MIM in B-cell development and suggest that predisposition of MIM-null mice to lymphomagenesis may involve aberrant interactions between B lineage cells and the lymphoid microenvironment.

High frequencies of leukemia stem cells in poor-outcome childhood precursor-B acute lymphoblastic leukemias.

In order to develop a xenograft model to determine the efficacy of new therapies against primary human precursor-B acute lymphoblastic leukemia (ALL) stem cells (LSCs), we used the highly immunodeficient non-obese diabetic (NOD).Cg-Prkdc(scid)IL2rg(tmlWjl)/SzJ (NOD-severe combined immune deficient (scid) IL2rg(-/-)) mouse strain. Intravenous transplantation of 2 of 2 ALL cell lines and 9 of 14 primary ALL cases generated leukemia-like proliferations in recipient mice by 1-7 months after transplant. Leukemias were retransplantable, and the immunophenotypes, gene rearrangements and expression profiles were identical or similar to those of the original primary samples. NOD-scid mice transplanted with the same primary samples developed similar leukemias with only a slightly longer latency than did NOD-scid-IL2Rg(-/-) mice. In this highly sensitive NOD-scid-IL2Rg(-/-)-based assay, 1-100 unsorted primary human ALL cells from five of five tested patients, four of whom eventually experienced leukemia relapse, generated leukemias in recipient mice. This very high frequency of LSCs suggests that a hierarchical LSC model is not valuable for poor-outcome ALL.

Identification of a region on the outer surface of the CBFbeta-SMMHC myeloid oncoprotein assembly competence domain critical for multimerization.

In the core binding factor (CBF)beta-smooth muscle myosin heavy chain (SMMHC) acute myeloid leukemia (AML) oncoprotein, CBFbeta lies N-terminal to the alpha-helical rod domain of SMMHC. Deletion of the SMMHC assembly competence domain (ACD), conserved among skeletal, smooth and nonmuscle myosins, prevents multimerization, inhibition of CBF and inhibition of cell proliferation. To define the amino acids critical for ACD function, three outer surface residues of ACD helices A-D, the subsequent helices E-H or the more N-terminal X or Z helices were now mutated. Variants were assessed for multimerization in low ionic strength in vitro and for nuclear localization as a measure of in vivo multimerization. Mutation of individual helices C-H reduced multimerization, with alteration of the outer surface of helices D or E having the greatest effect. The ability of these SMMHC variants to slow murine myeloid progenitor proliferation largely paralleled their effects on multimerization. Divergence at the boundaries of the ACD may reflect quantitative differences between in vitro and in vivo filament assembly. Each helix mutant retained the ability to bind the mSin3A corepressor. Agents interacting with the outer surface of the CBFbeta-SMMHC ACD that prevent multimerization may be effective as novel therapeutics in AML.

CBFbeta-SMMHC slows proliferation of primary murine and human myeloid progenitors.

CBFbeta-SMMHC is expressed in 8% of acute myeloid leukemias and inhibits AML1/RUNX1. In this study, murine marrow or human CD34(+) cells were transduced with retroviral or lentiviral vectors expressing CBFbeta-SMMHC or two mutant variants. CBFbeta-SMMHC reduced murine or human myeloid cell proliferation three- to four-fold in liquid culture relative to empty vector-transduced cells, during a period when vector-transduced cells accumulated five-fold and human cells 20-fold. CBFbeta-SMMHC decreased the formation of myeloid, but not erythroid, colonies two- to four-fold, and myeloid colonies expressing CBFbeta-SMMHC were markedly reduced in size. However, CBFbeta-SMMHC did not slow differentiation to granulocytes or monocytes. Neither CBFbeta-SMMHC(Delta2-11), which does not bind AML1, nor CBFbeta-SMMHC(DeltaACD), which does not multimerize or efficiently bind corepressors, slowed proliferation or reduced myeloid colonies. CBFbeta-SMMHC increased the G1/S ratio 1.4-fold. AML1 had an effect opposite to CBFbeta-SMMHC, stimulating proliferation of murine myeloid progenitors 2.0-fold in liquid culture. Thus, CBFbeta-SMMHC directly inhibits the proliferation of normal myeloid progenitors via inhibition of AML1 and dependent upon the integrity of its assembly competence domain. These findings support the development of therapeutics that target the ability of CBFbeta-SMMHC to interact with AML1 or to multimerize via its assembly competence domain.

Ex vivo zidovudine (AZT) treatment of CD34+ bone marrow progenitors causes decreased steady state mitochondrial DNA (mtDNA) and increased lactate production.

Haematopoietic suppression is one of the dose-limiting side effects of chronic zidovudine (AZT) therapy. We tested the hypothesis that AZT would reduce mitochondrial DNA (mtDNA) content in haematopoietic progenitors causing impaired haematopoiesis and mitochondrial dysfunction. We studied the effects of AZT 0-50 microM in vitro, on normal human CD34+ haematopoietic progenitor cells cultured ex vivo for up to 12 days. The mean AZT IC50 for granulocyte (phenotype CD15+/CD14-) and erythroid (phenotype glycophorin+/CD45-) cell proliferation was 2.5 microM (SD+/-0.7) and 0.023 microM (SD+/-0.005), respectively. In myeloid-rich cell cultures, the mean lactate content of the media, compared to untreated controls, increased by 86% (SD+/-23) at 10 microM AZT and in erythroid-rich cultures it increased by 134% (SD+/-24) in the presence of 0.5 microM AZT. In myeloid-rich cultures the AZT IC50 for the reduction in the mitochondrial/nuclear DNA content ratio was 5.6 microM, whereas in erythroid rich cultures this AZT IC50 was < 0.0005 microM. AZT produced concentration-dependent inhibition of CD34+ progenitor proliferation into both myeloid and erythroid lineages; erythropoiesis was more sensitive than myelopoiesis. Concurrently, AZT reduced steady state mtDNA content, while increasing lactate production. These findings support the hypothesis that mtDNA is one of the intracellular targets involved in the pathogenesis of AZT-associated bone marrow progenitor cell toxicity.

Human AML cells in NOD/SCID mice: engraftment potential and gene expression.

Most cases of human acute myeloid leukemia (AML) engraft in irradiated non-obese diabetic/severe combined immunodeficient (NOD/SCID) mice. Intravenous transfer of as few as 10(5) human AML cells resulted in engraftment. Cases with poor prognosis clinical features, including FLT3 mutations, tended to engraft efficiently. Nevertheless, AML cells obtained from patients at relapse did not engraft more efficiently than cells obtained from the same patients at initial diagnosis. One passage of human AML cells in NOD/SCID mice did not appear to select for increased virulence, as measured by serial transplantation efficiency. Finally, cDNA microarray analyses indicated that approximately 95% of genes were expressed at similar levels in human AML cells immunopurified after growth in mice, as compared to cells assessed directly from patients. Thus, the growth of human AML cells in NOD/SCID mice could yield large numbers of human AML cells for direct experimental use and could also function as a renewable, potentially unlimited source of leukemia cells, via serial transplantation.

Intensive timed sequential remission induction chemotherapy with high-dose cytarabine for childhood acute myeloid leukemia.

BACKGROUND: Timed sequential chemotherapy and high-dose cytarabine (cytosine arabinoside, Ara-C; HDAC) are both effective treatments for acute myeloid leukemia (AML). We review our institutional experience with timed sequential induction chemotherapy consisting of daunorubicin/Ara-C/-thioguanine (DAT) or idarubicin/Ara-C/-thioguanine (IAT) followed on day 14 by HDAC regardless of the degree of marrow aplasia for children with newly diagnosed AML. PROCEDURE: Children presenting with newly diagnosed AML were treated with induction chemotherapy consisting of idarubicin (12 mg/m/day on days 1-3 or daunorubicin at 45 mg/m(2)/day for the first five patients), Ara-C (100 mg/m(2)/day by continuous infusion on days 1-7), and thioguanine (100 mg/m(2)/day on days 1-7). HDAC (1 g/m(2)/dose every 12 hr for 10 doses) was administered beginning on day 14, regardless of the results of bone marrow examination. RESULTS: Thirteen children received timed sequential HDAC. Only one child received HDAC later than Day 18. Eleven of the children achieved a complete remission. All patients experienced grade 4 hematologic toxicity, and all had fever as well. There were 11 children with documented infections. Ten had grade 3 or 4 GI toxicity. One patient died of sepsis. CONCLUSIONS: HDAC administered as a part of timed sequential therapy yields an excellent remission induction rate with manageable toxicity.

Human CD34+ cell preparations contain over 100-fold greater NOD/SCID mouse engrafting capacity than do CD34- cell preparations.

OBJECTIVE: The CD34 cell surface marker is used widely for stem/progenitor cell isolation. Since several recent studies reported that CD34(-) cells also have in vivo engrafting capacity, we quantitatively compared the engraftment potential of CD34(+) vs CD34(-) cell preparations from normal human placental/umbilical cord blood (CB), bone marrow (BM), and mobilized peripheral blood (PBSC) specimens, using the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse model. METHODS: CD34(+) and CD34(-) cell preparations were purified by four different approaches in 14 individual experiments involving 293 transplanted NOD/SCID mice. In most experiments, CD34(+) cells were depleted twice (CD34(=)) in order to obtain efficient depletion of CD34(+) cells from the CD34(-) cell preparations. RESULTS: Dose-dependent levels of human hematopoietic cells were observed after transplantation of CD34(+) cell preparations. To rigorously assess the complementary CD34(-) cell preparations, cell doses 10- to 1000-fold higher than the minimum dose of the CD34(+) cell preparations necessary for engraftment were transplanted. Nevertheless, of 125 NOD/SCID mice transplanted with CD34(-) cell preparations purified from the same starting cells, only six mice had detectable human hematopoiesis, by flow cytometric or PCR assay. CONCLUSIONS: CD34(-) cells provide only a minor contribution to hematopoietic engraftment in this in vivo model system, as compared to CD34(+) cells from the same samples of noncultured human cells. Hematopoiesis derived from actual CD34(-) cells is difficult to distinguish from that due to CD34(+) cells potentially contaminating the preparations.

Inhibition of FLT3-mediated transformation by use of a tyrosine kinase inhibitor.

FLT3 is a member of the type III receptor tyrosine kinase (RTK) family. These receptors all contain an intrinsic tyrosine kinase domain that is critical to signaling. Aberrant expression of the FLT3 gene has been documented in both adult and childhood leukemias including AML, ALL and CML. In addition, 17-27% of pediatric and adult patients with AML have small internal tandem duplication mutations in FLT3. Patients expressing the mutant form of the receptor have been shown to have a decreased chance for cure. Our previous study, using a constitutively activated FLT3, demonstrated transformation of Ba/F3 cells and leukemic development in an animal model. Thus, there is accumulating evidence for a role for FLT3 in human leukemias. This has prompted us to search for inhibitors of FLT3 as a possible therapeutic approach in these patients. AG1296 is a compound of the tyrphostin class that is known to selectively inhibit the tyrosine kinase activity of the PDGF and KIT receptors. Since FLT3 is a close relative of KIT, we wanted to test the possible inhibitory activity of AG1296 on FLT3. In transfected Ba/F3 cells, AG1296 selectively and potently inhibited autophosphorylation of FL-stimulated wild-type and constitutively activated FLT3. Treatment by AG1296 abolished IL-3-independent proliferation of Ba/F3 cells expressing the constitutively activated FLT3 and thus, reversed the transformation mediated by activated FLT3. Inhibition of FLT3 activity by AG1296 in cells transformed by activated FLT3 resulted in apoptotic cell death, with no deleterious effect on their parental counterparts. Addition of IL-3 rescued the growth of cells expressing activated FLT3 in the presence of AG1296. This demonstrates that the inhibition is specific to the FLT3 pathway in that it leaves the kinases of the IL-3 pathway and other kinases further downstream involved in proliferation intact. Several proteins phosphorylated by the activated FLT3 signaling pathway, including STAT 5A, STAT 5B and CBL, were no longer phosphorylated when these cells were treated with AG1296. The activity against FLT3 suggests a potential therapeutic application for AG1296 or similar drugs in the treatment of leukemias involving deregulated FLT3 tyrosine kinase activity and as a tool for studying the biology of FLT3.

The adapter protein CrkL associates with CD34.

CD34 is a cell-surface transmembrane protein expressed specifically at the stem/progenitor stage of lymphohematopoietic development that appears to regulate adhesion. To elucidate intracellular signals modified by CD34, we designed and constructed glutathione-S-transferase (GST)- fusion proteins of the intracellular domain of full-length CD34 (GST-CD34i(full)). Precipitation of cell lysates using GST-CD34i(full) identified proteins of molecular mass 39, 36, and 33 kd that constitutively associated with CD34 and a 45-kd protein that associated with CD34 after adhesion. By Western analysis, we identified the 39-kd protein as CrkL. In vivo, CrkL was coimmunoprecipitated with CD34 using CD34 antibodies, confirming the association between CrkL and CD34. CD34 peptide inhibition assays demonstrated that CrkL interacts at a membrane-proximal region of the CD34 tail. To identify the CrkL domain responsible for interaction with CD34, we generated GST-fusion constructs of adapter proteins including GST-CrkL3' (C-terminal SH3) and GST-CrkL5' (N-terminal SH2SH3). Of these fusion proteins, only GST-CrkL3' could precipitate endogenously expressed CD34, suggesting that CD34 binds the C-terminal SH3 domain of CrkL. Interestingly, there appears to be differential specificity between CrkL and CrkII for CD34, because GST-CD34i(full) did not precipitate CrkII, a highly homologous Crk family member. Furthermore, GST-CD34i(full) did not bind c-Abl, c-Cbl, C3G, or paxillin proteins that are known to associate with CrkL, suggesting that CD34 directly interacts with the CrkL protein. CD34i(full) association with Grb or Shc adapter proteins was not detected. Our investigations shed new light on signaling pathways of CD34 by demonstrating that CD34 couples to the hematopoietic adapter protein CrkL. (Blood. 2001;97:3768-3775)

High levels of transgene expression following transduction of long-term NOD/SCID-repopulating human cells with a modified lentiviral vector.

Both oncoretroviral and lentiviral vectors have been shown to transduce CD34(+) human hematopoietic stem cells (HSC) capable of establishing human hematopoiesis in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mice that support partially human hematopoiesis. We and others have reported that murine stem cell virus (MSCV)-based oncoretroviral vectors efficiently transduced HSC that had been cultured ex vivo for 4-7 days with cytokines, resulting in transgene expression in lymphoid and myeloid progenies of SCID-engrafting cells 4-8 weeks post-transplantation. Although lentiviral vectors have been demonstrated to transduce HSC under minimal ex vivo culture conditions, concerns exist regarding the level of transgene expression mediated by these vectors. We therefore evaluated a novel hybrid lentiviral vector (GIN-MU3), in which the U3 region of the HIV-1 long terminal repeat was replaced by the MSCV U3 region (MU3). Human cord blood CD34(+) cells were transduced with vesicular stomatitis virus G envelope protein-pseudotyped lentiviruses during a 48-hour culture period. After a total of 4 days in culture, transduced cells were transplanted into NOD/SCID mice to examine gene transfer and expression in engrafting human cells. Fifteen weeks post-transplantation, 37% +/- 12% of engrafted human cells expressed the green fluorescence protein (GFP) gene introduced by the lentiviral vector. High levels of GFP expression were observed in lymphoid, myeloid and erythroid progenies, and in engrafted human cells that retained the CD34(+) phenotype 15 weeks post-transplantation. This study provides evidence that lentiviral vectors transduced both short-term and long-term engrafting human cells, and mediated persistent transgene expression at high levels in multiple lineages of hematopoietic cells.

Characterization of the tyrosine kinase Tnk1 and its binding with phospholipase C-gamma1.

Tnk1 is a nonreceptor tyrosine kinase cloned from CD34+/Lin-/CD38- hematopoietic stem/progenitor cells. The cDNA predicts a 72-kDa protein containing an NH(2)-terminal kinase, a Src Homology 3 (SH3) domain, and a proline-rich (PR) tail. We generated rabbit antiserum to a GST-Tnk1(SH3) fusion protein. Affinity-purified anti-Tnk1 antibodies specifically recognized a 72-kDa protein in Tnk1-transfected COS-1 cells and cells which express Tnk1 mRNA. Western blot analysis indicated that Tnk1 is expressed in fetal blood cells, but not in any other hematopoietic tissues examined. Tnk1 immunoprecipitated from cell lysates possessed kinase activity and was tyrosine phosphorylated. In binding experiments with a panel of GST-fusion constructs, only GST-PLC-gamma1(SH3) interacted with in vitro translated Tnk1. GST-protein precipitations from cell lysates confirmed that GST-PLC-gamma1(SH3) associated with endogenously expressed Tnk1. Conversely, GST-Tnk1(PR) protein constructs complexed with endogenously expressed PLC-gamma1. The association of Tnk1 with PLC-gamma1 suggests a role for Tnk1 in phospholipid signal transduction.

Ex vivo culture of cord blood CD34+ cells expands progenitor cell numbers, preserves engraftment capacity in nonobese diabetic/severe combined immunodeficient mice, and enhances retroviral transduction efficiency.

Ex vivo culture of hematopoietic stem/progenitor cells could potentially improve the efficacy of human placental/umbilical cord blood (CB) in clinical hematopoietic stem cell (HSC) transplantation and allow gene transduction using conventional retroviral vectors. Therefore, we first examined the effects of a 7-day period of ex vivo culture on the hematopoietic capacity of CB CD34+ cells. Medium for the ex vivo cultures contained either serum and six recombinant human hematopoietic growth factors (GFs), including Flt-3 ligand (FL), Kit ligand (KL = stem cell factor), thrombopoietin (Tpo), interleukin 3 (IL-3), granulocyte colony-stimulating factor (G-CSF), and interleukin 6 (IL-6), or a serum-free medium containing only FL, KL, and Tpo. After culture under both ex vivo conditions, the total numbers of viable cells, CD34+ cells, colony-forming cells (CFCs), and long-term culture initiating cells (LTC-ICs) were increased. In contrast, the severe combined immunodeficiency (SCID) mouse engrafting potential (SEP) of cultured cells was slightly decreased, as compared with fresh cells. Nevertheless, cultured human CB CD34+ cells were able to generate engraftment, shown to persist for up to 20 weeks after transplantation. We next tested the efficacy of retroviral transduction of cultured cells. Transduced cultured human cells were able to engraft in NOD/SCID mice, as tested 4 weeks after transplantation, and EGFP+CD34+ cells and EGFP+ CFCs were isolated from the chimeras. Thus, although additional improvements in ex vivo culture are still needed to expand the numbers and function of human HSCs, the current conditions appear to allow gene transduction into hematopoietic SCID engrafting cells, while at least qualitatively preserving their in vivo engraftment potential.

Human hematopoietic stem/progenitor cells generate CD5+ B lymphoid cells in NOD/SCID mice.

The nonobese diabetic/severe combined immunodeficient (NOD/SCID) xenotransplantation model is increasingly utilized to study both human lymphohematopoietic stem/progenitor cells and committed cell types. Human B lymphoid cells develop and proliferate in this model. We found high numbers of CD19+CD5+ B lymphoid cells in the bone marrows and spleens of NOD/SCID mice transplanted with human CD34+ stem/progenitor cells. The CD5+ cells accounted for a particularly large percentage of the B lymphoid cells in the spleens of chimeras analyzed three months after transplantation. CD19+CD5+ cells from all the analyzed chimeras coexpressed HLA-DR, surface IgM, CD20, CD38, CD43, and CD45. However, CD19+CD5+ cells were negative for kappa light chain, CD10, CD11a, CD11b, CD15, CD21, CD22, CD23, CD25, CD34, CD35, CD44, CD62L, CD69, and CD71. Cell surface expression of the lambda light chain, surface IgD, CD9, and CD40 antigens was detected in some but not all chimeras. Thus, the CD19+CD5+ cell population detected in our study has the phenotype of previously described CD5+ B lymphoid cells in humans and other species. The origin and role of the B lymphoid cells which express CD5 cell surface glycoprotein are poorly understood. The malignant cells in B lymphoid chronic lymphocytic leukemia express CD5, and the numbers of CD5+ B lymphoid cells are elevated in several autoimmune conditions. The human-NOD/SCID chimera system may provide an in vivo model to investigate the maturation and development of this cryptic human CD5+ B lymphoid cell subpopulation.

Comparisons of alloreactive potential of clinical hematopoietic grafts.

BACKGROUND: We sought to compare the immunoreactive potential of human cord blood (CB) versus normal adult bone marrow (BM) versus mobilized blood (peripheral blood stem cells; PBSC) from cancer patients. METHODS: Forty mice were randomized to receive a range of doses of T cell-replete cell preparations from one of the above three cell sources. Twenty-eight control mice underwent transplantation with T cell-depleted cells. Mice were observed for 60 days for the development of fatal xenogeneic graft-versus-host disease-like syndrome (GVHDLS). RESULTS: For the mice that had received T cell-replete grafts of CB or BM or PBSC, the duration of GVHDLS-free survival of the chimeras was inversely proportional to the number of T cells transplanted. After adjustment for the number of T cells transplanted, the relative hazard of developing fatal GVHDLS was 62-fold higher for PBSC and 210-fold higher for BM as compared with CB. Flow cytometric and histologic analyses of selected chimeras that died of GVHDLS showed extensive proliferation of human T cells in multiple organs. In contrast, mice that survived to day 60 were engrafted with human myeloid and B lymphoid cells. CONCLUSIONS: The immunoreactive potential, as measured by this in vivo assay, differed among clinical grafts: BM > PBSC > CB.