Loss of Stress Sensor GADD45a and GADD45b Accelerates BCR-ABL-Driven Leukemogenesis Via Distinct Signaling and Cellular Pathways.

Abstract Abstract 1217 The bcr/abl oncogene causes chronic myelogenous leukemia (CML) in humans. BCR/ABL is known to localize to the cytoskeleton and to display a constitutively active tyrosine kinase activity that leads to the recruitment of downstream effectors of cell proliferation and survival. This is accomplished via several adapter proteins and signaling pathways, including Ras, PI3K-AKT, PkD2-NFkB and JAK-STAT5, all of which are believed to participate in the pathogenesis of CML. The complex nature of these signaling pathways and how they contribute to the initiation and progression of CML is only partially understood. The Gadd45 family of genes (Gadd45a, Gadd45b & Gadd45g) encode for small (18 kd) nuclear proteins that are rapidly induced by multiple stressors, including genotoxic and oncogenic stress. They are involved in G2/M cell cycle arrest and apoptosis in response to exogenous stress stimuli through MAPK and JNK/SAPK pathways. Furthermore Gadd45a has been identified as a mediator of oncogenic Ras signaling. GADD45 proteins are upregulated during myeloid lineage terminal differentiation. To investigate if and how GADD45A and GADD45B play a role in the development of CML, syngeneic wild type lethally irradiated mice were reconstituted with wild type, gadd45a or gadd45b null myeloid progenitors transduced with a retrovirally expressed 210-kD BCR/ABL fusion oncoprotein. It was observed that loss of gadd45a or gadd45b accelerates the development of BCR/ABL driven leukemia in wild type recipients. BCR/ABL transformed gadd45a or gadd45b deficient progenitor recipients exhibited significantly accelerated kinetics of increase in the number of WBC and percentage of myeloid blasts in blood compared to mice reconstituted with the same number of wild type bone marrow cells transduced with BCR/ABL. There was also increase in the rate of accumulation of CD11b+Gr1+ cells in the bone marrow and spleen. Using in vitro and in vivo BrdU assays, enhanced proliferation capacity was observed for BCR/ABL transduced gadd45a, but not gadd45b, deficient myeloid progenitors. However, impaired apoptosis was observed both in BCR/ABL transduced gadd45a and gadd45b deficient myeloid progenitors. These results indicate that both gadd45a and gadd45b function as suppressors of the development of BCR/ABL driven CML, where gadd45a appears to suppress CML via a mechanism involving both inhibition of cell proliferation and enhancement of apoptosis, whereas gadd45b appears to effect only apoptosis. Enhanced JNK signaling was observed in both gadd45a and gadd45b deficient progenitors, whereas enhanced p38 and AKT signaling was observed only in gadd45a deficient myeloid progenitors. Taken together, these data indicate that loss of either gadd45a or gadd45b accelerates BCR-ABL driven CML via distinct signaling and cellular pathways. Further elucidating the role Gadd45 stress sensors play in suppressing the development of leukemia should increase understanding of the molecular/cellular pathology BCR/ABL mediated leukemogenesis, and has the potential to lead to the development of new/improved modalities for treatment of leukemia. Disclosures: No relevant conflicts of interest to declare.

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Loss of Growth Arrest DNA Damage 45a,b (GADD45a,b) Enhances Oncogenicity in BCR/ABL-Driven Chronic Myelogenous Leukemia.

Blood ◽

10.1182/blood.v114.22.3264.3264 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3264-3264

Author(s):

Xiaojin Sha ◽

Dan A. Liebermann ◽

Barbara Hoffman

Keyword(s):

Bone Marrow ◽

Dna Damage ◽

Chronic Myelogenous Leukemia ◽

Cellular Proliferation ◽

Conflicts Of Interest ◽

Growth Arrest ◽

Myelogenous Leukemia ◽

Wild Type ◽

Myeloid Lineage ◽

Myeloid Progenitors

Abstract 3264 Poster Board III-1 The bcr/abl oncogene causes chronic myelogenous leukemia (CML) in human. BCR/ABL induces the transformation of myeloid lineage through MAPK, JNK/SAPK, PI3K signaling pathways. Growth arrest DNA damage 45A (GADD45A) and GADD45B are upregulated during myeloid lineage terminal differentiation. They are involved in G2/M cell cycle arrest and apoptosis in response to exogenous stress stimuli through MAPK and JNK/SAPK pathways. To investigate the effect of GADD45A and GADD45B in the development of CML, syngeneic wild type lethally irradiated mice were reconstituted with wild type, gadd45a or gadd45b null myeloid progenitors transduced with a retrovirally expressed 210-kD BCR/ABL fusion oncoprotein. We found that loss of gadd45a or gadd45b accelerated the development of CML-like disease in wild type recipients. BCR/ABL transformed gadd45a or gadd45b deficient progenitor recipients exhibited a significantly accelerated kinetics of increase in the number of WBC and percentage of myeloid blasts in blood compared to mice reconstituted with the same number of wild type bone marrow cells transduced with BCR/ABL. There was also increase in the rate of accumulation of CD11b+Gr1+ cells in the bone marrow and spleen. Using in vitro and in vivo BrdU assays, enhanced proliferation capacity was observed for both BCR/ABL transduced gadd45a and gadd45b deficient myeloid progenitors. BCR/ABL transduced gadd45a and gadd45b deficient primary myeloid progenitors formed more and bigger colonies compared to BCR/ABL transformed wild type progenitors. Impaired apoptosis was showed in BCR/ABL transduced gadd45a deficient myeloid progenitors. These results indicate that both gadd45a and gadd45b function as suppressors of the development of BCR/ABL driven CML, where gadd45a appears to suppress CML via mechanism involving inhibition of cell proliferation enhancement of apoptosis, whereas gadd45b appears to only inhibit cellular proliferation. Dissecting the molecular nature of signaling paths involved in the suppressive function of gadd45a and gadd45b in BCR/ABL driven CML, as well as analysis of Gadd45 in CML patients, is underway. Disclosures: No relevant conflicts of interest to declare.

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JAK2V617F Mutation Selectively Exerts the STAT3 Pathway for Enhancing a Neutrophil Activation Marker.

Blood ◽

10.1182/blood.v114.22.1901.1901 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1901-1901

Author(s):

Seido Oku ◽

Katsuto Takenaka ◽

Kotaro Shide ◽

Takashi Kumano ◽

Kikushige Yoshikane ◽

...

Keyword(s):

Cell Proliferation ◽

Conflicts Of Interest ◽

Neutrophil Activation ◽

Myelogenous Leukemia ◽

Jak2v617f Mutation ◽

Wild Type ◽

Stat3 Pathway ◽

Nb4 Cells ◽

Pi3k Inhibitor Ly294002 ◽

Activation Marker

Abstract Abstract 1901 Poster Board I-924 Leukocyte alkaline phosphatase (LAP) is considered as a neutrophil activation marker. The level of LAP is quantitated as the LAP score. It is well known that patients with chronic myelogenous leukemia (CML) usually have low LAP scores, whereas those with BCR-ABL negative chronic myeloproliferative disorders (MPD) have elevated LAP scores. In CML patients, the premature release of granulocytes from the bone marrow into the peripheral blood is considered as the cause of low LAP scores. However, the reason for elevated LAP scores in BCR-ABL negative MPD patients has been unclear. An acquired JAK2V617F mutation is observed in most patients with BCR-ABL negative MPD. It has been shown that the JAK2V617F mutation induces constitutive activation of its downstream signaling pathways such as STAT3/STAT5, Ras/MAPK and PI3K pathways. We speculated that an elevated LAP score might be due to the activation of Jak2 downstream pathways through the JAK2V617F mutation. We analyzed LAP expression in BCR-ABL negative MPD patients. JAK2V617F homozygous patients had higher LAP expression than JAK2V617F heterozygous or negative patients. AG490, the Jak2 inhibitor, was shown to significantly decrease the LAP expression in neutrophils of JAK2V617F positive patients. The myeloid cell line NB4 was transfected with the JAK2V617F mutation and a wild-type Jak2 using lentivirus vectors. It was observed that the JAK2V617F mutation, but not wild-type Jak2, enhanced cell proliferation. Then the LAP expression in NB4 cells was evaluated after these cells were differentiated by all-trans retinoic acid and granulocyte colony-stimulating factor. It was observed that the JAK2V617F mutation, but not wild-type Jak2, increased LAP expression. Next, we examined which of the Jak2 downstream pathways played a major role in increasing LAP expression and prompting cell proliferation. By using MEK1/2 inhibitor U0126, PI3K inhibitor LY294002, STAT3 siRNA and STAT5 siRNA, we demonstrated that the JAK2V617F mutation primarily used the STAT3 pathway to increase LAP expression. On the other hand, the JAK2V617F mutation used the STAT5, the Ras/MAPK and the PI3K pathways, but not the STAT3 pathway, to prompt proliferation of NB4 cells. In conclusion, we obtained direct evidence that the JAK2V617F mutation induced elevation of LAP scores via the STAT3 pathway, and prompted proliferation of NB4 cells via the STAT5, the Ras/MAPK and the PI3K pathways. Our findings showed the possibility that the JAK2V617F mutation might use specific downstream pathways depending on various phenotypic manifestations of BCR-ABL negative MPD. Disclosures: No relevant conflicts of interest to declare.

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Loss of Stress Sensor GADD45a Accelerates BCR-ABL-Driven Leukemogenesis

Blood ◽

10.1182/blood.v118.21.1668.1668 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1668-1668

Author(s):

Kaushiki Mukherjee ◽

Xiaojin Sha ◽

Ravi Bhatia ◽

Barbara Hoffman ◽

Dan Liebermann

Keyword(s):

Conflicts Of Interest ◽

Multiple Stressors ◽

Chronic Phase ◽

Human Growth ◽

Myelogenous Leukemia ◽

Pcr Analysis ◽

Wild Type ◽

M Cell ◽

Cycle Arrest ◽

The Status

Abstract Abstract 1668 The bcr-abl fusion oncogene causes chronic myelogenous leukemia (CML) in human. Growth arrest DNA damage 45a (Gadd45a) gene, a member in the gadd45 family of genes including Gadd45b & Gadd45g, is upregulated during myeloid lineage terminal differentiation. It is involved in G2/M cell cycle arrest and apoptosis in response to multiple stressors, including genotoxic and oncogenic stress. To investigate the effect of GADD45A in the development of CML, syngeneic wild type lethally irradiated mice were reconstituted with either wild type or gadd45a null myeloid progenitors transduced with a retrovirally expressed 210-kD BCR-ABL fusion oncoprotein. It was observed that loss of gadd45a accelerates BCR-ABL driven CML resulting in the development of a more aggressive AML like disease. BCR-ABL transformed GADD45A deficient progenitors exhibit increased proliferation and decreased apoptosis, associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling and upregulation of p30C/EBPα and MCL-1 expression. Since Gadd45a functions as a tumor suppressor in murine BCR-Abl driven leukemia, the status of Gadd45a mRNA expression levels was also investigated in human CML samples by utilizing real time PCR analysis. It was found that Gadd45a transcript levels were significantly upregulated in chronic phase CML samples. However in accelerated and blast phase samples, expression was significantly downregulated relative to normal controls. Thus, Gadd45a expression was observed to be altered in human CML samples correlating with disease progression. These results provide novel evidence that gadd45a functions as a suppressor of BCR/ABL driven myeloid leukemogenesis. These data also provide the impetus to further elucidate the role Gadd45a plays in suppressing the development of CML, and explore how its loss contributes to the progression of CML to a more aggressive leukemic phenotype. Disclosures: No relevant conflicts of interest to declare.

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GADD45a Is a Tumor Suppressor in BCR-ABL-Driven Leukemogenesis

Blood ◽

10.1182/blood.v124.21.4530.4530 ◽

2014 ◽

Vol 124 (21) ◽

pp. 4530-4530

Author(s):

Kaushiki Mukherjee ◽

Xiaojin Sha ◽

Tomasz Skorski ◽

Ravi Bhatia ◽

Barbara Hoffman ◽

...

Keyword(s):

Tumor Suppressor ◽

Conflicts Of Interest ◽

Multiple Stressors ◽

Blast Crisis ◽

Chronic Phase ◽

Myelogenous Leukemia ◽

Pcr Analysis ◽

Wild Type ◽

M Cell ◽

The Status

Abstract The bcr-abl oncogene causes chronic myelogenous leukemia (CML). Growth arrest DNA damage 45a (Gadd45a) gene, a member in the gadd45 family of genes including Gadd45b & Gadd45g, is upregulated during myeloid lineage terminal differentiation. It is involved in G2/M cell cycle arrest and apoptosis in response to multiple stressors, including genotoxic and oncogenic stress . To investigate the effect of GADD45A in the development of CML, syngeneic wild type lethally irradiated mice were reconstituted with either wild type or gadd45a null myeloid progenitors transduced with a retrovirally expressed 210-kD BCR-ABL fusion oncoprotein. It was observed that loss of gadd45a accelerates BCR-ABL driven CML resulting in the development of a more aggressive AML like disease. Loss of Gadd45a resulted in increase of BCR-ABL expressing leukemic Stem/progenitor cells (GFP+Lin- cKit+Sca+). BCR-ABL transformed GADD45A deficient progenitors exhibit increased proliferation and decreased apoptosis, associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling and upregulation of p30C/EBPα and MCL-1 expression. Moreover, loss of Gadd45a was observed to facilitate transformation of BCR-ABL expressing BM cells to factor independence. These results provide novel evidence that gadd45a functions as a suppressor of BCR/ABL driven myeloid leukemogenesis. Since Gadd45a functions as a tumor suppressor in murine BCR-Abl driven leukemia, the status of Gadd45a mRNA expression levels was also investigated in human CML samples by utilizing real time PCR analysis. It was found that Gadd45a transcript levels were significantly up-regulated in chronic phase I CML samples. However in chronic phase-II, accelerated phase and blast crisis phase samples, expression was significantly down-regulated relative to normal controls. Thus, Gadd45a expression was observed to be altered in human CML samples correlating with disease progression. These results provide novel evidence that gadd45a functions as a suppressor of BCR/ABL driven myeloid leukemogenesis. These data also provide the impetus to further elucidate the role Gadd45a plays in suppressing the development of CML, and explore how its loss contributes to the progression of CML to a more aggressive leukemic phenotype. Disclosures No relevant conflicts of interest to declare.

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Lymphocytes Are Dispensable In Neutrophil Homeostasis.

Blood ◽

10.1182/blood.v116.21.2613.2613 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2613-2613 ◽

Cited By ~ 1

Author(s):

Stefanie Bugl ◽

Tina Wiesner ◽

Lothar Kanz ◽

Hans-Georg Kopp ◽

Stefan Wirths

Keyword(s):

Bone Marrow ◽

Conflicts Of Interest ◽

Red Cell ◽

Wild Type ◽

Absolute Increase ◽

Hematopoietic Stem ◽

Csf Levels ◽

Neutrophil Depletion ◽

Peripheral Neutrophil ◽

Myeloid Progenitors

Abstract Abstract 2613 Introduction: In contrast to red cell or platelet homeostasis, the feedback mechanisms involved in the control of peripheral neutrophil numbers are incompletely understood. Granulocyte-Colony Stimulating Factor (G-CSF) is generally accepted to be the most important determinant of neutrophil production and release from the bone marrow. Recently, a feedback loop including a specialized subset of T-lymphocytes (Tn cells) has been established to explain the correlation between peripheral neutrophil clearance and increased G-CSF production. Methods: Wild type C57bl/6 mice or NODSCIDcγ−/− received anti-Gr1 or anti-1A8 antibodies to deplete neutrophils. Hematopoietic tissues and peripheral blood were harvested at different times and analyzed on cellular, protein and RNA level. Results: Both anti-Gr1 and 1A8 antibodies reduced neutrophils effectively and persistently in vivo. The reaction on neutrophil depletion in the marrow uniformely consisted of an absolute increase in lin-/Sca1+/c-kit+ (LSK) cells and a shift of myeloid progenitors towards granulocyte-macrophage precursors (GMP) and common myeloid progenitors (CMP) at the expense of megakaryocyte-erythrocyte precursors (MEP). Of note, exogenous G-CSF resulted in identical changes. We therefore hypothesized that neutrophil depletion increases G-CSF through a feedback regulatory loop. Neutrophil depletion with anti-Gr1 antibody in wt mice increased G-CSF levels up to approximately 8-fold. While previous observations suggest that G-CSF may be passively regulated through receptor binding and internalization by mature neutrophils, we also found a 10-fold increase of G-CSF mRNA in the marrow. These findings are consistent with active feedback. Interestingly, the effects of 1A8 antibody on G-CSF were less pronounced. Instead, mice depleted of neutrophils with 1A8 antibody displayed predominant increases of M-CSF, suggesting redundant feedback pathways. Our results in wildtype mice treated with 1A8 antibody confirm previous data by Stark et al. (Immunity 2005; 22: 285–294), including increases of IL-23 and IL-17 after neutrophil depletion. However, when neutrophils were depleted in NODSCIDcγ−/− mice, who lack lymphocytes and NK-cells, both IL-23 and IL-17 remained unchanged, but G-CSF levels still increased markedly. Conclusions: Effective neutrophil depletion by antibodies directed against different neutrophil antigens uniformly results in major shifts in the hematopoietic bone marrow showing an increase in the number of LSK hematopoietic stem cells accompanied by a significant increase in myeloid progenitors at the expense of thrombopoietic, red cell, and lymphoid precursors. Our results demonstrate regulatory feedback loops of neutrophil granulopoiesis culminating in increased production of myelopoiesis stimulating cytokines such as G-CSF, GM-CSF, and M-CSF. The underlying chain of events includes IL-23 and IL-17 in wild type mice as previously described. However, additional redundant pathways exist in lymphocytopenic NODSCIDcγ−/− mice. Disclosures: No relevant conflicts of interest to declare.

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Ras and Wnt Interaction Contribute in Prostate Cancer Bone Metastasis

Molecules ◽

10.3390/molecules25102380 ◽

2020 ◽

Vol 25 (10) ◽

pp. 2380 ◽

Cited By ~ 1

Author(s):

Shian-Ren Lin ◽

Ntlotlang Mokgautsi ◽

Yen-Nien Liu

Keyword(s):

Prostate Cancer ◽

Bone Marrow ◽

Bone Metastasis ◽

Signaling Pathways ◽

Late Stage ◽

Ras Signaling ◽

Cancer Death ◽

Metastatic Site ◽

Cancer Types ◽

Cytological Changes

Prostate cancer (PCa) is one of the most prevalent and malignant cancer types in men, which causes more than three-hundred thousand cancer death each year. At late stage of PCa progression, bone marrow is the most often metastatic site that constitutes almost 70% of metastatic cases of the PCa population. However, the characteristic for the osteo-philic property of PCa is still puzzling. Recent studies reported that the Wnt and Ras signaling pathways are pivotal in bone metastasis and that take parts in different cytological changes, but their crosstalk is not well studied. In this review, we focused on interactions between the Wnt and Ras signaling pathways during each stage of bone metastasis and present the fate of those interactions. This review contributes insights that can guide other researchers by unveiling more details with regard to bone metastasis and might also help in finding potential therapeutic regimens for preventing PCa bone metastasis.

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CBFβ-SMMHC Inhibits G1 to S Progression in Primary Murine and Human Myeloid Progenitors Dependent upon Its AML1-Interaction and Assembly Competence Domains.

Blood ◽

10.1182/blood.v104.11.1971.1971 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1971-1971

Author(s):

Jenice D’Costa ◽

Shamik Chaudhuri ◽

Curt I. Civin ◽

Alan D. Friedman

Keyword(s):

Cell Cycle ◽

Cell Proliferation ◽

Cell Lines ◽

Cell Cycle Progression ◽

Myeloid Cell ◽

Cyclin D3 ◽

Wild Type ◽

Hematopoietic Stem ◽

Human Cd34 ◽

Myeloid Progenitors

Abstract CBFβ-SMMHC, encoded by the inv(16) or t(16;16) translocations in approximately 8% of acute myeloid leukemia (AML) cases, is a fusion protein containing amino acids 1-165 of the 182 residue core binding factor β (CBFβ) and the rod domain of smooth muscle myosin heavy chain (SMMHC). The CBFβ domain of CBFβ-SMMHC retains the ability to interact with AML1/RUNX1. The SMMHC domain both mediates multimerization and interacts directly with corepressors, including mSin3A. CBFβ-SMMHC inhibits the expression of AML1-regulated genes, by sequestering AML1 in multimeric complexes and by directly repressing AML1-regulated genes. CBFβ-SMMHC was previously found to slow G1 to S cell cycle progression in hematopoietic cell lines, reflecting repression of AML1-regulated genes required for cell cycle, including cyclin D3. This effect was overcome be exogenous c-Myc or cdk4. In this study, murine marrow or human CD34+ cells were transduced with retroviral or lentiviral vectors, respectively, expressing CBFβ-SMMHC or two mutant variants. CBFβ-SMMHC reduced murine or human myeloid cell proliferation 3- to 4-fold in liquid culture, during a period when control murine cells accumulated 5-fold and human cells 20-fold. CBFβ-SMMHC decreased the formation of myeloid, but not erythroid, colonies 2- to 4-fold, and myeloid colonies expressing CBFβ-SMMHC were markedly reduced in size. Lack of effect on erythroid colonies reflects their lack of expression of AML1. The mutant variant CBFβ-SMMHC(Δ2-11) does not bind AML1 due to a deletion near its N-terminus, and CBFβ-SMMHC(ΔACD) does not multimerize or efficiently bind corepressors due to a 28 residue deletion near its C-terminus. Neither of these mutants, which were expressed at levels similar to wild-type, slowed proliferation or reduced myeloid colonies. CBFβ-SMMHC increased the G1/S ratio in wild-type murine and human progenitors. Proliferation was still slowed in p15(−/−) murine marrow cells transduced with CBFβ-SMMHC, suggesting that additional mutations, such as activation of growth factor receptors and consequent c-myc induction, are required in primary AMLs to allow enhanced proliferation. AML1-ER(T), which contains full-length AML1 and accelerates G1 to S progression in cell lines when activated by 4HT, had an effect opposite to CBFβ-SMMHC, stimulating proliferation of murine or human myeloid progenitors 2-fold. In summary, CBFβ-SMMHC inhibits the proliferation of myeloid progenitors dependent upon inhibition of AML1 and integrity of its Assembly Competence Domain. Targeting the CBFβ-SMMHC ACD or its CBFβ domain may uncover novel therapeutics useful for AML cases expressing this oncoprotein. Furthermore, these findings support a model we have proposed previously which states that mutations which accelerate G1 are required during leukemogenesis by CBFβ-SMMHC and other CBF oncoproteins. Finally, our results lend support to the conclusion that AML1 participates in the regulation of normal myeloid stem-progenitor cell proliferation. Exogenous AML1 may therefore be useful for expansion of hematopoietic stem-progenitor cells.

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Slug Plays an Essential Role in the Radioprotection of Hematopoietic Progenitors In Vivo by Antagonizing p53-Mediated Apoptotic Pathways.

Blood ◽

10.1182/blood.v104.11.31.31 ◽

2004 ◽

Vol 104 (11) ◽

pp. 31-31

Author(s):

Wen-Shu Wu ◽

Dong Xu ◽

Stefan Heinrichs ◽

A. Thomas Look

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Leukemia Cells ◽

Dependent Manner ◽

Wild Type ◽

Γ Irradiation ◽

Aberrant Expression ◽

Hematopoietic Stem ◽

Myeloid Progenitors

Abstract An antiapoptotic role for Slug/Snail in mammals was suggested by studies in C. elegans, where CES-1/Scratch, a member of the Slug/Snail superfamily, was found to control the apoptotic death of NSM sister neurons by acting as a transcriptional repressor of EGL-1, a BH3-only proapoptotic protein. Identification of Slug as the target gene of the E2A-HLF oncoprotein in human pro-B leukemia cells led us to demonstrate its antiapoptotic function in IL-3-dependent murine pro-B cells. In contrast to its aberrant expression in pro-B leukemia cells, endogenous Slug is normally expressed in both LT-HSC and ST-HSC, as well as committed progenitors of the myeloid series, but not in pro-B and pro-T cells, implying its function in myelopoiesis. Using Slug−/− mice produced in our laboratory, we showed that these knockouts are much more radiosensitive than Slug+/− and wild-type mice, and that apoptotic cells increase significantly in the hematopoietic progenitor cells of Slug−/− mice as compared to wild-type mice following γ-irradiation, indicating a radioprotective function in vivo. We showed here that although the development of myeloid progenitors is not impaired under steady-state conditions, their repopulation is incomplete γ-irradiated in in Slug−/− mice. We demonstrate further the radiation-induced death of Slug−/− mice is exclusively a result of bone marrow failure with no apparent contribution from systemic injures to other tissues. By two-way bone marrow transplantation, we provide firm evidence that Slug protects mice from γ-irradiation-induced death in a cell-autonomous manner. Interestingly, regenerative capacity of hematopoietic stem cells (HSC) was retained in irradiated Slug−/− mice, which could be rescued by wild-type bone marrow cells after irradiation, indicating that Slug exerts its radioprotective function in myeloid progenitors rather than HSCs. Furthermore, we establish that Slug radioprotects mice by antagonizing downstream of the p53-mediated apoptotic signaling through inhibition of the p53-resposive proapoptotic gene Puma, leading in turn to inhibition of the mitochondria-dependent apoptotic pathway activated by γ-irradiation in myeloid progenitors. More interestingly, we observed that Slug is inducible by γ-irradiation in a p53-dependent manner. Together, our findings implicate a novel Slug-mediated feedback mechanism by which p53 control programmed cell death in myeloid progenitor cells in vivo in response to γ-irradiation.

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A Model of Severe Congenital Neutropenia Reveals Signaling Pathways Mediating Mutant Elastase-Triggered Agranulocytosis.

Blood ◽

10.1182/blood.v106.11.3070.3070 ◽

2005 ◽

Vol 106 (11) ◽

pp. 3070-3070

Author(s):

Andrew A. Aprikyan ◽

Tomas Vaisar ◽

Vahagn Makaryan ◽

Jay Heinecke

Keyword(s):

Bone Marrow ◽

Signaling Pathways ◽

Myelogenous Leukemia ◽

Severe Neutropenia ◽

Myeloid Differentiation ◽

Cellular Model ◽

Severe Congenital Neutropenia ◽

Congenital Neutropenia ◽

Histone H2b ◽

Maturation Arrest

Abstract Severe congenital neutropenia (SCN; Kostmann’s syndrome or infantile genetic agranulocytosis) defines an inheritable hematopoietic disorder of impaired neutrophil production due to a “maturation arrest” at the promyelocytic stage of differentiation in the bone marrow. SCN patients have recurring severe infections and often develop acute myelogenous leukemia. We and others reported accelerated apoptosis and cell cycle arrest of bone marrow-derived myeloid progenitor cells in SCN patients with autosomal dominant and autosomal recessive inheritance. Heterozygous mutations in the neutrophil elastase (NE) gene encoding a serine protease, are present in a majority of SCN patients, but not in healthy members of the family, thus indicating a key role of mutant NE in pathogenesis of this disorder. To date, there are no animal or cellular models of SCN as both the knock-in of mutant NE as well as the knock-out of normal NE failed to result in neutropenia phenotype in mice. The molecular mechanisms of mutant NE-mediated severe neutropenia remain largely unknown. We hypothesized that mutations in NE expose the protease to a new range of substrates. To explore this proposal, we established a cellular model of SCN based on tetracycline-regulated expression of mutant NE in human promyelocytic tet-off HL-60 cells that very closely recapitulated the human phenotype. Mutant NE expression resulted in a characteristic block of myeloid differentiation - the cellular hallmark of SCN. Expression of the mutant product was associated with a significant reduction in phosphatidylinosytol-3-kinase and phosphorylated PKB/Akt levels and an imbalance of anti-apoptotic Bcl-2 and pro-apoptotic Bax. These alterations contributed to observed dissipation of mitochondrial membrane potential as determined by FACS analysis, aberrant release of cytochrome C, and accelerated apoptosis. Marked changes in actin cytoskeleton that made the cells more rigid appeared to stem from a reduced level of alpha-actinin and elevated level of Rho GTPase. Immunoprecipitation of cell lysates with elastase-specific monoclonal antibodies followed by mass spectrometric analysis revealed that NE interacted with histone H2B, one of the key components of the nucleosome core of the chromatin. Interestingly, the expression level of histone H2B was substantially reduced in cells expressing mutant NE, therefore supporting the notion of altered substrate specificity of mutant NE. Thus, these observations provide the first evidence that mutant NE affects specific signaling pathways that lead to alterations in cytoskeleton and chromatin reorganization, subsequent apoptosis, and a block of myeloid differentiation in SCN. This cellular model of SCN should provide an invaluable tool for screening potential therapeutic agents capable of preventing maturation arrest and leukemogenesis in subjects suffering from severe congenital neutropenia.

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Requirement of CREB in Normal and Malignant Hematopoiesis.

Blood ◽

10.1182/blood.v108.11.1168.1168 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1168-1168

Author(s):

Jerry C. Cheng ◽

Deepa Shankar ◽

Stanley F. Nelson ◽

Kathleen M. Sakamoto

Keyword(s):

Bone Marrow ◽

Cell Proliferation ◽

Western Blot ◽

Blot Analysis ◽

Hematopoietic Cells ◽

Wild Type ◽

Qrt Pcr ◽

T315i Mutation

Abstract CREB is a nuclear transcription factor that plays an important role in regulating cellular proliferation, memory, and glucose homeostasis. We previously demonstrated that CREB is overexpressed in bone marrow cells from a subset of patients with acute leukemia at diagnosis. Furthermore, CREB overexpression is associated with an increased risk of relapse and decreased event-free survival in adult AML patients. Transgenic mice that overexpress CREB in myeloid cells developed myeloproliferative/myelodysplastic syndrome after one year. To further understand the role of CREB in leukemogenesis and in normal hematopoiesis, we employed RNA interference methods to inhibit CREB expression. To achieve sustained, CREB-specific gene knockdown in leukemia and normal hematopoietic cells, a lentiviral-based small hairpin (shRNA) approach was taken. Three CREB specific shRNAs were generated and tested for efficiency of gene knockdown in 293T cells. Knockdown efficiency approached 90 percent by Western blot analysis compared to vector alone and luciferase controls. Human myeloid leukemia cell lines, K562, TF1, and MV411, were then infected with CREB shRNA lentivirus, sorted for GFP expression, and analyzed using quantitative real time (qRT)-PCR, Western blot analysis, and growth and viability assays. Lentiviral CREB-shRNA achieved between 50 to 90 percent knockdown of CREB compared to control shRNAs at the protein and mRNA levels. To control for non-specific effects, we performed qRT-PCR analysis of the interferon response gene, OAS1, which was not upregulated in cells transduced with CREB shRNA constructs. Within 72 hours, cells transduced with CREB shRNA had decreased proliferation and survival. Similar results were obtained with murine leukemia cells (NFS60 and BA/F3 bcr-abl).To study the role of CREB in normal hematopoiesis, both primary murine and human hematopoietic cells were transduced with our shRNA constructs, and methylcellulose-based colony assays were performed. Primary hematopoietic cells infected with CREB shRNA lentivirus demonstrated a 5-fold decrease in colony number compared to control virus-infected cells (p<0.05). Bone marrow colonies consisted of myeloid progenitor cells that were mostly Mac-1+ by FACs analysis. Interestingly, there were fewer differentiated cells in the CREB shRNA transduced cells compared to vector control or wild type cells, suggesting that CREB is critical for both myeloid cell proliferation and differentiation. To study the in vivo effects of CREB knockdown on leukemia progression, we studied mice injected with BA/F3 cells that express both bcr/abl with the T315I mutation and a luciferase reporter gene. BA/F3 cells expressing the T315I mutation have a 2-fold increase in CREB overexpression compared to wild-type cells. Disease progression was monitored using bioluminescence imaging with luciferin. CREB knockdown was 90 percent after transduction and prior to injection into SCID mice. We observed improved survival of mice injected with CREB shRNA transduced BA/F3 bcr-abl (T315I) compared to vector control cells. To understand the mechanism of growth suppression resulting from CREB downregulation, we performed microarray analysis with RNA from CREB shRNA transduced K562 and TF1 cells. Several genes were downregulated using a Human Affymetrix chip. Most notable was Beclin1, a tumor suppressor gene often deleted in prostate and breast cancer that has been implicated in autophagy. Our results demonstrate that CREB is required for normal and leukemic cell proliferation both in vitro and in vivo.

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