ALBERT

Description: This study demonstrates in both stable and inducible BCR-ABL–expressing hematopoietic cells a down-regulation of the major mammalian DNA repair protein DNA-PKcs by BCR-ABL. Similar results were found in BCR-ABL CD34+ cells from patients with chronic myelogenous leukemia (CML). DNA-PKcs down-regulation is a proteasome-dependent degradation that requires tyrosine kinase activity and is associated with a marked DNA repair deficiency along with increased sensitivity to ionizing radiation. The conjunction of a major DNA repair deficiency and a resistance to apoptosis, both induced by BCR-ABL, provides a new mechanism to explain how secondary genetic alterations can accumulate in CML, eventually leading to blast crisis. The down-regulation of DNA-PKcs was reversible in CD34+ CML cells suggesting that this approach might offer a novel and powerful therapeutic strategy in this disease, especially to delay the blast crisis.

Description: In studies aimed at further characterizing the cellular immunodeficiency of the Wiskott-Aldrich syndrome (WAS), we found that T lymphocytes from WAS patients display abnormal chemotaxis in response to the T-cell chemoattractant stromal cell–derived factor (SDF)-1. The Wiskott- Aldrich syndrome protein (WASP), together with the Rho family GTPase Cdc42, control stimulus-induced actin cytoskeleton rearrangements that are involved in cell motility. Because WASP is an effector of Cdc42, we further studied how Cdc42 and WASP are involved in SDF-1–induced chemotaxis of T lymphocytes. We provide here direct evidence that SDF-1 activates Cdc42. We then specifically investigated the role of the interaction between Cdc42 and WASP in SDF-1–responsive cells. This was achieved by abrogating this interaction with a recombinant polypeptide (TAT-CRIB), comprising the Cdc42/Rac interactive binding (CRIB) domain of WASP and a human immunodeficiency virus–TAT peptide that renders the fusion protein cell-permeant. This TAT-CRIB protein was shown to bind specifically to Cdc42-GTP and to inhibit the chemotactic response of a T-cell line to SDF-1. Altogether, these data demonstrate that Cdc42-WASP interaction is critical for SDF-1–induced chemotaxis of T cells.

Description: Ex-vivo expansion of mature hematopoietic cells and progenitors is a major cell therapy technology aiming to shorten chemotherapy-induced cytopenias. The goal of amplifying the most primitive hematopoietic stem cell populations in clinically acceptable conditions is currently difficult, due to the absence of clinical grade early acting-cytokines and lack of adequate stem cell assays. Stem cell factor (SCF) and granulocyte colony stimulating factor (G-CSF) are both clinically applicable and they have been shown to induce a dose-response effect with regard to the expansion of mature cell populations when used in combination with early- acting cytokines. The combined effects of high concentrations of G-CSF and SCF on hematopoietic cells and progenitors and the effects of this procedure on the more primitive stem cell subsets have not been studied. We have developed a preclinical protocol to evaluate the feasibility of expansion of granulomonocytic cells using SCF and G-CSF with the goal of translating this protocol to the clinical application. In preliminary assays, the effects of low and high concentrations of both cytokines on total nucleated cell numbers and progenitors from purifed CD34+ cells (50 104cells/ml) have been tested. As compared to low concentrations (G-CSF 10 ng/ml and SCF 50 ng/ml) high concentrations of G-CSF and SCF (100 ng/ml and 300 ng/ml respectively) induced a two-fold increase of the nucleated cells and progenitors after 9 days of culture (n=7 experiments). High concentrations were therefore chosen for further experiments. CD34+ cells purified from mobilized peripheral blood grafts (purity 99%) have been cultured in a clinically acceptable medium containing clinical grade fetal calf serum ( 10 %), G-CSF (100 ng/ml) and SCF (300 ng/ml) for 9 days. Cells were analyzed using apoptosis tests, immunophenotyping and clonogenic assays at day 0 and day+9. In several experiments ( n= 9) a mean 21-fold expansion of total viable cell numbers was obtained, with a mean 4-fold expansion of clonogenic progenitors. Expanded cells had consistently CD11b+, CD13+, CD15+ phenotype and 2–4% of them remained CD34+. In a scale-up experiment started with 23.106 CD34+ cells, the total cell numbers expanded 16-fold at day+9, with generation of high numbers of CD11b + (18%), CD13+(98%) and CD15+ cells (91%) demonstrating the feasibility of the protocol in clinical scale. To determine if ex-vivo expansion would lead to a significant exhaustion of more primitive stem cells, we have evaluated the NOD/SCID-repopulating cell (RC) contents of the cultures before (day 0) and after ex-vivo expansion (day 9), in mice transplanted with day 0 and day+9 cells. In two experiments, the numbers of NOD/SCID mice engrafted with CD45+ human cells have been found increase from 30% ( 3/10) at day 0 to 100 % ( 10/10 ) at day 9 (Exp1 ) and from 50 % (3/6) at day 0 to 90 % ( 7/8 ) at day9 (Exp 2), demonstrating the persistence of NOD/SCID-RC potential after ex-vivo expansion. Overall the large-scale granulomonocytic cell production protocol that we developed could be of major interest in order to maintain the dose-intensity in high dose chemotherapy regimens and to shorten neutropenia after autologous PBSC transplantation while maintaing the stem cell potential of the graft.

Description: Imatinib mesylate (IM) is a tyrosine kinase inhibitor which is highly efficient in chronic myelogenous leukemia (CML), especially in the firts chronic phase of the disease. Recent data showed, however, that resistance to IM can develop in patients in more aggressive phases of their disease, which mainly occurs through mutations within the ABL kinase domain that interfere with IM binding, leading to IM-resistant relapses. The mechanisms of the occurrence of ABL kinase domain mutations in patients on IM therapy are not well understood, and in some of them, a mutation pre-existing to the introduction of IM was described, suggesting the possibility of a clonal selection under IM therapy. To determine if ABL kinase domain mutations could be induced de novo in primary marrow cells, we used an ecotropic BCR-ABL retrovirus (MIGR-p210 vector, 5.105 viral particles / ml) and infected 5-FU-treated bone marrow cells from C57BL/6 mice. Retrovirally transduced cells (30% GFP+) were transplanted in lethally irradiated animals in which they induced lethal leukemia in 3 weeks. Both BCR-ABL-transduced and control C57BL/6 bone marrow cells were seeded in liquid cultures (104 cells/well) in the presence of 0.25 μM IM with weekly half medium changes during which the concentration of IM was increased gradually from 0.25 to 2 μM over 2 months. In these conditions, no growth could be obtained from normal bone marrow cells (0/192 wells) whereas in 10/192 wells containing BCR-ABL-transduced cells, we observed significant growth on IM. These cells were then amplified in the presence of murine stromal MS-5 cells and 2 μM IM for over 6 months and 2 clones (C3 and C10) exhibiting persistent growth were further characterized. At cytological analysis both cell lines had a typical mast cell morphology. Flow cytomery analyses demonsrated the presence of CD41 marker on both cell lines, with absence of myeloid (Gr1), erythroid (Ter119) and B-cell (B220) markers. Cells were not polyploid and interestingly, they exhibited higher growth rates in the presence of IM, with reduced growth upon IM deprivation. Both cell lines had evidence of BCR-ABL vector integration by PCR analysis and were highly GFP+. To explore the mechanisms of IM-resistance in these cells, we extracted high molecular weight genomic DNA and amplified a BCR-ABL fragment of 1236 bp encompassing the ABL kinase domain of the integrated construct. We then sequenced the ABL kinase domain using internal primers in both 5′-3′ directions. Plasmid DNA from the original MIGR-p210 BCR-ABL vector served as control. In both clones (C3 and C10) ABL kinase point mutations were readily detectable which were not found in the BCR-ABL retroviral vector. C3 carried two mutations interesting the C helix (E300K) and the SH2 contact region (E371K) of ABL kinase domain, whereas C10 carried a single mutation in the C helix (D295N). These mutations were previously detected in a random in vitro mutagenesis assay of BCR-ABL in bacterial systems. Thus, our model is the first demonstration of the occurrence of ABL kinase domain mutations and the concomittant generation of an IM-resistant phenotype in primary marrow stem cells transduced with BCR-ABL vector DNA and selected in the presence of IM. The mechanisms of the occurrence of these mutations in vitro are currently under study but these results suggest that de novo ABL kinase mutations could also occur in vivo in CML patients treated with IM.

Description: A defect in cell trafficking and chemotaxis plays an important role in the immune deficiency observed in Wiskott-Aldrich syndrome (WAS). In this report, we show that marrow cells from WAS protein (WASP)–deficient mice also have a defect in chemotaxis. Serial transplantation and competitive reconstitution experiments demonstrated that marrow cells, including hematopoietic progenitors and stem cells (HSCs), have decreased homing capacities that were associated with a defect in adhesion to collagen. During development, HSCs migrate from the liver to the marrow and the spleen, prompting us to ask if a defect in HSC homing during development may explain the skewed X-chromosome inactivation in WAS carriers. Preliminary evidence has shown that, in contrast to marrow progenitor cells, fetal liver progenitor cells from heterozygous females had a random X-chromosome inactivation. When fetal liver cells from WASP-carrier females were injected into irradiated recipients, a nonrandom inactivation of the X-chromosome was found at the level of hematopoietic progenitors and HSCs responsible for the short- and long-term hematopoietic reconstitution. Therefore, the mechanism of the skewed X-chromosomal inactivation observed in WAS carriers may be related to a migration defect of WASP-deficient HSCs.

Description: Several studies suggest an implication of transforming growth factor-β1 (TGF-β1) in the promotion of myelofibrosis associated with hematopoietic malignancies, but the involvement of this cytokine is not fully investigated. To test directly the impact of TGF-β1 in the pathogenesis of myelofibrosis, bone marrow stem cells from homozygous TGF-β1 null (TGF-β1−/−) and wild-type (WT) littermates were infected with a retrovirus encoding the murine thrombopoietin (TPO) protein and engrafted into lethally irradiated wild-type hosts for long-term reconstitution. Over the 4 months of follow-up, TPO levels in plasma were markedly elevated in both groups of mice, and animals typically developed a myeloproliferative syndrome characterized by thrombocytosis, leukocytosis, splenomegaly, increased numbers of progenitors in blood, and extramedullary hematopoiesis. Severe fibrosis was observed in spleen and marrow from all the mice engrafted with WT cells. In contrast, none of the mice repopulated with TGF-β1−/− cells (chimerism 〉 70%) showed deposition of reticulin fibers at any time during the follow-up. In accordance with the development of fibrosis, latent TGF-β1 levels in plasma and extracellular fluid of the spleen from mice engrafted with WT cells were increased 6-fold and 4-fold, respectively, over levels found in normal hosts, whereas no increase over baseline levels could be demonstrated in animals undergoing transplantation with TGF-β1−/− cells. These data provide evidence that TGF-β1 produced by hematopoietic cells is pivotal for the pathogenesis of myelofibrosis that develops in mice with TPO overexpression.

Description: Mature dendritic cells (mDCs) can trigger the effector functions of natural killer (NK) cells. Knock-out, small-interfering RNA or neutralizing antibodies targeting interleukin 12 (IL-12) subunits revealed a critical role for IL-12 in NK cell interferon γ (IFN-γ) secretion promoted by mDCs. However, NK cell activation by DCs also required direct cell-to-cell contacts. DC-mediated NK cell activation involved the formation of stimulatory synapses between DCs and NK cells. The formation of DC/NK cell conjugates depended on cytoskeleton remodeling and lipid raft mobilization in DCs. Moreover, the disruption of the DC cytoskeleton using pharmacologic agents or the loss-of-function mutation of the Wiskott-Aldrich syndrome protein abolished the DC-mediated NK cell activation. Synapse formation promoted the polarized secretion of preassembled stores of IL-12 by DCs toward the NK cell. The synaptic delivery of IL-12 by DCs was required for IFN-γ secretion by NK cells, as assessed using inhibitors of cytoskeleton rearrangements and transwell experiments. Therefore, the cross-talk between DCs and NK cells is dictated by functional synapses. (Blood. 2004;104:3267-3275)

Description: Myelofibrosis and osteosclerosis are prominent features arising in mice overexpressing thrombopoietin (TPO). The pivotal role of transforming growth factor β1 (TGF-β1) in the pathogenesis of myelofibrosis has been documented, but the mechanisms mediating osteosclerosis remain unclear. Here, we used mice deficient in osteoprotegerin (OPG), a secreted inhibitor of bone resorption, to determine whether osteosclerosis occurs through a deregulation of osteoclastogenesis. Marrow cells from opg-deficient mice (opg−/−) or wild-type (WT) littermates were infected with a retrovirus encoding TPO and engrafted into anopg−/− or WT background for long-term reconstitution. The 4 combinations of graft/host (WT/WT,opg−/−/opg−/−,opg−/−/WT, and WT/opg−/−) were studied. Elevation of TPO and TGF-β1 levels in plasma was similar in the 4 experimental groups and all the mice developed a similar myeloproliferative syndrome associated with severe myelofibrosis. Osteosclerosis developed in WT hosts engrafted with WT or opg−/− hematopoietic cells and was associated with increased OPG levels in plasma and decreased osteoclastogenesis. In contrast,opg−/− hosts exhibited an osteoporotic phenotype and a growth of bone trabeculae was rarely seen. These findings suggest that osteosclerosis in mice with TPO overexpression occurs predominantly via an up-regulation of OPG in host stromal cells leading to disruption of osteoclastogenesis.

Description: Myelofibrosis is a clinical feature of several hematopoietic disorders and is most prominent in idiopathic myelofibrosis. It is characterized by excessive deposits of extracellular matrix proteins which occur as a marrow microenvironment reactive response to cytokines released from the clonal malignant myeloproliferation. The observation that mice exposed to high systemic levels of thrombopoietin (TPO) invariably develop a myelofibrosis within a period of 4 to 8 weeks has allowed the demonstration of the crucial role of TGF-b1 released by hematopoietic cells in the promotion of myelofibrosis (Chagraoui et al Blood, 2002 ; 100 :3495). The aim of this study was to investigate whether TGF-b1 inhibition could directly inhibit the fibrosis development in a curative approach of this murine model. An adenovirus encoding for TGF-b1 soluble receptor was constructed. SCID mice were infected with this adenovirus to measure the TGF-b1 inhibitory effect. We measure TGF-b1-Rs (TGF-b1 soluble receptor) concentration and TGF-b1 levels in 5 groups of mice injected with 0- 6.5 106- 6.5 107- 6.5 108-and 6.5 109 pfu/mice. We demonstrated that 6.5 108 pfu/mice was able to completely inhibit 10 fold increase of circulating TGF-b1 and that 1 microgram of soluble receptor was able to inhibit 1 ng of TGF-b1. All subsequent experiments were performed by injecting 1 109 pfu/mice either shortly after transplantation (preventive) or after 30 days (curative). Briefly, 46 mice (2 individuals experiments n= 16 and n= 30) were transplanted with 5-FU treated syngeneic bone marrow cells transduced with a retrovirus encoding for murine TPO. Retroviral transduction efficiency varied from 67% to 87%. All mice developed a myeloproliferative syndrome characterized by an increase in platelet count, a leukocytosis, an increase in progenitor cells in the blood circulation and a 1000-fold increase in TPO level. TGF-b Rs (13 to 32 micrograms/ml) was detected in the blood of all mice injected with the TGF-b1-Rs Adeno. A dramatic decrease in TGF-b1 level was noticed in all these treated mice in comparison to the control groups. Histological analysis demonstrated that the two approaches (curative or preventive) were identical to abolish fibrosis development in bone marrow as well as in the spleen. However, all mice which received the TGF-b1-RS adenovirus developed a hepatitis leading to death 6 weeks to 3 months after infections. In conclusion our approach support the major role of TGF-b1 in myelofibrosis development as previously demonstrated by our group using TGF-b1 KO mice. Moreover, the present results suggest that TGF-b1 inhibition could be a relevant approach to treat patients presenting a TGF-b1 mediated bone marrow fibrosis.

Description: Injuries to the vessel wall and subsequent exposure of collagen from the subendothelial matrix result in thrombus formation. In physiological conditions, the platelet plug limits blood loss. However, in pathologic conditions, such as rupture of atherosclerotic plaques, platelet–collagen interactions are associated with cardiovascular and cerebral vascular diseases. Platelet glycoprotein VI (GPVI) plays a crucial role in collagen-induced activation and aggregation of platelets, and people who are deficient in GPVI suffer from bleeding disorders. Based on the fact that GPVI is coupled to the Fc receptor (FcR)-γ chain and thus should share homology with the FcR chains, the genes encoding human and mouse GPVI were identified. They belong to the immunoglobulin (Ig) superfamily and share 64% homology at the protein level. Functional evidence demonstrating the identity of the recombinant protein with GPVI was shown by binding to its natural ligand collagen; binding to convulxin (Cvx), a GPVI-specific ligand from snake venom; binding of anti-GPVI IgG isolated from a patient; and association to the FcR-γ chain. The study also demonstrated that the soluble protein blocks Cvx and collagen-induced platelet aggregation and that GPVI expression is restricted to megakaryocytes and platelets. Finally, human GPVI was mapped to chromosome 19, long arm, region 1, band 3 (19q13), in the same region as multiple members of the Ig superfamily. This work offers the opportunity to explore the involvement of GPVI in thrombotic disease, to develop alternative antithrombotic compounds, and to characterize the mechanism involved in GPVI genetic deficiencies.