Raf1 Is Required for Induction of a Bcr-Abl Positive CML Like Myeloproliferative Disease in Mice

Abstract Introduction: Chronic myelogenous leukemia (CML) results from neoplastic transformation of hematopoietic stem cells (HSC), characterized by a chromosomal translocation t(9;22)(q34;q11). This aberration leads to the expression of the oncogenic tyrosine kinase Bcr-Abl, which mediates signals for proliferation, transformation and anti-apoptosis via various different pathways including the Raf/MEK/ERK cascade. The cytoplasmic protein Raf1 is a key molecule within this cascade. Recent studies have revealed an additional function of the Raf-1 kinase that is independent of the activation of the MAPK cascade and whose effect is to increase resistance to apoptosis. Therefore Raf1 is an interesting target for molecular therapies and more effective Raf1 inhibitors have recently been developed by the pharmaceutical industry. Here we report the impact of Raf1 signalling for Bcr-Abl mediated transformation. Methods: We exerted a siRNA based approach in combination with a murine bone marrow transplantation model. To this end we designed a MSCV based retrovirus encoding both the Raf1 microRNA and the Bcr-Abl oncogene on a single construct. This approach ensured knockdowns of more than 90% of Raf1 in every Bcr-Abl transformed cell. Results: Methylcellulose assays demonstrated that bone marrow coexpressing Raf1 microRNA and Bcr-Abl had a 2 fold decreased colony forming ability compared to control cells. We then transduced bone marrow (BM) with retrovirus coexpressing Raf1 microRNA and p185 Bcr-Abl and transplanted lethally irradiated recipient Balb/C mice. The onset and progression of leukemia was significantly delayed in mice transplanted with Raf1 microRNA and Bcr-Abl compared with the Bcr- Abl transduced control microRNA group. Raf1 knockdown mice showed only a moderate rise of white blood cell (WBC) counts and prolonged overall survival (median survival 39 ± 7.1 days) in comparison to control mice (23.3 ± 2.4 days). However, we were not able to completely avoid the development of leukemia by Raf1 knockdown. Conclusion: Taken together our data demonstrate that Raf1 is important for the development of a myeloproliferative disease by Bcr-Abl in mice. Therefore Raf1 inhibition in combination with Bcr-Abl kinase inhibition depicts an interesting approach towards eradication of Bcr- Abl positive leukemia. In addition, this study describes a novel and versatile approach to express an oncogene and a microRNA using a single retroviral construct. Thus this powerful tool can be used to systematically screen drugable signalling targets involved in oncogenesis.

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Grb10 Mediated Akt Activation Is Required for Induction of CML Like Myeloproliferative Disease in Mice by BCR-ABL.

Blood ◽

10.1182/blood.v110.11.1012.1012 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1012-1012

Author(s):

Corinna Albers ◽

Anna L. Illert ◽

Cornelius Miething ◽

Christian Peschel ◽

Justus Duyster

Keyword(s):

Bone Marrow ◽

Tyrosine Kinases ◽

Chromosomal Translocation ◽

Control Group ◽

Myeloproliferative Disease ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Vitro Analysis ◽

The Impact

Abstract Chronic myelogenous leukaemia (CML) results from the neoplastic transformation of hematopoietic stem cells (HSC) and is characterized by a chromosomal translocation t(9;22)(q34;q11). This aberration leads to the expression of the oncogenic tyrosine kinase BCR-ABL, which mediates signals for proliferation, transformation and anti-apoptosis via various signalling pathways. Grb10, a member of the growth factor bound proteins, is known to bind activated tyrosine kinases like BCR-ABL and might be involved in the activation of the Akt signalling pathway. Here we report the impact of Grb10 for BCR-ABL mediated transformation. We exerted a siRNA based approach in combination with a murine bone marrow transplantation model. To this end we designed a MSCV based retrovirus encoding both a Grb10 microRNA and the BCR-ABL oncogene on a single construct. This approach ensured knockdowns of more than 90% in every BCR-ABL transformed cell. Methylcellulose assays demonstrated that bone marrow coexpressing Grb10 microRNA and BCR-ABL had a 4-fold decreased colony forming ability compared to control cells. We then transduced bone marrow (BM) with retrovirus coexpressing Grb10 microRNA and p185 BCR-ABL and transplanted lethally irradiated recipient Balb/C mice. The onset and progression of leukaemia was significantly delayed in mice transplanted with Grb10 microRNA and BCR-ABL compared with the BCR-ABL transduced control microRNA group. However, we were not able to completely avoid the development of leukaemia by Grb10 knockdown. Mice transplanted with the Grb10 knockdown construct showed a delayed lymphoblastic disease, positive for B220, whereas the control group developed a rapid myeloproliferative disease, characterized by CD11b and Gr-1. In vitro analysis of BaF/3 and 32D cells showed that Grb10 knockdown in combination with BCR-ABL expression leads to a reduced phosphorylation of Akt. Taken together our data demonstrate that Grb10 is required for the development of a myeloproliferative disease by BCR-ABL in mice. Hereby, Grb10 seems to be critical for the BCR-ABL induced activation of the Akt pathway. In addition, this study describes a novel approach to express an oncogene and a microRNA using a single retroviral construct. This tool can be used to systematically screen for drugable signalling targets involved in oncogenesis.

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Myeloproliferative disease induced by TEL-PDGFRB displays dynamic range sensitivity to Stat5 gene dosage

Blood ◽

10.1182/blood-2006-07-036335 ◽

2007 ◽

Vol 109 (9) ◽

pp. 3906-3914 ◽

Cited By ~ 33

Author(s):

Jennifer A. Cain ◽

Zhifu Xiang ◽

Julie O'Neal ◽

Friederike Kreisel ◽

AnnaLynn Colson ◽

...

Keyword(s):

Dynamic Range ◽

Fusion Gene ◽

Gene Dosage ◽

Fetal Liver ◽

Chromosomal Translocation ◽

Myeloproliferative Disease ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Cell Culture Systems ◽

Stem And Progenitor Cells

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Constitutively active AKT depletes hematopoietic stem cells and induces leukemia in mice

Blood ◽

10.1182/blood-2009-06-229443 ◽

2010 ◽

Vol 115 (7) ◽

pp. 1406-1415 ◽

Cited By ~ 184

Author(s):

Michael G. Kharas ◽

Rachel Okabe ◽

Jared J. Ganis ◽

Maricel Gozo ◽

Tulasi Khandan ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Bone Marrow Cells ◽

Leukemic Stem Cells ◽

Myeloproliferative Disease ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Akt Activation ◽

Marrow Cells

Abstract Human cancers, including acute myeloid leukemia (AML), commonly display constitutive phosphoinositide 3-kinase (PI3K) AKT signaling. However, the exact role of AKT activation in leukemia and its effects on hematopoietic stem cells (HSCs) are poorly understood. Several members of the PI3K pathway, phosphatase and tensin homolog (Pten), the forkhead box, subgroup O (FOXO) transcription factors, and TSC1, have demonstrated functions in normal and leukemic stem cells but are rarely mutated in leukemia. We developed an activated allele of AKT1 that models increased signaling in normal and leukemic stem cells. In our murine bone marrow transplantation model using a myristoylated AKT1 (myr-AKT), recipients develop myeloproliferative disease, T-cell lymphoma, or AML. Analysis of the HSCs in myr-AKT mice reveals transient expansion and increased cycling, associated with impaired engraftment. myr-AKT–expressing bone marrow cells are unable to form cobblestones in long-term cocultures. Rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR) rescues cobblestone formation in myr-AKT–expressing bone marrow cells and increases the survival of myr-AKT mice. This study demonstrates that enhanced AKT activation is an important mechanism of transformation in AML and that HSCs are highly sensitive to excess AKT/mTOR signaling.

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SOCS1 cooperates with FLT3-ITD in the development of myeloproliferative disease by promoting the escape from external cytokine control

Blood ◽

10.1182/blood-2010-08-301416 ◽

2012 ◽

Vol 120 (8) ◽

pp. 1691-1702 ◽

Cited By ~ 23

Author(s):

Pavankumar N. G. Reddy ◽

Bülent Sargin ◽

Chunaram Choudhary ◽

Stefan Stein ◽

Manuel Grez ◽

...

Keyword(s):

Bone Marrow ◽

Target Genes ◽

Colony Growth ◽

Interferon Γ ◽

Myelogenous Leukemia ◽

Cytokine Signaling ◽

Interferon Α ◽

Myeloproliferative Disease ◽

Clinical Prognosis ◽

Murine Bone Marrow

Abstract Activating mutations in the receptor tyrosine kinase FLT3 are frequently found in acute myelogenous leukemia patients and confer poor clinical prognosis. It is unclear how leukemic blasts escape cytokine control that regulates normal hematopoiesis. We have recently demonstrated that FLT3-internal tandem duplication (ITD), when localized to the biosynthetic compartment, aberrantly activates STAT5. Here, we show that one of the target genes induced by STAT5 is suppressor of cytokine signaling (SOCS)1—a surprising finding for a known tumor suppressor. Although SOCS1 expression in murine bone marrow severely impaired cytokine-induced colony growth, it failed to inhibit FLT3-ITD–supported colony growth, indicating resistance of FLT3-ITD to SOCS1. In addition, SOCS1 coexpression did not affect FLT3-ITD–mediated signaling or proliferation. Importantly, SOCS1 coexpression inhibited interferon-α and interferon-γ signaling and protected FLT3-ITD hematopoietic cells from interferon-mediated growth inhibitory effects. In a murine bone marrow transplantation model, the coexpression of SOCS1 and FLT3-ITD significantly shortened the latency of a myeloproliferative disease compared with FLT3-ITD alone (P < .01). Mechanistically, SOCS proteins shield FLT3-ITD from external cytokine control, thereby promoting leukemogenesis. The data demonstrate that SOCS1 acts as a conditional oncogene, providing novel molecular insights into cytokine resistance in oncogenic transformation. Restoring cytokine control may provide a new way of therapeutic intervention.

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Cdx4 Differs from Cdx2 in Its Oncogenic Potential and Its Regulation of Hox Gene Expression in the Murine Bone Marrow Transplantation Model.

Blood ◽

10.1182/blood.v112.11.1187.1187 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1187-1187

Author(s):

Silvia Thoene ◽

Vijay P.S. Rawat ◽

Vegi M. Naidu ◽

Wolfgang Hiddemann ◽

Michaela Feuring-Buske ◽

...

Keyword(s):

Bone Marrow ◽

Stem Cell ◽

Expression Profile ◽

Ectopic Expression ◽

Hematopoietic Progenitors ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Integration Sites ◽

The Impact

Abstract Cdx4 is known to be of importance for specification of cell fate in embryonic hematopoiesis with defects leading to severe perturbation of blood formation. When overexpressed in a murine hematopoietic stem cell line, Cdx4 is capable to enhance progenitor formation in vitro and promote lymphoid reconstitution of lethally irradiated, transplanted mice in vivo. In line with this important function of Cdx4 in early hematopoiesis, we analyzed expression of Cdx4 in highly purified subpopulations isolated from murine bone marrow (BM) cells by TaqMan qPCR. Cdx4 showed an expression profile known from other stem cell regulatory genes with high expression in early hematopoietic progenitors followed by decreasing expression towards the more differentiated stages of hematopoiesis, with a more than 1200-fold lower expression in total BM cells compared to progenitor enriched 5-FU BM cells (n=3). To test the impact of Cdx4 on murine progenitors, we retrovirally transduced 5-FU BM cells with Cdx4. Overexpression of Cdx4 induced growth of BM cells in liquid expansion assay (Cdx4 5.7×108±2.2×108 SEM, EGFP 2.6×106±9×105 SEM, p=0.020; cell numbers after 14 days in cytokine supplemented medium, n=5). In addition, expression of Cdx4 conferred serial replating capacity to murine BM progenitors compared to empty vector control (CFU total after 3rd replating: 4.5×109±1.3×109 SEM/500 input cells in 1st CFC, n=5). This effect was significantly stronger compared to hematopoietic progenitors overexpressing the leukemogenic Cdx2 (p=0.008). Immunophenotyping of cells after 3rd replating showed expression of mainly myeloid antigens and cytospin preparation revealed a mature myeloid morphology. Interestingly, these colonies were able to engraft lethally irradiated mice and showed multilineage engraftment (lymphoid:myloid ratio week 16 after transplantation: 0.5:1, n=2), indicating the ability of Cdx4 expressing colonies to maintain stem cell properties in vitro. In contrast to Cdx2-transplanted mice which showed a severe myeloid bias, regular peripheral blood analysis of mice transplanted with Cdx4 overexpressing BM cells showed multilineage engraftment confirmed by immunophenotyping and normal hematological parameters (RBC 6.7×109±4.2×108, WBC 5.8×106±5.19×105; lymphoid:myeloid ratio 1.4:1; week 8–28). Of note, with a median latency of 309 days after transplantation, nine out of ten mice transplanted with Cdx4-transduced BM cells died of transplantable leukemia. In six out of seven cases we found single retroviral integration sites, indicating a monoclonal origin of the disease. We could determine three different integration sites located between 200 and 700 bp upstream of coding sequences (n=4; Opa3, Akap1, Sema4d). The integration sites of two other mice were located intragenic (Zfyve2, Zfp407), indicating that insertional mutagenesis might be a necessary factor for Cdx4 induced leukemogenesis. Moreover, qRT-PCR revealed that Cdx4 in contrast to Cdx2 did not induce ectopic expression of the leukemogenic Hoxb8 and was associated with a significant lower (7.8-fold) expression of the leukemogenic Hoxb6 in transduced murine BM cells. Taken together, these data indicate that Cdx4 plays a major role in the regulation of early hematopoiesis. Its expression profile and its hematopoietic activity in different hematopoietic assays clearly differs from Cdx2, which was shown to be highly leukemogenic in mice and to be ectopically expressed in human AML. Murine models analyzing the impact of Cdx4 and Cdx2 expression on hematopoietic development will help to delineate critical differences between the two related genes.

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β-Catenin Suppression Targets Imatinib Resistant Leukemia Stem Cells In Mice with BCR-ABL Induced Myeloproliferative Disease

Blood ◽

10.1182/blood.v116.21.93.93 ◽

2010 ◽

Vol 116 (21) ◽

pp. 93-93 ◽

Cited By ~ 1

Author(s):

Florian H Heidel ◽

Tobias A Neff ◽

Zhaohui Feng ◽

Scott A. Armstrong

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Combination Therapy ◽

Leukemia Stem Cells ◽

Published Data ◽

Imatinib Treatment ◽

Myeloproliferative Disease ◽

Hematopoietic Stem ◽

Genetic Inactivation ◽

The Impact

Abstract Abstract 93 CML is driven by the oncogenic fusion kinase BCR-ABL which is capable of corrupting and transforming hematopoietic stem cells (HSC) and thus inititating myeloproliferative disease (MPD). While imatinib (IM) has become an effective treatment for CML, the leukemia stem cells (LSC) which reside in the bone marrow (BM) remain largely unaffected by imatinib treatment. As LSC rely on their ability to self-renew, several genes/pathways responsible for self-renewal properties have been investigated as potential targets of LSCs in myeloid neoplasia. Recent studies clearly indicate that canonical Wnt/β-catenin-signaling plays a pivotal role in initiation and development of BCR-ABL induced MPD (Zhao, Cancer Cell 2009; Hu, Leukemia 2009). However, the impact of β-catenin (Ctnnb1) on the maintenance of LSC in established disease, which more accurately models the clinical scenario, has not been extensively studied. To address the impact of targeting Ctnnb1 in CML-LSC, we used a conditional Ctnnb1 knockout mouse with an estrogen-receptor driven Cre-recombinase as BM-donors. CML was induced by retroviral transduction of either Ctnnb1flox/flox ER-Cre+, Ctnnb1flox/wt ER-Cre+, or Ctnnb1wt/wt ER-Cre+ murine bone marrow with a virus encoding BCR-ABL (MSCV-BCR-ABLp210-GFP), and injection of transduced cells into wildtype syngeneic recipient mice. Administration of tamoxifen to recipient mice with CML led to excision of Ctnnb1 in the Ctnnb1flox/flox homozygous or Ctnnb1flox/wt heterozygous cells. Interestingly genetic inactivation of Ctnnb1 after onset of CML in primary recipient mice resulted only in minor prolongation of survival. The animals died due to organ infiltration (primarily lung) by differentiated progenitor cells that were presumably not affected by Ctnnb1 deletion. However, in the BM and peripheral blood, a clear reduction of GFP+ cells was evident in heterozygous - and even more pronounced - in homozygous animals, indicating a potential effect on more immature cells and perhaps LSC. To determine if LSC were compromised and to investigate the efficacy of a combination treatment with IM, we used a serial BM-transplantation assay. Consistent with previously published data, we found that leukemia cells in the Sca-1/GFP+ fraction are able to re-establish CML in secondary recipient mice and thus contain LSC. IM treatment alone led to a relative increase of Sca-1/GFP+ LSC over time. However, we found a significant reduction of Sca-1/GFP+ bone marrow cells after inactivation of Ctnnb1 during IM-treatment. Mice transplanted with Ctnnb1flox/flox ER-Cre+ CML cells had on average 0.025% Sca-1/GFP+ (total of 7,375 cells/mouse) cells in the bone marrow after treatment of the recipient mice with IM and tamoxifen, mice that received Ctnnb1flox/wt ER-Cre+ CML had 0.036% Sca-1/GFP+ cells (total of 10,584 cells/mouse) and mice that received Ctnnb1wt/wt ER-Cre+ CML had 0.075% Sca-1/GFP+ cells (total of 24,375 cells/mouse). As indomethacin has recently been shown to inhibit Ctnnb1 in hematopoietic and AML stem cells (Goessling, Cell 2009; Wang, Science 2010), we aimed to investigate the efficacy of indomethacin combined with IM. We were able to demonstrate a decrease in Ctnnb1 protein after indomethacin treatment of human cell lines and transformed murine BM cells by either immunoblot, flow-cytometry or immunofluorescence. Moreover, we were able to show synergy between indomethacin and IM co-treatment which produced almost complete abrogation of Ctnnb1 protein. IM/Indomethacin co-treatment of mice with CML decreased the Kit+Sca1+ fraction of the Lin-GFP+ bone marrow population to 0.92% (1799 cells/mouse) whereas treatment with IM alone was similar to untreated mice with 2.8% of the Lin-GFP+ population being Kit+Sca1+ (22,601 cells/mouse). Importantly, transplantation of the bone marrow from treated mice into tertiary recipients showed significantly prolonged survival of tertiary recipient mice that received bone marrow from donors treated with the combination therapy (IM/INDO) compared to IM/DMSO controls (p=0.0038*). In summary, we demonstrate that inhibiting Ctnnb1 by genetic inactivation or drug treatment is an effective combination therapy with imatinib mesylate and targets CML leukemia stem cells. This provides strong evidence for the development and clinical use of β-catenin-targeted therapies in CML. Disclosures: No relevant conflicts of interest to declare.

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AML1/Runx1 Negatively Regulates the Number of Quiescent Hematopoietic Stem Cells in Adult Hematopoiesis.

Blood ◽

10.1182/blood.v108.11.4204.4204 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4204-4204

Author(s):

Motoshi Ichikawa ◽

Takashi Asai ◽

Masahiro Nakagawa ◽

Masahito Kawazu ◽

Susumu Goyama ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Side Population ◽

Hematopoietic Cells ◽

Myelogenous Leukemia ◽

Human Leukemia ◽

Myeloproliferative Disease ◽

Hematopoietic Stem ◽

Quiescent Hscs

Abstract Transcription factor AML1 (also called Runx1), which was initially isolated from the t(8;21) chromosomal translocation frequently found in the acute myelogenous leukemia FAB M2 subtype, is essential for the development of multilineage hematopoiesis in mouse embryos. By analyzing conditional AML1 knockout mice, we have previously shown that AML1 negatively regulates the number of immature hematopoietic cells defined as lineage-negative, CD34− Sca-1+ c-Kit+ (34KSL) cells in adult hematopoiesis, while it is required for megakaryocytic maturation and lymphocytic development. The former is a significant observation because an increase in hematopoietic stem/progenitor cells due to defective AML1 function may be closely related to the development of human leukemia. In support of this is the fact that mice in which leukemic chimeric protein AML1/ETO is expressed in hematopoietic cells are subject to myeloproliferative disease and develop leukemia after additional mutation. However, it has remained yet to be determined how AML1 contributes to homeostasis of hematopoietic stem cells (HSCs). To address this issue, we analyzed in detail HSC function in the absence of AML1. Notably, cells in the Hoechst 33342 side population (SP) fraction are increased in number in AML1-deficient bone marrow, which suggests enrichment of quiescent HSCs. We quantitatively evaluated HSCs by bone marrow transplantation assays using limiting dilution and found a significant increase in HSC number within the AML1-deficient bone marrow. These results indicate that the number of quiescent HSCs is negatively regulated by AML1, loss of which may result in accumulation of leukemic stem cell pool in AML1-related leukemia.

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Myeloproliferative Disease Induced by TEL-PDGFRB Displays Dynamic Range Sensitivity to Stat5 Gene Dosage in Mice.

Blood ◽

10.1182/blood.v108.11.3620.3620 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3620-3620

Author(s):

Jennifer A. Cain ◽

Jing Chen ◽

Zhifu Xiang ◽

Julie O’Neal ◽

Benjamin H. Lee ◽

...

Keyword(s):

Bone Marrow ◽

Median Survival ◽

Dynamic Range ◽

Gene Dosage ◽

Chromosomal Translocation ◽

Myeloproliferative Disease ◽

Murine Bone Marrow ◽

Relative Contribution ◽

Stat5a Gene ◽

Transplantation Experiments

Abstract Expression of the constitutively activated TEL/PDGFβR fusion protein is associated with the t(5;12)(q33;p13) chromosomal translocation found in a subset of patients with chronic myelomonocytic leukemia (CMML). TEL/PDGFβR activates multiple signal transduction pathways in cell culture systems and induces myeloproliferative disease (MPD) in a murine bone marrow transduction/transplantation model of disease. Two TEL-PDGFβR juxtamembrane tyrosines, corresponding to tyrosines activating Stat5 and Src signaling molecules in native PDGFβR, are required for TEL-PDGFRB mediated MPD in mice. We used gene-targeted mice as donors in bone marrow transduction/transplantation experiments to characterize the contribution of Stat and Src genes in the development of TEL-PDGFRB disease. Mice transplanted with cells harboring targeted deletions of both Stat5a and Stat5b genes (TPiGFP→Stat5ab−/−) were protected from rapidly fatal MPD (median survival >125 vs. 25 days in TPiGFP→Stat5ab+/+mice, P<0.0001). In contrast, TPiGFP→Lyn−/−Hck−/−Fgr−/− mice developed rapidly fatal MPD characterized by splenomegaly and leukocytosis. Similarly, TEL-PDGFRB induced MPD in TPiGFP→Stat1−/− mice and TPiGFP→Stat1+/+ strain-matched mice alike (median survival 45 vs. 53 days). These data suggest that Stat5, but not Src family kinases Lyn, Hck and Fgr nor Stat family member Stat1, is functionally relevant in the development of TEL-PDGFRB mediated disease. To assess the relative contribution of the Stat5a gene in TEL-PDGFRB mediated disease, we used mice harboring either homozygous (Stat5a−/−) or heterozygous (Stat5a+/−) deletion as donors in bone marrow transduction/transplantation experiments with TEL-PDGFRB. Surprisingly, survival was significantly prolonged in TPiGFP→Stat5a−/− mice (144 vs. TPiGFP→Stat5a+/+ 31 days, P <0.0001) and TPiGFP→Stat5a+/− mice (145 versus TPiGFP→Stat5a+/+ 31 days, P <0.001), demonstrating significant sensitivity of TEL-PDGFRB-induced MPD to loss of even a single Stat5a allele. TEL-PDGFRB induced severe leukocytosis in TPiGFP→Stat5a+/+ mice (median WBC 547K/μL), but disease was attenuated in TPiGFP→Stat5a+/− mice mice (median WBC 56K/μL) and undetectable in TPiGFP→Stat5a−/−mice (median WBC 5K/μL), suggesting that TEL-PDGFRB disease is exquisitely sensitive to Stat5a gene dosage. On the other hand, TPiGFP→Stat5b−/− mice developed robust MPD with similar latency to wild-type mice. However, 4 of 13 TPiGFP→Stat5b−/− mice failed to develop MPD and were longer-lived, suggesting a role for Stat5b in TEL-PDGFRB disease penetrance. To address the possibility that Stat5a and Stat5b were distinct in their ability to transmit myeloproliferative signals in TEL-PDGFRB mediated MPD, we designed add-back constructs to co-express TEL-PDGFRB with either Stat5a (TPiStat5a) or Stat5b (TPiStat5b). Addback Stat5 restored MPD with similar disease latency in TPiStat5a→Stat5a−/− and TPiStat5b→Stat5a−/− mice (median survival 96 and 97 days, respectively). TPiStat5a→Stat5a+/− mice developed MPD with shorter disease latency (28 days), again correlating with Stat5a gene dosage. These data provide functional evidence for the importance of the Stat5 pathway in a model of TEL-PDGFRB-induced CMML and suggest that Stat5a and Stat5b play unique roles in the development of disease in this model.

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A Single Retroviral Vector Design for the Simultaneous Expression of a Mir30 Based Shrna with An Oncogene – Identification of Raf-1 but Not BRAF as a Crucial Mediator for BCR-ABL Mediated Leukemogenesis.

Blood ◽

10.1182/blood.v116.21.3392.3392 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3392-3392

Author(s):

Corinna Albers ◽

Anna Lena Illert ◽

Hannes Leischner ◽

Cornelius Miething ◽

Richard Huss ◽

...

Keyword(s):

Bone Marrow ◽

Overall Survival ◽

Retroviral Vector ◽

Myelogenous Leukemia ◽

Vector System ◽

Signaling Cascade ◽

In Vivo Models ◽

Murine Bone Marrow ◽

The Impact ◽

Transplantation Model

Abstract Abstract 3392 Introduction: Chronic myelogenous leukemia (CML) is characterized by the t(9;22)(q34;q11) chromosomal translocation and the expression of BCR-ABL, a fusion protein with tyrosine kinase activity. BCR-ABL activates various signaling cascades mediating signals for proliferation, transformation and anti-apoptosis. The BCR-ABL inhibitor imatinib is the standard therapy for CML. However, this treatment is assumed to be not curative since leukemia initiating cells cannot be completely eradicated by solely BCR-ABL inhibition. Identification of key mediators within the BCR-ABL signaling cascade thus remains crucial. The MEK/ERK cascade is one of the major promitogenic pathways activated in CML. Whether Raf-1, BRAF or both Raf isoforms are required for BCR-ABL mediated activation of this pathway is not known. As both Raf-1 and BRAF knockout mice are embryonic lethal, the role of Raf-1 and BRAF in BCR-ABL mediated leukemogenesis has not been investigated in appropriate in vivo models so far. Here we studied the impact of Raf-1 and BRAF for BCR-ABL dependent transformation by using a retroviral vector system, which allows to directly couple shRNA based target suppression to oncogene expression in a CML mouse model. Methods: We exerted an shRNA-based approach in combination with a murine bone marrow transplantation model. To this end we designed a MSCV based retrovirus encoding both the BCR-ABL oncogene and miR-30 based shRNAs (miR) for BRAF and Raf-1 respectively on a single construct resulting in one shared RNA transcript. This approach ensured knockdowns of more than 80–90% for the respective Raf protein in every BCR-ABL transformed cell. Result: Methylcellulose assays showed that primary bone marrow cells coexpressing Raf-1 miR and BCR-ABL had a 2 fold decreased colony forming ability, whereas BRAF knockdown had no impact on colony forming ability compared to control cells. We then transplanted murine bone marrow (BM), transduced with retrovirus coexpressing Raf-1 or BRAF miR and p185 BCR-ABL, to lethally irradiated recipient Balb/C mice. The onset and progression of leukemia was significantly delayed in mice transplanted with Raf-1 miR but not BRAF miR and BCR-ABL compared with the BCR-ABL transduced control miR group. Raf-1 knockdown mice showed only a moderate rise of white blood cell (WBC) counts and prolonged overall survival in comparison to control mice. However, BRAF knockdown had no significant effect on overall survival or disease progression in the bone marrow transduction transplantation model. We hypothesized that this impact of Raf-1 knockdown might be due to incomplete activation of the MEK/ERK cascade in the absence of Raf-1. We could demonstrate that Raf-1 is necessary for BCR-ABL dependent ERK activation in primary murine bone marrow as well as in cell lines. In contrast in BRAF knockdown BCR-ABL positive cells levels of phosphorylated and thereby activated ERK remained unchanged compared to control cells, indicating that BRAF is dispensable for BCR-ABL dependent ERK phosphorylation. Conclusion: Taken together our data demonstrate that primarily Raf-1 is responsible for BCR-ABL mediated activation of the promitogenic MEK/ERK signaling cascade. Raf-1 but not BRAF is also crucial for the development of a myeloproliferative disease by BCR-ABL in mice. Therefore, Raf-1 but not BRAF inhibition may be a potential interesting additional therapeutic approach in CML.The coexpression of an oncogene and a target specific miR-30 based shRNA from a single retroviral construct displays a powerful tool that can be used to systematically screen drugable signaling targets involved in CML and other leukemic malignancies. Disclosures: No relevant conflicts of interest to declare.

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The Impact of Age and Gender on Hematopoietic Stem Cells and Immune Contexture of the Bone Marrow Microenvironment

Cells Tissues Organs ◽

10.1159/000510774 ◽

2020 ◽

pp. 1-6

Author(s):

Rebar N. Mohammed

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Hematopoietic Stem Cells ◽

Immune Cells ◽

Absolute Number ◽

Cell Number ◽

Hematopoietic Stem ◽

The Absolute ◽

And Gender ◽

The Impact

Hematopoietic stem cells (HSCs) are a rare population of cells that reside mainly in the bone marrow and are capable of generating and fulfilling the entire hematopoietic system upon differentiation. Thirty-six healthy donors, attending the HSCT center to donate their bone marrow, were categorized according to their age into child (0–12 years), adolescence (13–18 years), and adult (19–59 years) groups, and gender into male and female groups. Then, the absolute number of HSCs and mature immune cells in their harvested bone marrow was investigated. Here, we report that the absolute cell number can vary considerably based on the age of the healthy donor, and the number of both HSCs and immune cells declines with advancing age. The gender of the donor (male or female) did not have any impact on the number of the HSCs and immune cells in the bone marrow. In conclusion, since the number of HSCs plays a pivotal role in the clinical outcome of allogeneic HSC transplantations, identifying a younger donor regardless the gender is critical.

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