scholarly journals Roles of tyrosine 589 and 591 in STAT5 activation and transformation mediated by FLT3-ITD

Blood ◽  
2006 ◽  
Vol 108 (4) ◽  
pp. 1339-1345 ◽  
Author(s):  
Jennifer L. Rocnik ◽  
Rachel Okabe ◽  
Jin-Chen Yu ◽  
Benjamin H. Lee ◽  
Neill Giese ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Bone Marrow Cells ◽  
Marrow Transplant ◽  
Myeloproliferative Disease ◽  
Juxtamembrane Domain ◽  
Murine Bone Marrow ◽  
Downstream Effectors

Abstract Acquired mutations in the FLT3 receptor tyrosine kinase are common in acute myeloid leukemia and result in constitutive activation. The most frequent mechanism of activation is disruption of the juxtamembrane autoregulatory domain by internal tandem duplications (ITDs). FLT3-ITDs confer factor-independent growth to hematopoietic cells and induce a myeloproliferative syndrome in murine bone marrow transplant models. We and others have observed that FLT3-ITD activates STAT5 and its downstream effectors, whereas ligand-stimulated wild-type FLT3 (FLT3WT) does not. In vitro mapping of tyrosine phosphorylation sites in FLT3-ITD identified 2 candidate STAT5 docking sites within the juxtamembrane domain that are disrupted by the ITD. Tyrosine to phenylalanine substitution of residues 589 and 591 in the context of the FLT3-ITD did not affect tyrosine kinase activity, but abrogated STAT5 activation. Furthermore, FLT3-ITD–Y589/591F was incapable of inducing a myeloproliferative phenotype when transduced into primary murine bone marrow cells, whereas FLT3-ITD induced myeloproliferative disease with a median latency of 50 days. Thus, the conformational change in the FLT3 juxtamembrane domain induced by the ITD activates the kinase through dysregulation of autoinhibition and results in qualitative differences in signal transduction through STAT5 that are essential for the transforming potential of FLT3-ITD in vivo.

Direct Binding of Grb2 Is Required for Efficient Induction of Myeloproliferative Disease in Mice by ETV6/FLT3.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2903-2903
Author(s):  
Kazuhisa Chonabayashi ◽  
Masakatsu Hishizawa ◽  
Shin Kawamata ◽  
Masashi Matsui ◽  
Tatsuharu Ohno ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Binding Sites ◽  
Bone Marrow Cells ◽  
Myeloproliferative Disease ◽  
Juxtamembrane Domain ◽  
Murine Bone Marrow ◽  
Direct Binding ◽  
Marrow Cells

Abstract Abstract 2903 Poster Board II-879 FMS-like tyrosine kinase 3 (FLT3), a class III receptor tyrosine kinase, is one of the most frequently mutated genes in hematological malignancies. The most common mutations of FLT3 are internal tandem duplications (ITDs) within the juxtamembrane domain: these mutations occur in 20% to 30% of patients with AML and are closely associated with a poor prognosis. In a small number of patients with myeloproliferative neoplasms (MPNs), FLT3 has been reported to fuse to ETV6 (TEL) and contribute to leukemogenesis, but the leukemogenic mechanism of ETV6/FLT3 remains unclear. We encountered a case of ETV6/FLT3 fusion in a patient with MPN complicated with T-cell lymphoblastic lymphoma. In this case, both myeloid and lymphoma cells shared the same chromosomal translocation, t(12;13)(p13;q12), and allogeneic hematopoietic stem cell transplantation led to complete remission for 3 years. Full-length ETV6/FLT3 fusion cDNA was cloned from the patient's bone marrow cells. Sequence analysis of the PCR product revealed that, in contrast to the finding of previously reported two cases of ETV6/FLT3-positive MPN, ETV6 exon 6 was fused to FLT3 exon 14 and that the fused portion of ETV6 contained 2 potential Grb2-binding sites (Vu et al., Leukemia 2006; Walz et al., Blood 2007a). The ETV6/FLT3 conferred IL-3-independent growth to Ba/F3 and 32Dcl3 cells. Using a dominant negative approach, we showed that both STAT5 and Ras played important roles in ETV6/FLT3-mediated transformation of the hematopoietic cell lines. To investigate the role of the ETV6/FLT3 fusion protein in vivo, we used a murine bone marrow transplant model. Retroviral transduction of the ETV6/FLT3 into primary murine bone marrow cells resulted in a CML-like myeloproliferative disease (MPD) with complete penetrance in the transplanted mice. The disease progressed to cause death at a median of 18 days after transplantation (n = 16). The transplanted mice developed severe leukocytosis (159 × 103 /μl to 417 × 103 /μl), splenomegaly, and extensive infiltration of myeloid cells in the bone marrow, spleen, liver, and peripheral blood. ETV6/FLT3-induced MPD was oligoclonal and only 2 of the 9 secondary transplant recipients developed similar MPD when 5 × 106 spleen cells from 3 independent diseased mice were used as donors. We assayed the mutant forms of the ETV6/FLT3 to test their ability to transform hematopoietic cells. Induction of MPD required the oligomerization domain of ETV6 and the tyrosine kinase activity of FLT3. Mice that received the double tyrosine-to-phenylalanine mutant of ETV6/FLT3 at sites 589 and 591 (Y589/591F) in the juxtamembrane domain of FLT3, which are critical for FLT3-ITD-induced MPD, also developed a similar MPD phenotype. Unlike FLT3-ITDs, Y589/591F mutation did not abrogate STAT5 activation in Ba/F3 and 32Dcl3 cells transformed by ETV6/FLT3. A recent study has shown that direct binding of Grb2 to tyrosine 768, 955, and 969 of FLT3 is important for FLT3-ITD-mediated proliferation and survival of hematopoietic cells. Tyrosine 314 in exon 5 of ETV6 has also been reported as the principal Grb2-binding site that contributes to leukemogenesis via oncogenic ETV6 fusion proteins such as ETV6/ABL. Thus, we next investigated the role of Grb2 binding in ETV6/FLT3-mediated leukemogenesis. Using coimmunoprecipitation assays, we demonstrated that Grb2 also binds to the tyrosine 314 and 354 of ETV6 of the ETV6/FLT3, in addition to the tyrosine 768, 955, and 969 of FLT3. Both ETV6/FLT3-Y314/354F and ETV6/FLT3-Y768/955/969F retained their interaction with Grb2 and induced rapidly fatal MPD when they were transduced into primary murine bone marrow cells. On the other hand, the ETV6/FLT3 mutant at all the binding sites of Grb2 (Y314/354/768/955/969F) significantly attenuated MPD development in mice. Simultaneous mutation of these 5 tyrosine residues completely abolished the binding of Grb2 and resulted in a marked decrease in the binding and phosphorylation of Gab2 and impaired activation of STAT5 and Akt in Ba/F3 cells. These results indicate that tyrosine 589 and 591 of FLT3 are dispensable for the ETV6/FLT3-induced MPD phenotype, and suggest that both ETV6 and FLT3 portions contribute to the ETV6/FLT3-mediated leukemogenesis by binding directly to Grb2. Our observations provide deep insights into the oncogenic signaling induced by active FLT3 mutants as well as provide a potential target for therapies. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Expression of Jak2V617F causes a polycythemia vera–like disease with associated myelofibrosis in a murine bone marrow transplant model

Blood ◽  
2006 ◽  
Vol 107 (11) ◽  
pp. 4274-4281 ◽  
Author(s):  
Gerlinde Wernig ◽  
Thomas Mercher ◽  
Rachel Okabe ◽  
Ross L. Levine ◽  
Benjamin H. Lee ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Polycythemia Vera ◽  
Extramedullary Hematopoiesis ◽  
Marrow Transplant ◽  
Myeloproliferative Disease ◽  
Murine Bone Marrow ◽  
Retroviral Integration ◽  
Vitro Analysis

AbstractAn acquired somatic mutation, Jak2V617F, was recently discovered in most patients with polycythemia vera (PV), chronic idiopathic myelofibrosis (CIMF), and essential thrombocythemia (ET). To investigate the role of this mutation in vivo, we transplanted bone marrow (BM) transduced with a retrovirus expressing either Jak2 wild-type (wt) or Jak2V617F into lethally irradiated syngeneic recipient mice. Expression of Jak2V617F, but not Jak2wt, resulted in clinicopathologic features that closely resembled PV in humans. These included striking elevation in hemoglobin level/hematocrit, leukocytosis, megakaryocyte hyperplasia, extramedullary hematopoiesis resulting in splenomegaly, and reticulin fibrosis in the bone marrow. Histopathologic and flow cytometric analyses showed an increase in maturing myeloid lineage progenitors, although megakaryocytes showed decreased polyploidization and staining for acetylcholinesterase. In vitro analysis of primary cells showed constitutive activation of Stat5 and cytokine-independent growth of erythroid colony-forming unit (CFU-E) and erythropoietin hypersensitivity, and Southern blot analysis for retroviral integration indicated that the disease was oligoclonal. Furthermore, we observed strain-specific differences in phenotype, with Balb/c mice demonstrating markedly elevated leukocyte counts, splenomegaly, and reticulin fibrosis compared with C57Bl/6 mice. We conclude that Jak2V617F expression in bone marrow progenitors results in a PV-like syndrome with myelofibrosis and that there are strain-specific modifiers that may in part explain phenotypic pleiotropy of Jak2V617F-associated myeloproliferative disease in humans.

scholarly journals Protection of bone marrow transplant recipients from lethal doses of methotrexate by the generation of methotrexate-resistant bone marrow.

1987 ◽  
Vol 166 (1) ◽  
pp. 210-218 ◽  
Author(s):  
D A Williams ◽  
K Hsieh ◽  
A DeSilva ◽  
R C Mulligan
Keyword(s):  
Bone Marrow ◽  
Mammalian Cells ◽  
Bone Marrow Cells ◽  
Marrow Transplant ◽  
Murine Bone Marrow ◽  
Vitro Analysis ◽  
High Level ◽  
Lethal Doses

To develop a highly efficient means for generating methotrexate resistant (MTXr) hematopoietic cells in vivo, a recombinant retroviral genome was constructed that encodes a MTXr dihydrofolate reductase (DHFRr). Cell lines producing high titers of virus capable of transmitting the DHFR gene were generated and used to infect mammalian cells in vitro. Analysis of infected fibroblasts indicated that the DHFRr gene was transmitted intact and conferred a high level of MTXr upon cells. Based on these findings, DHFRr-containing virus was used to infect murine bone marrow cells in vitro. Following infection, the transduced cells were introduced into lethally irradiated recipients via bone marrow transplantation techniques. The presence of the proviral sequences in cells of the spleen and bone marrow of engrafted recipients was associated with significantly increased survival of mice treated with otherwise lethal doses of MTX.

Expression of human adenosine deaminase in murine hematopoietic cells

1988 ◽  
Vol 8 (12) ◽  
pp. 5116-5125
Author(s):  
J W Belmont ◽  
G R MacGregor ◽  
K Wager-Smith ◽  
F A Fletcher ◽  
K A Moore ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Adenosine Deaminase ◽  
High Titer ◽  
Virus Production ◽  
Marrow Transplant ◽  
Mouse Bone Marrow ◽  
Regulation Of Expression ◽  
Murine Bone Marrow

Multiple replication-defective retrovirus vectors were tested for their ability to transfer and express human adenosine deaminase in vitro and in vivo in a mouse bone marrow transplantation model. High-titer virus production was obtained from vectors by using both a retrovirus long terminal repeat promoter and internal transcriptional units with human c-fos and herpes virus thymidine kinase promoters. After infection of primary murine bone marrow with one of these vectors, human adenosine deaminase was detected in 60 to 85% of spleen colony-forming units and in the blood of 14 of 14 syngeneic marrow transplant recipients. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.

JAK2 Rearrangements, Including the Novel SEC31A-JAK2 Fusion, Are Recurrent in Classical Hodgkin Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 140-140 ◽  
Author(s):  
Katrien Van Roosbroeck ◽  
Luk Cox ◽  
Idoya Lahortiga ◽  
Olga Gielen ◽  
Thomas Tousseyn ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chromosomal Translocation ◽  
Marrow Transplant ◽  
Chromosomal Translocations ◽  
The Novel ◽  
Oncogenic Potential ◽  
Murine Bone Marrow ◽  
Transplant Model

Abstract Abstract 140 Molecular mechanisms underlying the pathogenesis of classical Hodgkin lymphoma (cHL) are poorly understood. Although no characteristic chromosomal translocation has been identified in cHL, gain and amplification of the 9p24 region harbouring JAK2 has been observed in up to 50% of cHLs. JAK2 encodes a protein tyrosine kinase (PTK) that plays a key role in the JAK/STAT signalling pathway. Chromosomal translocations and gain-of-function mutations involving JAK2 occur in several haematological malignancies. The aim of this study was to characterize a novel t(4;9)(q21;p24) found in a case of nodular sclerosis HL (NSHL), and to determine the in vitro and in vivo consequences of the fusion associated with this translocation. FISH with BAC clones flanking JAK2/9p24 was used to identify the 9p breakpoint and demonstrated involvement of JAK2. A BAC- and fosmid-walking interphase FISH strategy was further applied to identify the 4q21 breakpoint which was eventually mapped in the region of SEC31A. SEC31A is ubiquitously expressed in human cells and is known to play a role in ER-to-Golgi vesicular transport. Further molecular studies led to the identification of a SEC31A-JAK2 in-frame fusion transcript in which exon 24 of SEC31A is fused to exon 17 of JAK2. Of note, our recent studies showed involvement of SEC31A as a partner of ALK in ALK+ LBCL (Van Roosbroeck et al., Haematologica 2009, in press). To determine the in vitro oncogenic potential of SEC31A-JAK2, a chimeric expression construct was designed and introduced into mouse haematopoietic IL3-dependent Ba/F3 cells. SEC31A-JAK2 was found to transform Ba/F3 cells to IL3-independent growth, demonstrating its implication in oncogenic transformation. The fusion protein is likely to function as a constitutively activated tyrosine kinase, due to SEC31A-mediated oligomerization of JAK2. Attempts to identify the SEC31A domain responsible for the constitutive JAK2 activation are ongoing. Initial experiments with deletion mutants containing or lacking the WD40-like repeats of SEC31A exclude these repeats to be the driving force of JAK2 activation. An in vivo role of the fusion was assessed with a murine bone marrow transplant model. All six recipients of SEC31A-JAK2 transduced bone marrow cells developed a fatal disease after 107 – 174 days, showing involvement of the blood, bone marrow and spleen, and in a subset of mice also of lymph nodes and thymus. FACS and histopathological examination of the involved tissues in 3 mice revealed the development of a T-lymphoblastic lymphoma. Analysis of the remaining mice is still ongoing. In addition, we showed that the T-lymphoblastic disease is transplantable to secondary recipients. Downstream of the SEC31A-JAK2 fusion we could demonstrate constitutive activation of the ERK pathway in Ba/F3 cells bearing the SEC31A-JAK2 construct as well as in the reconstituted mouse tissues. To determine the incidence of JAK2 rearrangements in cHL, we screened 60 unselected cHL cases, including 25 with NSHL, by FISH and cDNA-based nested PCR. Using this approach, we identified one additional case with a SEC31A-JAK2 fusion showing 4q21 and 9p24 breakpoints identical to these in the index case. Moreover, we found a third case with a JAK2 rearrangement and two extra copies of the 3'JAK2. As SEC31A is not involved in the latter aberration, further studies aiming at the identification of the JAK2 partner in this case of cHL are ongoing. The vast majority (80%) of the remaining cHL cases analyzed by FISH revealed recurrent gains/amplifications of JAK2. In summary, we proved that JAK2 is recurrently targeted by chromosomal translocations in cHL. We identified and molecularly characterized the novel t(4;9)(q21;p24) resulting in a SEC31A-JAK2 fusion found in two NSHL cases and identified another not yet characterized JAK2 rearrangement in the third cHL case. We demonstrated the oncogenic potential of the SEC31A-JAK2 fusion both in vitro in the mouse haematopoietic IL3-dependent Ba/F3 cell line and in vivo in a murine bone marrow transplant model. Of note, this is the first report of a recurrent translocation associated with cHL. Although aberrant expression of various PTKs including JAK2 has already been documented in cHL, our results indicate that at least in some cHL cases, this aberration can be driven by a chromosomal translocation. Disclosures: No relevant conflicts of interest to declare.

Meis1-mediated apoptosis is caspase dependent and can be suppressed by coexpression of HoxA9 in murine and human cell lines

Blood ◽  
2005 ◽  
Vol 105 (3) ◽  
pp. 1222-1230 ◽  
Author(s):  
Peter J. Wermuth ◽  
Arthur M. Buchberg
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Cell Types ◽  
Selective Advantage ◽  
Myeloid Differentiation ◽  
Homeodomain Protein ◽  
Murine Bone Marrow ◽  
Differentiation Pathways

AbstractCoexpression of the homeodomain protein Meis1 and either HoxA7 or HoxA9 is characteristic of many acute myelogenous leukemias. Although Meis1 can be overexpressed in bone marrow long-term repopulating cells, it is incapable of mediating their transformation. Although overexpressing HoxA9 alone transforms murine bone marrow cells, concurrent Meis1 overexpression greatly accelerates oncogenesis. Meis1-HoxA9 cooperation suppresses several myeloid differentiation pathways. We now report that Meis1 overexpression strongly induces apoptosis in a variety of cell types in vitro through a caspase-dependent process. Meis1 requires a functional homeodomain and Pbx-interaction motif to induce apoptosis. Coexpressing HoxA9 with Meis1 suppresses this apoptosis and provides protection from several apoptosis inducers. Pbx1, another Meis1 cofactor, also induces apoptosis; however, coexpressing HoxA9 is incapable of rescuing Pbx-mediated apoptosis. This resistance to apoptotic stimuli, coupled with the previously reported ability to suppress multiple myeloid differentiation pathways, would provide a strong selective advantage to Meis1-HoxA9 coexpressing cells in vivo, leading to leukemogenesis.

scholarly journals Expression of human adenosine deaminase in murine hematopoietic cells.

1988 ◽  
Vol 8 (12) ◽  
pp. 5116-5125 ◽  
Author(s):  
J W Belmont ◽  
G R MacGregor ◽  
K Wager-Smith ◽  
F A Fletcher ◽  
K A Moore ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Adenosine Deaminase ◽  
High Titer ◽  
Virus Production ◽  
Marrow Transplant ◽  
Mouse Bone Marrow ◽  
Regulation Of Expression ◽  
Murine Bone Marrow

Multiple replication-defective retrovirus vectors were tested for their ability to transfer and express human adenosine deaminase in vitro and in vivo in a mouse bone marrow transplantation model. High-titer virus production was obtained from vectors by using both a retrovirus long terminal repeat promoter and internal transcriptional units with human c-fos and herpes virus thymidine kinase promoters. After infection of primary murine bone marrow with one of these vectors, human adenosine deaminase was detected in 60 to 85% of spleen colony-forming units and in the blood of 14 of 14 syngeneic marrow transplant recipients. This system offers the opportunity to assess methods for increasing efficiency of gene transfer, for regulation of expression of foreign genes in hematopoietic progenitors, and for long-term measurement of the stability of expression in these cells.

scholarly journals Characterization of a 5-fluorouracil-enriched osteoprogenitor population of the murine bone marrow

Blood ◽  
1993 ◽  
Vol 82 (12) ◽  
pp. 3580-3591
Author(s):  
N Falla ◽  
Vlasselaer Van ◽  
J Bierkens ◽  
B Borremans ◽  
G Schoeters ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Collagen Matrix ◽  
Collagen Type I ◽  
Nodule Formation ◽  
Mouse Bone Marrow ◽  
Type I ◽  
Murine Bone Marrow

In the presence of beta-glycerophosphate and vitamin C, cultures of normal mouse bone marrow cells form three-dimensional structures that stain positive with the Von Kossa technique and express alkaline phosphatase (ALP), collagen type I, and osteocalcin. Little is known about the characteristics and frequency of the cells that contribute to this phenomenon. Most likely, mature osteoblastic cells do not contribute to the nodule formation because no osteocalcin expressing cells are detected in the flushed marrow by in situ hybridization. Limiting dilution analysis shows that, in normal bone marrow, 1 of 2.2 x 10(5) cells has the potency to form a bone nodule and to express ALP, collagen, and osteocalcin in a temporal fashion. Upon in vivo treatment with 5-fluorouracil (5-FU), this frequency increases 12-fold, eg, 1 in 1.75 x 10(4) cells shows osteogenic activity. In comparison, fibroblast colony forming cells occur at a frequency of 1 of 2.5 x 10(4) or 1 of 5 x 10(3) plated cells in normal or 5-FU-treated marrow, respectively. Using density centrifugation, the majority of the osteoprogenitor cells in 5-FU marrow are found in the low-density (1.066 to 1.067 g/mL) fractions. In addition, these cells bind to nylon wool but not to plastic and aggregate in the presence of wheat germ agglutinin and soybean agglutinin. Scanning and transmission electron microscopy shows that the bone nodules in 5-FU marrow cultures are composed of fibroblastoid cells embedded in a mineralized collagen matrix. In conclusion, our results show that a quiescent cell population in the murine bone marrow with fibroblastoid characteristics contributes to the formation of bone-like nodules in vitro.

scholarly journals C-Terminal Domain of ABL Family Kinases, ABL and ARG, Defines Their Distinct Leukemogenic Activities in Vivo

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2368-2368 ◽  
Author(s):  
Asumi Yokota ◽  
Hideyo Hirai ◽  
Tsukimi Shoji ◽  
Taira Maekawa ◽  
Keiko Okuda
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Tyrosine Kinase ◽  
Fusion Proteins ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Terminal Domain ◽  
C Terminus

Abstract ARG (ABL2) is a member of ABL family kinases and highly homologous to ABL (ABL1) except the C-terminal domain adjacent to the kinase domain. TEL/ARG that consists of ARG fused to TEL (ETV6) has been found in AML M3, M4 or T-ALL patients, with additional chromosomal abnormalities of t(15;17)(q12;q21), inv(16)(p13;q12) or t(1;10;12)(q25;q23;p13) translocation, respectively. The structure of TEL/ARG is similar to that of TEL/ABL, which has been found in patients with T-ALL, B-ALL, AML and CML. TEL mediates homo-oligomerization of these fusion proteins, TEL/ABL and TEL/ARG, resulting in constitutive activation of the tyrosine kinases. Although ABL fusion proteins such as BCR/ABL and TEL/ABL have been intensively investigated, the involvement of TEL/ARG in leukemogenesis is not fully elucidated yet. We have recently reported that in vitro transforming activity of TEL/ARG was significantly lower than that of TEL/ABL although their kinase activities were almost identical. Interestingly, the in vitro transforming activities of C-terminus-swapped mutants, TEL/ABL with C-terminal domain of ARG [TEL-ABL (ARG-C)] or TEL/ARG with C-terminal domain of ABL [TEL/ARG (ABL-C)], were comparable to those of TEL/ARG or TEL/ABL, respectively, while kinase activities in the swapped mutants were not altered. These results suggest that C-termini of ABL family kinases contain some functional domain that defines their distinct transforming activities. The purpose of this study is to compare the in vivo leukemogenic activities of TEL/ABL and TEL/ARG, and evaluate the impact of the C-terminal domains. First, we investigated whether TEL/ABL or TEL/ARG caused leukemia in mice. Each fusion gene together with GFP gene was retrovirally transduced into the bone marrow cells harvested from C57BL/6 mice treated with 5-fluorouracil, and the transduced cells were transplanted into lethally irradiated mice. Similar to BCR/ABL, transplantation of TEL/ABL-transduced cells induced rapid myeloproliferative status accompanied by hepatomegaly and/or splenomegaly, and all the recipient mice died within 33 days after transplantation, indicating the development of myeloid leukemia. In contrast, the recipient mice transplanted with TEL/ARG-transduced cells did not develop myeloid leukemia but infiltrative mastocytosis, and died around 200 days after transplantation (Figure 1). Hemophagocytic mast cells accumulating in the bone marrow, and mast cells circulating in the peripheral blood were also observed in these mice. Next we investigated the roles of C-terminal domains of ABL and ARG in their in vivo leukemogenic activities. C-terminus-swapped mutants, TEL/ABL (ARG-C) and TEL/ARG (ABL-C) were retrovirally transduced into bone marrow cells and the transduced cells were transplanted as described above. Intriguingly, TEL/ABL (ARG-C) mutant failed to cause myeloproliferative status or leukemia at day 153 (Figure 2A). On the other hand, TEL/ARG (ABL-C) induced lethal myeloid leukemia in 4 out of 13 mice (30.8%) within 111 days after transplantation (Figure 2B). Collectively, the in vivo phenotypes induced by TEL/ABL (ARG-C) or TEL/ARG (ABL-C) resembled those induced by TEL/ARG or TEL/ABL, respectively. Mastocytosis, a characteristic of TEL-ARG-induced phenotype, has not been observed so far in any of the recipients of TEL/ABL (ARG-C) or TEL/ARG (ABL-C). In conclusion, these results indicate that C-terminal domain of ABL family kinases defines their distinct leukemogenic activities in vivo through modulating both proliferation and differentiation. Notably, C-terminus of ARG strongly suppressed the in vivo leukemogenic activity of TEL/ABL without impairing the tyrosine kinase activity. Further clarification of the molecular mechanisms underlying the suppressive activity of C-terminus of ARG will lead to development of a novel therapeutic strategy, especially for patients with CML harboring mutations, which are resistant to tyrosine kinase inhibitors. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

High Level Expression of a Membrane-Anchored Inhibitor of HIV Infection in Murine Hematopoietic Stem and Progenitor Cells Does Not Pertubate Serial Multilineage Repopulation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1758-1758
Author(s):  
Axel Schambach ◽  
Bernhard Schiedlmeier ◽  
Jens Bohne ◽  
Dorothee von Laer ◽  
Geoff Margison ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Transgene Expression ◽  
Bone Marrow Cells ◽  
Hematopoietic Stem ◽  
Murine Bone Marrow ◽  
Hiv 1 ◽  
Marrow Cells

Abstract T20 is a 36-amino-acid peptide that binds to HIV-1 gp41 and thereby acts as a fusion inhibitor, thus mediating potent and selective inhibition of HIV-1 entry in vitro and in vivo. An extended peptide expressed as an artificial, membrane-bound molecule (mbC46) efficiently inhibits HIV infection of primary human T-cells following retroviral vector mediated gene transfer (Egelhofer et al., J Virol, 2004). To develop an even more stringent approach to HIV gene therapy, we targeted hematopoietic stem cells. In 3 experimental groups of C57BL/6 mice (9 animals/group), we investigated the long-term toxicity of murine bone marrow cells transduced with M87o, a therapeutic vector designed to coexpress mbC46 and an HIV-derived RNA RRE-decoy to inhibit HIV replication. As controls we used the same vector containing an inactive C46 peptide and mock-transduced cells. Blood samples were collected monthly. Donor chimerism and transgene expression in multiple lineages were determined by FACS analysis and transgene integration was measured by real time PCR. Six months after transplantation, 4 mice per group were sacrificed and the remaining 5 mice per group were observed for another 6 months. In addition to the parameters mentioned above, we performed complete histopathology, blood counts and clinical biochemistry. Donor chimerism in all groups ranged from 82 – 94% (day 190 and day 349). In the M87o group, 60% of donor cells expressed mbC46. FACS data showed persisting transgene expression in T-cells (CD4, CD8, 65%), B-cells (B220, 46%), myeloid cells (CD11b, 68%), platelets (CD41, 19%), and RBC (60%) of the peripheral blood and bone marrow cells. Highly sustained gene marking (2–4 copies/genome) was noticed on day 190. To reveal latent malignant clones potentially originating from side effects of the genetic manipulation, 1x106 bone marrow cells from 4 primary recipients were transplanted into lethally irradiated secondary recipients (3 recipients/primary mouse) and these mice were observed for 8 months. All together, we could not observe any evidence for leukemogenic capacity. Analysis of peripheral blood and bone marrow showed a similar transgene expression pattern compared to the primary mice. To generate a complete chimerism of transgenic cells, we chose the human drug resistance gene methylguanine-methyltransferase (MGMT, P140K) to select for mbC46-transduced stem cells in vitro and in vivo. Different coexpression strategies were tested. Function of the MGMT protein was confirmed in a quantitative alkyltransferase assay and in a cytotoxicity assay using BCNU or temozolomide. In vitro selection of transduced 32D and PM1 cells with benzylguanine and BCNU showed >95% positive cells with evidence of polyclonal survival. Transduced PM1 cells underwent an HIV challenge assay. In vivo experiments in a murine bone marrow transplantation setting are ongoing to determine the potency and safety of combined retroviral expression of mbC46 and MGMT in relevant preclinical models. Successful conclusion of these studies will hopefully result in a phase I clinical trial testing the concept of generating an HIV-resistant autologous hematopoiesis.

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