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

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.

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Potentiation of antileukemic therapies by the dual PI3K/PDK-1 inhibitor, BAG956: effects on BCR-ABL– and mutant FLT3-expressing cells

Blood ◽

10.1182/blood-2007-09-114454 ◽

2008 ◽

Vol 111 (7) ◽

pp. 3723-3734 ◽

Cited By ~ 60

Author(s):

Ellen Weisberg ◽

Lolita Banerji ◽

Renee D. Wright ◽

Rosemary Barrett ◽

Arghya Ray ◽

...

Keyword(s):

Bone Marrow ◽

Tyrosine Kinase ◽

Cell Lines ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Akt Signaling ◽

Inhibitory Effects ◽

Marrow Cells

AbstractMediators of PI3K/AKT signaling have been implicated in chronic myeloid leukemia (CML) and acute myeloid leukemia (AML). Studies have shown that inhibitors of PI3K/AKT signaling, such as wortmannin and LY294002, are able to inhibit CML and AML cell proliferation and synergize with targeted tyrosine kinase inhi-bitors. We investigated the ability of BAG956, a dual PI3K/PDK-1 inhibitor, to be used in combination with inhibitors of BCR-ABL and mutant FLT3, as well as with the mTOR inhibitor, rapamycin, and the rapamycin derivative, RAD001. BAG956 was shown to block AKT phosphorylation induced by BCR-ABL–, and induce apoptosis of BCR-ABL–expressing cell lines and patient bone marrow cells at concentrations that also inhibit PI3K signaling. Enhancement of the inhibitory effects of the tyrosine kinase inhibitors, imatinib and nilotinib, by BAG956 was demonstrated against BCR-ABL expressing cells both in vitro and in vivo. We have also shown that BAG956 is effective against mutant FLT3-expressing cell lines and AML patient bone marrow cells. Enhancement of the inhibitory effects of the tyrosine kinase inhibitor, PKC412, by BAG956 was demonstrated against mutant FLT3-expressing cells. Finally, BAG956 and rapamycin/RAD001 were shown to combine in a nonantagonistic fashion against BCR-ABL– and mutant FLT3-expressing cells both in vitro and in vivo.

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CREB Plays a Critical Role in the Regulation of Normal and Malignant Hematopoiesis.

Blood ◽

10.1182/blood.v110.11.1224.1224 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1224-1224

Author(s):

Jerry C. Cheng ◽

Dejah Judelson ◽

Kentaro Kinjo ◽

Jenny Chang ◽

Elliot Landaw ◽

...

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

Progenitor Cells ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Leukemia Cells ◽

Mouse Bone Marrow ◽

T315i Mutation

Abstract The cAMP Response Element Binding Protein, CREB, is a transcription factor that regulates cell proliferation, memory, and glucose metabolism. We previously demonstrated that CREB overexpression is associated with an increased risk of relapse in a small cohort of adult acute myeloid leukemia (AML) patients. Transgenic mice that overexpress CREB in myeloid cells develop myeloproliferative/myelodysplastic syndrome after one year. Bone marrow cells from these mice have increased self-renewal and proliferation. To study the expression of CREB in normal hematopoiesis, we performed quantitative real-time PCR in both mouse and human hematopoietic stem cells (HSCs). CREB expression was highest in the lineage negative population and was expressed in mouse HSCs, common myeloid progenitors, granulocyte/monocyte progenitors, megakaryocyte/erythroid progenitors, and in human CD34+38- cells. To understand the requirement of CREB in normal HSCs and myeloid leukemia cells, we inhibited CREB expression using RNA interference in vitro and in vivo. Bone marrow progenitor cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in CFU-GM but increased Gr-1/Mac-1+ cells compared to vector control infected cells (p<0.05). There were fewer terminally differentiated Mac-1+ cells in the CREB shRNA transduced cells (30%) compared to vector control (50%), suggesting that CREB is critical for both myeloid cell proliferation and differentiation. CREB downregulation also resulted in increased apoptosis of mouse bone marrow progenitor cells. Given our in vitro results, we transplanted sublethally irradiated mice with mouse bone marrow cells transduced with CREB or scrambled shRNA. At 5 weeks post-transplant, we observed increased Gr-1+/Mac-1+ cells in mice infused with CREB shRNA transduced bone marrow compared to controls. After 12 weeks post-transplant, there was no difference in hematopoietic reconstitution or in the percentage of cells expressing Gr-1+, Mac-1+, Gr-1/Mac-1+, B22-+, CD3+, Ter119+, or HSCs markers, suggesting that CREB is not required for HSC engraftment. To study the effects of CREB knockdown in myeloid leukemia cells, K562 and TF-1 cells were infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed for CREB expression and proliferation. Within 72 hours, cells transduced with CREB shRNA demonstrated decreased proliferation and survival with increased apoptosis. In cell cycle experiments, we observed increased numbers of cells in G1 and G2/M with CREB downregulation. Expression of cyclins A1 and D, which are known target genes of CREB, was statistically significantly decreased in TF-1 and K562 cells transduced with CREB shRNA lentivirus compared to controls. To study the in vivo effects of CREB knockdown on leukemic progression, we injected SCID mice with Ba/F3 cells expressing bcr-abl or bcr-abl with the T315I mutation and the luciferase reporter gene. Cells were transduced with either CREB or scrambled shRNA. Disease progression was monitored using bioluminescence imaging. The median survival of mice injected with CREB shRNA transduced Ba/F3 bcr-abl or bcr-abl with the T315I mutation was increased with CREB downregulation compared to controls (p<0.05). Our results demonstrate that CREB is a critical regulator of normal and neoplastic hematopoiesis both in vitro and in vivo.

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Familial Mastocytosis: Identification Of KIT K509I Mutation and Its In Vitro Sensitivity To Imatinib, Dasatinib and PKC412

Blood ◽

10.1182/blood.v122.21.5267.5267 ◽

2013 ◽

Vol 122 (21) ◽

pp. 5267-5267 ◽

Cited By ~ 2

Author(s):

Paula De Melo Campos ◽

João Agostinho Machado-Neto ◽

Adriana Silva Santos Duarte ◽

Renata Scopim-Ribeiro ◽

Flavia Fonseca de Carvalho Barra ◽

...

Keyword(s):

Bone Marrow ◽

Mast Cells ◽

Tyrosine Kinase ◽

Bone Marrow Cells ◽

Systemic Mastocytosis ◽

History Of ◽

Primary Bone ◽

Primary Bone Marrow ◽

Apoptosis Ratio

Abstract Background Mast cell diseases are myeloproliferative neoplasms characterized by an abnormal proliferation and accumulation of mast cells in different tissues. The clinical presentation of mastocytosis is heterogeneous, ranging from skin-limited disease to more aggressive variants that may be associated with multiorgan dysfunction/failure and shortened survival. In a relatively high proportion of cases, the clonal nature of the disease can be established on the basis of the demonstration of gain-of-function mutations involving the tyrosine kinase (TK) domain of KIT in skin lesions and BM cells and by the factor-independent proliferation and transforming abilities of these mutations. The tyrosine kinase inhibitor Imatinib is a treatment available for mastocytosis patients; however, some KIT mutations, specially KIT D816V, confer resistance to this drug. Aims To characterize the clinical phenotype and molecular mutations of 2 relatives with diagnosis of systemic mastocytosis (WHO 2008). We also aimed to test the in vitro sensitivity of primary bone marrow (BM) cells from both patients to tyrosine kinase inhibitors. Patients and methods Four individuals were included in the study; two patients (case 1 [mother], and case 2 [daughter]), and the parents of case 1. DNA samples were obtained from total BM cells, CD3+ BM cells and oral mucosa of patients, and from peripheral blood of all individuals. KIT (exons 1 to 21) was submitted for Sanger sequencing analysis. Primary bone marrow cells (5X104) from the 2 patients were cultured and treated with Imatinib (5uM), Dasatinib (80nM) and PKC 412 (100nM) or with vehicle only (control cells) and submitted for proliferation (MTT) and apoptosis assays (Annexin-V/PI) at days 4, 8 and 12 of culture. Results Case 1 was a 33 year-old woman with a chronic history of pruritic skin rash who was referred to our outpatient service for evaluation of massive splenomegaly (25 centimeters in length) and pancytopenia. She had neither comorbidities nor any familial history of hematological malignancies. The patient had no siblings and had only one daughter (case 2). At biopsy, she showed extensive skin and bone marrow infiltration by mast cells. During follow up, the patient presented with spontaneous splenic rupture and had to undergo splenectomy, which led to the resolution of pancytopenia. She was diagnosed with Aggressive Systemic Mastocytosis. Her daughter (case 2), a 17 year-old woman, was also evaluated for an insidious history of diffuse skin rash. Skin and bone marrow biopsies showed massive infiltration by atypical mast cells and a diagnosis of Indolent Systemic Mastocytosis was made. The rare KIT K509I mutation was found in all DNA samples obtained from both patients, but not from the parents of case 1. This suggests that the KIT K509I was a germ line mutation acquired de novo by patient 1 that was subsequently transmitted to her daughter (patient 2). In vitro treatment of primary bone marrow cells harboring the KIT K509I mutation from patients 1 and 2 resulted in variable clinical response rates according to the drug used and the treatment duration. Imatinib treatment resulted in a significant reduction in proliferation (days 4, 8 and 12 of culture) and an increase in apoptosis (days 8 and 12) in cases 1 and 2 (all p≤0.03). Although Dasatinib resulted in decreased proliferation in both patients at day 12 (all p≤0.008), a significantly higher apoptosis ratio was observed only for patient 1 at day 12 of culture (p=0.03). PKC412 had a negative effect over cell growth in patient 1 (days 4 and 8) and in patient 2 (day 4) (all p≤0.03); however, no effect in apoptosis ratio was seen. Conclusions We herein provide a report of a KIT K509I mutation in familial mastocytosis. This mutation has been previously described in the literature in one case of familial mastocytosis. Although rare, the screening for KIT K509I mutation should be considered in all cases of familial mastocytosis. Based on in vitro studies, mastocytosis patients harboring the KIT K509I mutation could benefit from treatment with Imatinib, Dasatinib and PKC 412. However, Imatinib may be more effective in inducing neoplastic mast cells apoptosis. Both patients described were started on Imatinib in June 2013. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood-2005-11-011429 ◽

2006 ◽

Vol 108 (4) ◽

pp. 1339-1345 ◽

Cited By ~ 93

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.

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The Grb2 binding site is required for the induction of chronic myeloid leukemia-like disease in mice by the Bcr/Abl tyrosine kinase

Blood ◽

10.1182/blood.v96.2.664 ◽

2000 ◽

Vol 96 (2) ◽

pp. 664-670 ◽

Cited By ~ 91

Author(s):

Ryan P. Million ◽

Richard A. Van Etten

Keyword(s):

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Intracellular Signaling ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Myeloproliferative Disease ◽

Efficient Induction ◽

Direct Binding

Abstract The BCR/ABL oncogene results from a balanced translocation between chromosomes 9 and 22 and is found in patients with chronic myeloid leukemia (CML) and in some patients with acute B-lymphoid leukemia. The Bcr/Abl fusion protein is a constitutively active tyrosine kinase that stimulates several intracellular signaling pathways, including activation of Ras through direct binding of the SH2-containing adapter protein Grb2 to Bcr tyrosine 177. A tyrosine-to-phenylalanine mutation (Y177F) at this site blocks the co-association of Bcr/Abl and Grb2 in vivo and impairs focus formation by Bcr/Abl in fibroblasts. However, the Bcr/Abl Y177F mutant can transform hematopoietic cell lines and primary bone marrow cells in vitro, so the importance of the Bcr/Abl–Grb2 interaction to myeloid and lymphoid leukemogenesis in vivo is unclear. We have recently demonstrated the efficient induction of CML-like myeloproliferative disease by BCR/ABL in a murine bone marrow transduction/transplantation model system. The Y177F mutation greatly attenuates the myeloproliferative disease induced by BCR/ABL, with mice developing B- and T-lymphoid leukemias of longer latency. In addition, the v-abl oncogene of Abelson murine leukemia virus, whose protein product lacks interaction with Grb2, is completely defective for the induction of CML-like disease. These results suggest that direct binding of Grb2 is required for the efficient induction of CML-like myeloproliferative disease by oncogenic Abl proteins.

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Deficiency of the E3 Ligase Hoip Impairs Adult Hematopoiesis and Myeloid Leukemia

Blood ◽

10.1182/blood-2021-145298 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 1153-1153

Author(s):

Koji Jimbo ◽

Shuhei Koide ◽

Takahiro Ito ◽

Arinobu Tojo ◽

Katsuhiro Sasaki ◽

...

Keyword(s):

Bone Marrow ◽

Myeloid Leukemia ◽

Research Funding ◽

Bone Marrow Cells ◽

Science Research ◽

Leukemia Cells ◽

Japan Society ◽

Retroviral Transduction

Abstract Ubiquitination is a post-translational modification that plays important roles in the regulation of various cellular processes. The linear ubiquitin chain assembly complex (LUBAC) is the only E3 ubiquitin ligase, which can specifically generate linear ubiquitin chains. LUBAC is composed of three subunits: catalytic Hoip (Rnf31), Hoil-1l (RBCK1), and Sharpin. LUBAC regulates canonical NF-κB signaling pathway and apoptosis. Several studies have shown that LUBAC is required for fetal hematopoiesis and tumorigenesis in several solid cancers, but the role of Hoip, which is the catalytic component of the LUBAC, in adult hematopoiesis and myeloid leukemia is unclear. To address whether Hoip is required for adult hematopoiesis, we used tamoxifen induced Hoip deleted mice (Hoip fl/fl; Rosa26-CreERT). Deletion of Hoip reduced the numbers of almost all cell fractions in bone marrow and peripheral blood, except for long-term hematopoietic stem cell (LT-HSC) fraction (CD34 - Flk2 - Lineage - c-Kit + Sca-1 +: LSK). Deletion of Hoip treated with tamoxifen markedly impaired colony formation of LT-HSCs in vitro. Among competitive transplantation assay using bone marrow cells from Hoip fl/fl; Rosa26-CreERT mice, deletion of Hoip treated with tamoxifen following stable reconstitution 8 weeks after transplantation led to a significant reduction of Hoip-deficient chimerism compared to control in vivo (Figure A). Deletion of Hoip induced apoptosis in bone marrow cells and increased the frequency of CD34 - LSK cells in S/G2/M phase of the cell cycle. Collectively, these data indicated that Hoip is required for adult hematopoiesis. To evaluate the consequences of Hoip deletion in myeloid leukemia propagation and maintenance, we utilized aggressive murine myeloid leukemia models driven by retroviral transduction of oncogenes: MLL-AF9 and NRAS G12V-driven acute myeloid leukemia (AML) and BCR-ABL and NUP98-HOXA9-driven blast crisis of chronic myelogenous leukemia (CML-BC) models. Deletion of Hoip in established leukemia cells treated with tamoxifen markedly impaired colony formation of leukemia cells in vitro and led to a significantly longer survival with reduced disease burden in bone marrow and spleen in vivo in both of leukemic models (Figure B,C). Deletion of Hoip also induced apoptosis in leukemia stem cells (LSCs: c-Kit + Lineage - cells) (Figure D) and increased the frequency of LSCs in S-phase of the cell cycle. Furthermore, we evaluated the contribution of HOIP to human myeloid leukemia. Knockdown of HOIP by small hairpin RNA using lentivirus in several myeloid leukemia cell lines (THP-1, SKM-1, and K562) and primary patient-derived AML cells exhibited reduced numbers of colony forming cells in vitro, and increased apoptosis. LUBAC inhibitor gliotoxin impaired the growth of murine and human leukemia cells. These results indicate that Hoip is essential for propagation and maintenance in murine and human myeloid leukemia. Because LUBAC regulates canonical NF-κB signaling pathway, we evaluated protein levels of p65 phosphorylation on Ser536 (p-p65) and IκBα in leukemia cells by flow cytometric analysis. Reduction in p-p65 and inhibition of IκBα degradation were observed in Hoip-deficient leukemia cells (Figure E), indicating that Hoip plays an important role in regulation of NF-κB signaling in myeloid leukemia. Since Hoip is the central component of LUBAC and the only catalytically active subunit of E3 ubiquitin ligase, we examined the importance of each domain of Hoip for myeloid leukemia propagation using Hoip mutant isoforms. Loss of colony-forming ability of Hoip-deficient AML cells could not be rescued by retroviral transduction of the Hoip mutants with deletion of UBA domain (which is required for interaction with the other LUBAC subunits) and with deletion of RBR domain and with mutation in C879A (which lack ligase activity) (Figure F), indicating that LUBAC ligase activity and interaction with the other LUBAC subunits are critical for leukemia propagation. Thus, our data demonstrated that Hoip is essential for adult hematopoiesis and myeloid leukemia. Given that patients with myeloid leukemia have shown increased activity of NF-κB signaling, inhibition of LUBAC ligase activity could serve as a promising strategy for treating myeloid leukemia. Figure 1 Figure 1. Disclosures Jimbo: Japan Society for the Promotion of Science: Research Funding. Ito: Japan Society for the Promotion of Science: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding. Iwama: Nissan Chemical Corporation: Research Funding. Nannya: Otsuka Pharmaceutical Co., Ltd.: Consultancy, Speakers Bureau; Astellas: Speakers Bureau. Konuma: Japan Society for the Promotion of Science: Research Funding; The Japanese Society of Hematology: Research Funding; SGH Foundation: Research Funding; Institute for Frontier Life and Medical Sciences, Kyoto University: Research Funding.

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The effect of quercetin on oxidative DNA damage and myelosuppression induced by etoposide in bone marrow cells of rats.

Acta Biochimica Polonica ◽

10.18388/abp.2014_1915 ◽

2014 ◽

Vol 61 (1) ◽

Cited By ~ 2

Author(s):

Monika A Papież

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Cell Line ◽

Myeloid Leukemia ◽

Oxidative Dna Damage ◽

Bone Marrow Cells ◽

Common Side Effect ◽

Marrow Cells

There is increasing evidence for the existence of an association between the presence of etoposide phenoxyl radicals and the development of treatment-related acute myeloid leukemia (t-AML), which occurs in a few percent of patients treated with this chemotherapeutic agent. The most common side effect caused by etoposide is myelosuppression, which limits the use of this effective drug. The goal of the study was to investigate the influence of antioxidant querectin on myelosuppression and oxidative DNA damage caused by etoposide. The influence of quercetin and/or etoposide on oxidative DNA damage was investigated in LT-12 cell line and bone marrow cells of rats via comet assay. The effect of quercetin on myelosuppression induced by etoposide was invetsigated by cytological analysis of bone marrow smears stained with May-Grünwald-Giemsa stain. Etoposide caused a significant increase in oxidative DNA damage in bone marrow cells and LT-12 cell line in comparison to the appropriate controls. Quercetin significantly reduced the oxidative DNA damage caused by etoposide both in vitro and in vivo. Quercetin also significantly protected against a decrease in the percentage of myeloid precursors and erythroid nucleated cells caused by etoposide administration in comparison to the group treated with etoposide alone. The results of the study indicate that quercetin could be considered a protectively acting compound in bone marrow cells during etoposide therapy.

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The Grb2 binding site is required for the induction of chronic myeloid leukemia-like disease in mice by the Bcr/Abl tyrosine kinase

Blood ◽

10.1182/blood.v96.2.664.014k52_664_670 ◽

2000 ◽

Vol 96 (2) ◽

pp. 664-670 ◽

Cited By ~ 9

Author(s):

Ryan P. Million ◽

Richard A. Van Etten

Keyword(s):

Bone Marrow ◽

Chronic Myeloid Leukemia ◽

Tyrosine Kinase ◽

Intracellular Signaling ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

Myeloproliferative Disease ◽

Efficient Induction ◽

Direct Binding

The BCR/ABL oncogene results from a balanced translocation between chromosomes 9 and 22 and is found in patients with chronic myeloid leukemia (CML) and in some patients with acute B-lymphoid leukemia. The Bcr/Abl fusion protein is a constitutively active tyrosine kinase that stimulates several intracellular signaling pathways, including activation of Ras through direct binding of the SH2-containing adapter protein Grb2 to Bcr tyrosine 177. A tyrosine-to-phenylalanine mutation (Y177F) at this site blocks the co-association of Bcr/Abl and Grb2 in vivo and impairs focus formation by Bcr/Abl in fibroblasts. However, the Bcr/Abl Y177F mutant can transform hematopoietic cell lines and primary bone marrow cells in vitro, so the importance of the Bcr/Abl–Grb2 interaction to myeloid and lymphoid leukemogenesis in vivo is unclear. We have recently demonstrated the efficient induction of CML-like myeloproliferative disease by BCR/ABL in a murine bone marrow transduction/transplantation model system. The Y177F mutation greatly attenuates the myeloproliferative disease induced by BCR/ABL, with mice developing B- and T-lymphoid leukemias of longer latency. In addition, the v-abl oncogene of Abelson murine leukemia virus, whose protein product lacks interaction with Grb2, is completely defective for the induction of CML-like disease. These results suggest that direct binding of Grb2 is required for the efficient induction of CML-like myeloproliferative disease by oncogenic Abl proteins.

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Bmi-1 Is Dispensable for the Development of Acute Myeloid Leukemia Mediated by MLL-AF9

Blood ◽

10.1182/blood.v116.21.63.63 ◽

2010 ◽

Vol 116 (21) ◽

pp. 63-63

Author(s):

Tobias A Neff ◽

Florian H Heidel ◽

Scott Armstrong

Keyword(s):

Bone Marrow ◽

Myeloid Leukemia ◽

Bone Marrow Cells ◽

S Phase ◽

Median Latency ◽

Self Renewal ◽

Transplantation Experiments ◽

Phase Proportion

Abstract Abstract 63 The Polycomb group 1 gene Bmi1 has been shown to be required for several normal adult stem cell types, including hematopoietic stem cells. High expression of Bmi1 is correlated with adverse prognosis in human AML and MDS. Bmi1 null murine fetal liver cells transformed with HoxA9 and Meis1 give rise to a primary leukemia of expanding blasts that fails to expand in secondary recipient mice thus demonstrating a defect in leukemia self-renewal (Nature. 2003; 423:255). More recently, Bmi1 has also been shown to bind PML-RARA, and to be required for in vitro replating of PML-RARA immortalized murine colony forming cells (cfc). Our laboratory recently demonstrated that MLL-AF9, a recurrent translocation commonly found in human acute leukemia, more efficiently transforms murine granulocyte macrophage progenitors (GMP) compared to the combination of HoxA9 and Meis1. We decided to test if MLL-AF9 is able to bypass the requirement for Bmi1 previously reported in other murine leukemia models. Lineage marker negative (Lin-) bone marrow cells from WT and Bmi1 −/− mice were transduced with an MSCV-based ecotropic retroviral vector expressing MLL-AF9 and linked via an internal ribosomal entry site (IRES) the selectable marker, GFP. Cells were harvested and injected into 5 sublethally (200 cGy) irradiated NOD/SCID mice per group. All mice developed AML based on GFP expression, spleen size, forward/side scatter profile and surface marker staining (Mac1 positive/Gr1 positive, B220 negative, CD3 negative). Median latency in the WT group was 54 days, whereas mice receiving a graft consisting of Bmi1 −/− cells transduced with MLL-AF9 developed leukemia with a median latency of 89 days. The experiment was repeated with very similar results with median latencies of 50 days (WT group) and 70 days (Bmi1 −/−). To confirm sustained self-renewal, we then proceeded to secondary transplantation experiments. Secondary leukemia developed with similar latency in recipients of primary WT (31.5d) and Bmi1−/− (32d) leukemic bone marrow cells. The experiment was repeated with bone marrow from mice with primary leukemias from the second cohort and again similar latencies were observed (42.5 days in the WT group vs 29.5 days in the Bmi1 −/− group.). Finally we proceeded to tertiary transplantation experiments. All recipients of secondary AML cells developed AML with median latencies of 17.5 d (WT) and 21 d (KO). To compare our results with a system closer to the original studies of leukemogenesis in a Bmi1 −/− background, we chose to transduce lin- bone marrow from WT and Bmi1 −/− with retroviral vectors for HoxA9-IRES-GFP and Meis1-pgk-puro. Both, WT and HoxA9/Meis1 transduced cells replated in in vitro methylcellulose cultures for at least 4 rounds. In the first of 2 in vivo experiments, 5/5 recipients of wild type cells developed acute myeloid leukemia with a median latency of 88 days. In contrast, none of the five recipients of HoxA9/Meis1a transduced Bmi1−/− cells developed leukemia, and these mice were sacrificed 6 months after transplantation. In a second independent experiment, 5/5 recipients of WT HoxA9/Meis1 transduced cells developed AML with a median latency of 62 days. One recipient of Bmi1 −/− cells transduced with HoxA9/Meis1 developed leukemia. One mouse died from a spontaneous lymphoma. The remaining 3 mice in this cohort did not develop leukemia. These findings are consistent with the previously published data and suggest that MLL-AF9 is more efficient in generating AML in Bmi1−/− bone marrow compared to the combination of HoxA9 and Meis1. To further characterize the Bmi1 −/− leukemias, we analyzed expression of the primary Bmi1 target p16 by Western blot. In primary, secondary and tertiary AMLs from Bmi1 −/− mice, derepression of p16 could be demonstrated. We analyzed the cell cycle distribution of cultured tertiary leukemia cells. WT cells showed a S phase proportion of 44–45 percent (n=2), whereas the Bmi1 −/− cells demonstrated a S-phase proportion of 30–38% (n=2). These results demonstrate modest effects of p16 derepression on cell cycle without catastrophic failure to divide. This is mirrored by the in vivo development of secondary and tertiary leukemias in the presence of elevated p16 levels. Preliminary data suggest a similar but less pronounced derepression of p19. Future experiments will focus on the mechanisms that allow MLL-AF9 transduced cells to grow in the presence of derepressed p16 and p19. Disclosures: Armstrong: Epizyme, Inc: Consultancy.

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Thrombopoietin-Induced Stimulation of Megakaryocyte- Enriched Bone Marrow Cultures

10.1055/s-0039-1687198 ◽

1979 ◽

Author(s):

K. L. Kellar ◽

B. L. Evatt ◽

C. R. McGrath ◽

R. B. Ramsey

Keyword(s):

Bone Marrow ◽

Dna Synthesis ◽

Bone Marrow Cells ◽

Rabbit Plasma ◽

Bone Marrow Cultures ◽

Plasma Fractions ◽

Marrow Cultures ◽

Stimulation Of

Liquid cultures of bone marrow cells enriched for megakaryocytes were assayed for incorporation of 3H-thymidine (3H-TdR) into acid-precipitable cell digests to determine the effect of thrombopoietin on DNA synthesis. As previously described, thrombopoietin was prepared by ammonium sulfate fractionation of pooled plasma obtained from thrombocytopenic rabbits. A control fraction was prepared from normal rabbit plasma. The thrombopoietic activity of these fractions was determined in vivo with normal rabbits as assay animals and the rate of incorporation of 75Se-selenomethionine into newly formed platelets as an index of thrombopoietic activity of the infused material. Guinea pig megakaryocytes were purified using bovine serum albumin gradients. Bone marrow cultures containing 1.5-3.0x104 cells and 31%-71% megakaryocytes were incubated 18 h in modified Dulbecco’s MEM containing 10% of the concentrated plasma fractions from either thrombocytopenic or normal rabbits. In other control cultures, 0.9% NaCl was substituted for the plasma fractions. 3H-TdR incorporation was measured after cells were incubated for 3 h with 1 μCi/ml. The protein fraction containing thrombopoietin-stimulating activity caused a 25%-31% increase in 3H-TdR incorporation over that in cultures which were incubated with the similar fraction from normal plasma and a 29% increase over the activity in control cultures to which 0.9% NaCl had been added. These data suggest that thrombopoietin stimulates DNA synthesis in megakaryocytes and that this tecnique may be useful in assaying thrombopoietin in vitro.

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