scholarly journals Radiosensitivity of Fancd2-/- mouse Bone Marrow Stromal Cells Is Altered By Abrogation of TGF-β Signaling

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4796-4796
Author(s):  
Katherine Chen ◽  
Darcy Franicola ◽  
Donna Shields ◽  
Michael W. Epperly ◽  
Xichen Zhang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Survival Curve ◽  
Radiation Sensitivity ◽  
Mouse Bone Marrow ◽  
Wild Type ◽  
Increased Risk ◽  
Significant Difference

Abstract Both marrow-transplanted and non-transplanted Fanconi Anemia (FA) patients are often radiosensitive. Due to an increased risk of developing secondary malignancies, these patients require dose and volume modification during radiotherapy. To determine whether abrogation of TGF-β signaling alters the radiation sensitivity of Fancd2-/- mice, cell lines derived from double knockout (DKO) (SMAD3-/- Fancd2-/-) mice were compared with those from Fancd2-/-, SMAD3-/-, and wild-type mice for ionizing irradiation sensitivity. Bone marrow stromal cell lines were derived from long-term bone marrow cultures of DKO, Fancd2-/-, SMAD3-/-, and wild-type SMAD3+/+ (129/Sv) X Fancd2+/+ (B6) F1 mice. Radiation sensitivity was determined using clonogenic irradiation survival curves. There was no significant difference in radiosensitivity comparing DKO cells (Do = 1.95 ± 0.06 Gy, ň = 4.3 ± 0.7) to the wild type SMAD3+/+ (129/Sv) X Fancd2+/+ (B6) F1 cell line (Do = 2.00 ± 0.11 Gy, and ň = 5.1 ± 0.7, p = 0.7003 and 0.4820, respectively). The Fancd2-/- cell line was more radiosensitive with a Do of 1.37 ± 0.09 Gy compared to 1.95 ± 0.07 and 2.00 ± 0.11 for DKO and wild type cells (p = 0.0063 and 0.0360, respectively. In contrast, the SMAD3-/- cell line was more radioresistant with an increased shoulder on the irradiation survival curve (ň = 12.1 ± 2.9) compared to the DKO or wild type SMAD3+/+ (129/Sv) X Fancd2+/+ (B6) F1 cell lines (ň = 4.335 ± 0.7 or 5.1 ± 0.7, p = 0.00277 or 0.0426, respectively). This confirms and extends results with SMAD3-/- mouse derived cell lines on another background strain (C57BL/6J) (Epperly, et al., Radiation Research, 165:671-677, 2006). TGF-β signaling was abrogated in both DKO and SMAD3-/- mouse cell lines (measured by TGF-β inhibition of fresh marrow CFU-GEMM in vitro), confirming the phenotype of altered TGF-β signaling. Therefore, radiosensitivity associated with the Fancd2-/- genotype was abrogated by interruption of the TGF-β signaling pathway in the same cells. Supported by research grant NIAID/NIH, U19A168021. Disclosures No relevant conflicts of interest to declare.

Neuronal/Mitochondrial Nitric Oxide Synthase Homologous Deletion Recombinant Negative Mice (NOS1 −/−) Long-Term Bone Marrow Cultures (LTBMCs) Demonstrate Increased Longevity and Radioresistance of Derived Cell Lines.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1355-1355
Author(s):  
Michael W. Epperly ◽  
Joel S. Greenberger ◽  
Shaonan Cao ◽  
Darcy Franicola ◽  
Hongmei Shen ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Bone Marrow ◽  
Cell Lines ◽  
Stromal Cell ◽  
Survival Curve ◽  
G1 Arrest ◽  
No Production ◽  
Significant Difference ◽  
Bone Marrow Cultures ◽  
Mitochondrial Nitric Oxide Synthase

Abstract Neuronal NOS (NOS1) is localized to mitochondria. Ionizing irradiation results in influx of calcium into mitochondria stimulating production of nitric oxide (NO), and also increases production of superoxide which reacts with NO to produce peroxynitrite. Peroxidation of mitochondrial lipids, release of cytochrome C and apoptosis is directly related to mitochondrial peroxynitrite. We hypothesized that reduction of mitochondrial NO production should provide radioresistance. In addition, since ROS production is associated with aging NOS1−/− mouse LTBMCs should demonstrate greater hematopoietic longevity. LTBMCs established from NOS1 −/− mice demonstrated increased cumulative adherent cobblestone islands (adherent stem cell containing islands), production of total nonadherent cells, and cumulative day 7 and day 14 CFU-GEMM hematopoietic multi-lineage colony forming cells (over 65 weeks) compared to NOS1 +/+ controls (22 weeks) (p < 0.0001). Seven and 14 day CFU-GEMM production in nonadherent cell harvests from NOS1 −/− LTBMCs continued for 65 weeks compared to 15 weeks for NOS1 +/+ LTBMCs (p < 0.0001). Marrow stromal cell lines derived from NOS1 −/− and NOS1 +/+ culture were irradiated to doses from 0 to 800 cGy, plated in 4 well tissue culture plates, stained with crystal violet 7 days later and colonies of greater than 50 cells were counted. NOS1 −/− stromal cell lines had an increased shoulder on the survival curve compared to the NOS1 +/+ cells (n = 32.15 ± 1.21 and 10.47 ± 3.20, respectively, p=0.0026). Cell cycle analysis of NOS1 −/− and NOS1 +/+ cell lines following 10 Gy irradiation demonstrated a G1 arrest 6 hr after irradiation, in both; however, by 24 hr, NOS1 +/+ but not NOS1−/− cells resumed normal cycling. To determine whether the radioresistance of NOS1−/− cells was attributable to expected higher levels of antioxidants, cells were analyzed for glutathione (GSH) and glutathione peroxidase (GPX). NOS1 −/− cells demonstrated increased GSH compared to NOS1 +/+ cells at 0, 30 min and 24 hr following irradiation (p < 0.0001) with no significant difference in GPX before or after irradiation. NOS1 −/− compared to NOS1+/+. IL-3 dependent hematopoietic cells from NOS1 −/− LTBMCs had significantly decreased apoptosis, 24 hrs following 10 Gy irradiation (5.3 ± 2.4 vs. 14.8 ± 3.3 %, respectively, p = 0.049). Therefore, reduction of NOS1 in bone marrow increases hematopoietic longevity in LTBMCs and radioresistance of derived cell lines.

Cooperation of MLL/AF10(OM-LZ) with K-Ras Mutation Induced Myeloid Sarcoma in a Mouse Bone Marrow Transplantation Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1964-1964
Author(s):  
Jen-Fen Fu ◽  
Lee-Yung Shih
Keyword(s):  
Bone Marrow ◽  
Cell Lines ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Fusion Gene ◽  
Flow Cytometric Analysis ◽  
Myeloid Sarcoma ◽  
Mouse Bone Marrow ◽  
Myeloproliferative Disease ◽  
Multiple Tumor

Abstract Abstract 1964 Poster Board I-987 We analyzed genetic mutations in a large cohort of AML patients and found that two of the five patients with MLL/AF10 and N-/K-RAS mutations had cutaneous tumors (myeloid sarcomas). To study the cooperative role of MLL/AF10 and N-/K-RAS in the formation of myeloid sarcoma, we established two cell lines by retroviral transduction of MLL/AF10(OM-LZ) and K-RASG12C into GFP-B6 mouse bone marrow cells. Flow cytometric analysis revealed that the cells with MLL/AF10(OM-LZ) and K-RASG12C showed a decreased Mac-1 and CD115 expression when compared with the cells with a single MLL/AF10(OM-LZ) mutation. Microarray and RT-PCR analyses revealed an increased gene expression in Hoxa10 and Meis1, but not Hoxa9. In addition, the phagocytosis related genes, Cybb and Lyz were decreased in the cells harboring MLL/AF10(OM-LZ) and K-RASG12C. These results suggested that cooperation of MLL/AF10(OM-LZ) and K-RASG12C mutations blocked the cells in a more primitive hematopoietic stage. When the two cell lines were intra-peritoneally injected into B6 mice, the mice developed myeloproliferative disease-like myeloid leukemia as that of the mice transplanted with cells carrying a single MLL/AF10(OM-LZ) fusion gene. The median survival time were 33±4.2 and 31.6±5.1 days, respectively, which were shorter than that of the mice transplanted with cells carrying a single MLL/AF10(OM-LZ) fusion gene (49.8±5.0 days). We found that the majority (84%) of mice transplanted with cells harboring both MLL/AF10(OM-LZ) and K-RASG12C mutations formed multiple tumor masses involving gastrointestinal tract, kidney, peritoneum, paraspinal soft tissue, and/or skin. Cytological examination from the imprint smears of tumor masses showed massive infiltrates of leukemia blastic cells. Immunohistochemical stains of the paraffin-fixed histological sections of tumor masses were positive for GFP, confirmed that the tumor cells were generated from the transplanted cell lines. We have established a mouse model which can be used for further study of the myeloid sarcoma formation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Activation of Non-Canonical Immune Signalling Pathways Drives Marrow Failure in a Murine Model of MDS

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3246-3246
Author(s):  
Rawa Ibrahim ◽  
Joanna Wegrzyn ◽  
Linda Ya-Ting Chang ◽  
Patricia Umlandt ◽  
Jeff Lam ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Signalling Pathway ◽  
Gene Set Enrichment Analysis ◽  
Mouse Bone Marrow ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem

Abstract The Myelodysplastic Syndromes (MDS) are the most common hematological malignancies arising from stem/progenitor cells. MDS is characterized by ineffective hematopoiesis in one or more lineages of the bone marrow, resulting in peripheral cytopenias and the propensity to progress to either acute myeloid leukemia (AML) or bone marrow failure (BMF). The most common cytogenetic aberration associated with MDS is deletion of the long arm of chromosome 5. Many of the molecular events involved in the development of del(5q) MDS have been elucidated including haploinsufficiency of the gene encoding the ribosomal protein RPS14, responsible for the anemia observed, and haploinsufficency of the miRNAs miR-145 and miR-146a, which together target the innate immune signaling pathway, specifically, the Toll-like receptor-4 (TLR-4)signalling pathway. It has been demonstrated that overexpression of a target of miR-146a,TRAF6, in mouse bone marrow can recapitulate the phenotype of del(5q) MDS including the cytopenias and progression to BMF or AML. However, enforced expression of TIRAP, a miR-145 target gene, results in rapid BMF independent of TRAF6. The molecular and cellular mechanisms responsible for the differential outcome of overexpression of two genes that act within the same signalling pathway remain to be fully understood. We have identified several differentially expressed cytokines, including interferon gamma (IFNγ) and interleukin-10 (IL-10), following TIRAP overexpression compared with TRAF6 overexpression. Promoter methylation analysis has shown hypermethylation of key adaptors and signal transducers that lie between TIRAP and TRAF6 in the TLR-4 signalling pathway, suggesting activation of different pathways by TIRAP and TRAF6 overexpression. Indeed, blockade of TRAF6 and MyD88 did not inhibit TIRAP induced expression of these cytokines, suggesting that IFNγ and IL-10 production occurs in a TRAF6 and MyD88 independent manner. We identified IFNγ as the critical effector cytokine responsible for TIRAP mediated marrow failure. Gene set enrichment analysis has shown an enrichment of an IFNγ signature in MDS patients with a low risk of transformation to AML compared to healthy controls. Furthermore, interferon signatures were highly enriched in MDS patients compared to patients with AML, suggesting an important role for IFNγ signaling in driving MDS progression toward marrow failure as opposed to leukemic progression. IFNγ has been shown to inhibit components of the bone marrow niche by blocking RANK signalling in stromal cells such as osteoclast progenitors. Using coculture of TIRAP expressing bone marrow cells with the RAW264.7 monocyte cell line, a cell line that is capable of differentiation into osteoclasts, we found an inhibition in the ability of these cells to form osteoclasts compared to control. This provides the first line of evidence suggesting that immune signalling defects arising from genetic perturbations in the hematopoietic stem cell compartment can result in stem cell niche dysfunction leading to marrow failure. Disclosures No relevant conflicts of interest to declare.

scholarly journals Phenotypic heterogeneity among stromal cell lines from mouse bone marrow disclosed in their extracellular matrix composition and interactions with normal and leukemic cells

Blood ◽  
1985 ◽  
Vol 66 (2) ◽  
pp. 447-455 ◽  
Author(s):  
D Zipori ◽  
J Toledo ◽  
K von der Mark
Keyword(s):  
Bone Marrow ◽  
Extracellular Matrix ◽  
Cell Line ◽  
Cell Lines ◽  
Stromal Cell ◽  
Leukemic Cells ◽  
Matrix Composition ◽  
Mouse Bone Marrow ◽  
Type I

Abstract Study of a series of stromal cell lines from mouse bone marrow (MBA) verified and extended their classification as phenotypically distinct subtypes. Production of extracellular matrix proteins was examined using specific antibodies. Fibronectin and laminin were detected in all of the cell lines tested, yet 14F1.1 adipocytes exhibited particularly prominent extracellular deposition. This cell line and MBA-13.2 cells were positive to both collagen types I and IV, whereas MBA-1 and MBA- 2.1 were stained with anticollagen type I antibodies only. Coculture experiments revealed differences among the lines in their effects on normal myeloid cells and leukemic cell lines. In promoting the in vitro accumulation of myeloid progenitors (CFU-C), 14F1.1 cells surpassed the others. The MBA-2.1 cell line was particularly inhibitory to MPC-11 plasmacytoma and Friend erythroleukemia cells. However, the latter were refractory to other stromal cell lines, whereas MPC-11 cells were inhibited to various degrees by virtually all of the cell lines. Physical separation between the interacting cells reduced the inhibition in some but not all cases, and no inhibitory activity was detected in conditioned media. The MBA-13 stromal cells synergistically promoted the differentiation of dimethylsulfoxide (Me2SO)-induced Friend erythroleukemia. The latter cells themselves, at high concentrations, as well as some of the stromal cell lines and unrelated adherent cells, antagonized the Me2SO effect, revealing possible reversible stages in the Friend cell differentiation pathway.

Nucleophosmin Protects Hematopoietic Cells from Stress-Induced Apoptosis through Inhibition of P53.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2558-2558
Author(s):  
Qishen Pang ◽  
June Li ◽  
Xiaoling Zhang ◽  
Daniel P. Sejas ◽  
Grover C. Bagby
Keyword(s):  
Bone Marrow ◽  
Cell Survival ◽  
Cell Line ◽  
Hematopoietic Cells ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Mouse Bone Marrow ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Induced Apoptosis

Abstract Nucleophosmin (NPM) is a multifunctional protein frequently overexpressed in actively proliferating cells including tumor and hematopoietic stem cells. Here we report that NPM protects hematopoietic cells from stress-induced apoptosis through inhibition of the tumor suppressor p53. Specifically, we forced expression of wild-type (WT) NPM or a mutant variant with a deletion of the C-terminal 120 aa of NPM (NPMΔC) by retroviral gene transfer in the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent myeloid cell line MO7e (expresses low level of NPM) and the lymphoblast HSC536 cells derived from a Fanconi anemia (FA) patient in the C complementation group (expresses essentially undetectable NPM). Using a flow cytometric method for caspase 3 activation (early apoptosis), we demonstrated that overexpression of NPM but not the mutant NPMΔC confers MO7e and HSC536 cells resistance to apoptosis induced by growth factor deprivation and treatment with the chemotherapeutic drug etoposide. In addition, suppression of NPM expression by small interfering RNA targeting NPM in chronic myelogenous leukemia line K562 and FA-associated acute myelogenous leukemia cell line UoC-M1 increases etoposide-induced apoptosis, thus providing proof of concept evidence that the pathological elevations of NPM found in cancers and leukemias are important for maintaining cell survival and resistance to apoptosis. Because overexpression of the mutant NPMΔC, which lacks the p53-interacting domain, fails to confer cellular resistance to stress-induced apoptosis, we determined whether NPM protects cells from apoptotic cell death through a mechanism involving p53. We used the genetically matched p53 WT and null mouse bone marrow (BM) cells to show that overexpression of WT NPM protects against ionizing irradiation (IR)-induced apoptosis of wild-type but not p53-null BM cells. Moreover, NPM inhibits IR-induced p53 phosphorylation at Ser18 and transactivation, and interacts with p53 in bone marrow hematopoietic cells. Thus, this study not only demonstrates anti-apoptotic function of a proliferation-promoting protein but also suggests that cancer progression may require increased expression of NPM to suppress p53 activation and maintain cell survival.

Alpha-Catenin Is Dispensable for Normal Hematopoietic Stem Cell Function.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1438-1438
Author(s):  
Natallia Mikhalkevich ◽  
Michael W. Becker
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Conflicts Of Interest ◽  
Fusion Product ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Myeloid Neoplasms ◽  
Increased Risk ◽  
Significant Difference ◽  
The Impact

Abstract Abstract 1438 Poster Board I-461 We previously demonstrated the loss of expression of alpha-E-Catenin, the product of the CTNNA1 gene, in primary leukemic stem cells isolated from patients with advanced Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML) associated with loss of all or part of the long arm of chromosome 5. To formally assess the impact of loss of Ctnna1 expression on hematopoiesis, we employed a murine model for the hematopoietic specific conditional loss of Ctnna1 expression. We demonstrate that Ctnna1 deficiency is associated with normal hematopoietic maturation and proliferation as assessed by peripheral blood examination and methycellulose colony assays. We assessed stem cell and early progenitor frequencies using both flow cytometry and functional assays. Ctnna1 deficiency was associated with equivalent frequencies of Sca1+C-Kit+CD135-Lineage- HSCs in both experimental animals and controls. Short term HSC and MPP frequencies were likewise unaltered. We assessed HSC function using transplantation studies. In competitive repopulation experiments, HSCs deficient for Ctnna1 maintained stable engraftment of recipient mice for up to 1 year. Limiting dilution analyses detected no significant difference in HSC frequency between wild type and Ctnna1 deficient mice. We examined the potential role of Ctnna1 deficient hematopoietic stem cells in two murine models for myeloid neoplasms 1.) exposure to mutagen ENU and 2.) a model for murine AML driven by the HoxA9-Nup98 fusion product. Following exposure of HSCs to ENU, loss of Ctnna1 was not associated with an increased risk of development of a myeloid neoplasm. Expression of the HoxA9-Nup98 fusion product by retroviral infection of Ctnna1 deficient and wild type Sca1+C-Kit+Lineage- cells resulted in no difference in time to development of the previously characterized myeloproliferative disorder or acute leukemia. Taken together, these data demonstrate that in the absence of specific genetic abnormalities, loss of Ctnna1 expression in primary murine HSCs is not associated with aberrant HSC function or the development of myeloid neoplasms. Further studies are necessary to define a role for of loss of Ctnna1 expression in human myeloid malignancies. Disclosures No relevant conflicts of interest to declare.

Ionizing Irradiation Protection and Mitigation by Carbamazepine Is p53 and Autophagy Independent,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3400-3400
Author(s):  
Hyun Kim ◽  
Mark E. Bernard ◽  
Amy Farkas ◽  
Frank Houghton ◽  
Donna Shields ◽  
...  
Keyword(s):  
Cell Lines ◽  
Hind Limb ◽  
Conflicts Of Interest ◽  
Survival Curve ◽  
Tumor Growth Inhibition ◽  
Independent Manner ◽  
Mitochondrial Membrane Depolarization ◽  
Significant Difference

Abstract Abstract 3400 Introduction: Carbamazepine (CBZ), an established sodium channel blocker, used in treatment of epilepsy and trigeminal neuralgia, induces autophagy. CBZ has recently been demonstrated to be an ionizing radiation mitigator and protector (Kim,H.et. al IJRB-in press). CBZ protected C57BL/6NHsd female mice from irradiation if given 24 hr before total body irradiation (TBI) and mitigated if given 12 hours after irradiation. Materials and Methods: To elucidate the mechanism of CBZ action, autophagy incompetent (ATG5−/−) and competent (ATG5+/+) mouse embryo fibroblasts (MEF), p53−/− and p53+/+ bone marrow stromal cells and 32D cl 3 murine IL-3 dependent hematopoietic progenitor cells were tested for CBZ mediated radiation protection and mitigation in clonagenic irradiation survival curves. We also measured CBZ effect on irradiation-induced apoptosis, and depletion of antioxidant stores in vitro and after (TBI) in vivo in control mice and in hind limb irradiated mice with orthotopic tumors. Results: CBZ was a significant radiation protector and mitigator for both ATG5−/− and ATG5 +/+ cell lines by an increased ñ (a measurement of the shoulder on the clonogenic survival curve). CBZ treated ATG5 +/+ cells has an increased ñ of 11.1 +/− 0.2 or 8.8 +/− 0.2 for CBZ added before or after irradiation respectively compared to 5.4 +/− 0.9 for irradiation control cells (p = 0.0287 or 0.0119,respectively). ATG −/− cells were also protected and mitigated by CBZ (ñ of 16.1 +/− 2.6 as a radioprotector or 9.8 +/− 1.5 as a mitigator compared to 4.6 +/− 0.7 for irradiated control cells (p = 0.0002 or 0.0037, respectively). Thus, CBZ functions independent of autophagy. Incubation of p53 +/+ and p53−/− cell lines in 10 μM CBZ for one hour before irradiation protected (ñ of 5.6 +/− 0.9 compared to 1.9 +/− 0.5 for control irradiated p53 +/+ cells (p = 0.0126) and 3.7 +/− 1.9 compared to 1.8 +/− 0.4 for irradiated control p53 −/− cells (p = 0.0018). Thus, CBZ functions in a p53 independent manner. Other pro-autophagy drugs, Valproic Acid and Lithium Carbonate, were ineffective radiation protectors or mitigators. CBZ treatment of 32D cl 3 cells for one hour before or immediately following irradiation had no effect on mitochondrial membrane depolarization, apoptosis or viability but was protective and mitigative in clonagenic survival curve assays. Irradiation initially decreased antioxidant stores in both ATG5+/+ and ATG5−/− cells 10 to 30 min following irradiation. However, cells treated with CBZ had a faster recovery of antioxidant stores to pre-irradiation levels by 90 min compared to 120 min for control irradiated cells. To determine if CBZ protected both normal tissues and tumors in vivo mice with 3LL Lewis Lung carcinoma hind limb xenografts were treated with 10 mg/kg CBZ before or after 20 Gy limb irradiation. There was no significant difference in tumor growth inhibition by 20 Gy in mice treated with CBZ before or after irradiation (p = 0.2431 or 0.5439, respectively). Conclusion: Thus, CBZ is an effective radiation protector and mitigator of normal but not tumor tissues by a novel mechanism independent of autophagy, p53 and apoptosis. Acknowledgments: This project was supported by U191A168021–06. Disclosures: No relevant conflicts of interest to declare.

CSF3R T618I Mouse Bone Marrow Transplant Model Of Neutrophilic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 223-223
Author(s):  
Julia E Maxson ◽  
Angela G. Fleischman ◽  
Samuel B Luty ◽  
Lacey R Royer ◽  
Anupriya Agarwal ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Clinical Trials ◽  
Bone Marrow Transplant ◽  
Conflicts Of Interest ◽  
Marrow Transplant ◽  
Mouse Bone Marrow ◽  
Wild Type ◽  
Cell Counts ◽  
Financial Interest ◽  
White Blood Cell Counts

Abstract Background We have recently identified mutations in CSF3R in the majority of patients with chronic neutrophilic leukemia (CNL). The most common mutation identified thus far is the T618I membrane proximal mutation, which confers rapid transformation capacity and ligand-independence to the receptor. We also observed less common mutations that result in truncation of the cytoplasmic domain of CSF3R. These truncation mutations, which have been studied in the context of severe congenital neutropenia progression to acute myeloid leukemia, lack the capacity for inducing disease in transgenic mouse models but enhance the progression to leukemia in the presence of a second genetic driver. The CSF3R T618I mutation transforms cells at a substantially faster rate than the truncation mutation, but the capacity of this mutation to induce a hematologic disorder in mice is not yet known. We created a CSF3R T618I mouse bone marrow transplant model, which resulted in a lethal myeloproliferative disorder characterized by high levels of granulocytes. Methods Mice were transplanted with donor bone marrow infected with a murine retrovirus expressing either wild type CSF3R or the CSF3R T618I mutation. White blood cell counts were measured 1-2 times per week for 90 days. The percentages of monocytes, granulocytes, T-cells and B-cells were analyzed by flow cytometery. The levels of pSTAT3 were measured by phospho-flow. Hematoxylin and eosin staining was performed on fixed and sectioned spleens, livers and bone marrow. Results Mice transplanted with CSF3R T618I showed an initial transient granulocytic leukocytosis that normalized by 33 days post transplant. Subsequently, at day 47 the CSF3R T618I mice exhibited a dramatic and persistent rise in white blood cell counts. The elevated white blood cells were primarily mature granulocytes. By day 90, all of the mice transplanted with CSF3R T618I had died, while the mice transplanted with cells expressing wild type CSF3R did not exhibit morbidity or mortality. The granulocytes from CSF3R T618I mice also exhibited elevated levels of pSTAT3—a marker of JAK signaling downstream of CSF3R—relative to CSF3R wild type granulocytes. Histology revealed that both CSF3R wild type and T618I mice had hypercellular bone marrow, however the hypercellularity was more extreme in the CSF3R T618I mice. Mice harboring the CSF3R T618I and to a lesser extent wild type CSF3R exhibited infiltration of the spleen and liver by mature granulocytic cells and disrupted tissue architecture. Conclusion The CSF3R T618I mouse bone marrow transplant provides a tractable model of Neutrophilic Leukemia. In contrast to CSF3R truncation mutations, which do not cause leukemia in isolation, the T618I mutation causes a fatal expansion of granulocytes. This model provides a useful tool for studying the biology of granulocytes and myeloproliferative neoplasms. Disclosures: Off Label Use: Ruxolitinib - a JAK1/2 inhibitor that we propose can be used off-label for disease management of CSF3R-mutant neutrophilic leukemia. Fleischman:Incyte: Speakers Bureau. Druker:Incyte: PI or co-investigator on clinical trials., PI or co-investigator on clinical trials. Other; Novartis: PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU., PI or co-investigator on Novartis clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other; Bristol-Myers Squibb: PI or co-investigator on BMS clinical trials. OHSU and Dr. Druker have a financial interest in MolecularMD. OHSU has licensed technology used in some of these clinical trials to MolecularMD. Potential conflicts of interest are managed by OHSU. Other. Tyner:Incyte Corporation: Research Funding.

scholarly journals CRISPR-Mediated Loss of Immunoglobulin Heavy Chain in Multiple Myeloma Cell Line Results in Metabolic Pathway Alterations

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 1885-1885
Author(s):  
Denise K Walters ◽  
Renee C Tschumper ◽  
Dominique B Hoelzinger ◽  
Sophia J Quinton ◽  
Xiaojian Shi ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Cell Line ◽  
Cell Lines ◽  
Heavy Chain ◽  
Light Chain ◽  
Conflicts Of Interest ◽  
Dna Demethylation ◽  
Growth Patterns ◽  
Amino Acid Synthesis ◽  
Significant Difference

Abstract Aberrant over production of monoclonal immunoglobulin is a classic feature of the plasma cell (PC) malignancy multiple myeloma (MM). At diagnosis and more frequently upon relapse, some MM patients present with increased production of monoclonal free light chain (FLC) without a corresponding increase in intact immunoglobulin. This phenomenon is commonly referred to as light chain escape (LCE). As previously described (Walters et al. 2018, Experimental Hematology, Vol 57 p42-49), we have established a monoclonal IgA kappa MM cell line, designated as MC-B11/14, from the bone marrow (BM) aspirate of a 46 year old man diagnosed with MM. This cell line is non-hyperdiploid and possesses an 11;14 chromosomal translocation. Given our long standing interest in immunoglobulin function and secretion, we used CRISPR technology to knock out IgA heavy chain production in an effort to create a model of LCE. Successful knockout (KO) of IgA expression was demonstrated by immunofluorescence, flow cytometry, and western blot. As expected, secretion of intact IgA was undetectable in the MC-B11/14IgA- cells. Notably, no significant difference in morphology, phenotypic markers, or growth patterns was observed between the MC-B11/14WT and MC-B11/14IgA- cells. Examination of drug sensitivity between the MC-B11/14WT and MC-B11/14IgA- cells revealed that the response to pomalidomide treatment was similar between the MC-B11/14WT and MC-B11/14IgA- cells; however, the MC-B11/14IgA- cells were found to be more resistant to treatment with bortezomib. Given that increased bortezomib resistance was the only significant difference detected between the MC-B11/14WT and MC-B11/14IgA- cell lines in our initial study, we decided to take a metabolomics approach to examine whether loss of IgA heavy chain expression may alter cellular metabolism. Metabolomics or the profiling of small molecules is a powerful tool to characterize the metabolome of cells/biological systems and has proven to be useful in identifying alterations that occur in malignancy. In this regard, we employed pathway-specific targeted LC-MS/MS protocols in order to examine over 330 metabolites in 35 metabolic pathways in MC-B11/14WT and MC-B11/14IgA- methanolmetabolite extracts using an Agilent 1290 UPLC-6490 QQQ-MS liquid chromatography mass-spectrometer. Of the 330 metabolites examined, 28 were found to be significantly more abundant in the MC-B11/14IgA- cells compared to MC-B11/14 WT cells while 79 were found to be significantly less abundant. In general, the majority of significant metabolic differences between the two cell lines were found to involve metabolites from various amino acid synthesis pathways. More specifically, the MC-B11/14 WT cell line was found to possess significantly more citraconic acid, anthranilic acid, and homovanillic acid. By contrast, further analysis revealed that the MC-B11/14IgA- cell line was found to possess an 11.5 fold increase of the metabolite 2-hydroxyglutarate (2-HG). 2-HG has previously been shown to act as a competitive antagonist of α-ketoglutarate (α-KG), which results in the inhibition of α-KG-dependent dioxygenases including the JmjC domain-containing histone demethylases (KDMs) and the ten-eleven translocation (TET) family of DNA hydroxylases. Notably, these proteins are known to play a role in histone and DNA demethylation, respectively. Taken together, these data suggest that MM cells that have undergone LCE may also possess an accumulation of 2-HG which could lead to altered patterns of histone and DNA methylation. Given that MM patients who relapse with LCE typically have a poorer prognosis, further investigation of metabolomics in light-chain only expressing cell lines and patients who have relapsed with LCE is warranted. Disclosures No relevant conflicts of interest to declare.

Generation of a Factor Dependent Myeloid Cell Line from Nucleophosmin-1 Heterozygous (NPM-1+/−) Mouse Bone Marrow as a Model for 5q- MDS

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 852-852
Author(s):  
Arati Khanna-Gupta ◽  
Hong Sun ◽  
Jian Chen ◽  
Stephanie Halene ◽  
Paolo Sportoletti ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Retinoic Acid ◽  
Cell Line ◽  
Cell Lines ◽  
Myeloid Cell ◽  
Specific Gene ◽  
Ros Production ◽  
Granule Protein ◽  
Increased Risk ◽  
Myeloid Cell Line

Abstract Myelodysplastic syndromes (MDS) are a heterogeneous group of clonal disorders characterized by ineffective and disordered hematopoiesis with an increased risk of transformation to acute myeloid leukemia (AML). Interstitial deletion of 5q (del 5q) is the most frequent chromosomal abnormality seen in MDS. Two subgroups of MDS with del 5q have been described. The first, the so called “5q-syndrome” is defined by isolated del 5q and no excess blasts in the bone marrow, female predominance, typical dysmegakaryopoiesis, thrombocytosis and a favorable outcome. The second subgroup involves MDS with del 5q associated with an excess of marrow blasts and/or chromosomal abnormalities in addition to del 5q, that usually do not have the typical features of the 5q-syndrome and carry poorer prognosis. Haploinsufficiency for the ribosomal protein RPS14 gene has recently been identified as a compelling candidate gene for causing the 5q- syndrome, but the genes responsible for poor prognosis del 5q MDS remain to be determined. NPM-1, a highly conserved, ubiquitously expressed nucleolar phosphoprotein that belongs to the nucloeplasmin family of nuclear chaperones, maps to a region on chromosome 5q that is the target of deletions in both denovo and therapy-associated MDS in humans.. NPM-1 has been implicated in ribosome biogenesis and the transport of pre-ribosomal particles, maintenance of genomic stability by the control of cellular ploidy, in DNA repair and in the regulation of DNA transcription by controlling chromatin condensation/decondensation. NPM1−/− mice die at mid-gestion (E11.5) due to severe anemia. NPM-1 has been shown to be deleted or involved with chromosomal translocations in hematologic malignancies, and is one of the most frequently mutated genes in AML. NPM-1+/− mice develop a hematological syndrome very similar to that observed in MDS patients. The haploinsufficient NPM-1 model therefore provides an excellent platform to examine not only MDS but also to study AML progression. Studies on the molecular mechanisms underlying 5q- syndrome have been hampered by the lack of in vitro model cell lines. We have generated a factor dependent myeloid cell line from the bone marrow of NPM-1+/+ and NPM-1+/− mice using retroviral transduction of a truncated retinoic acid receptor alpha gene. The resulting cell lines are IL-3-dependent and can be induced to undergo neutrophil maturation by the addition of GM-CSF and/or all- trans retinoic acid (ATRA). We have shown that, like neutrophils derived from the bone marrow of NPM1+/− mice, the NPM-1+/− cell line-derived neutrophils display both defective neutrophil-specific gene expression and abnormal neutrophil function. We found in particular, that the expression of the myeloid master regulator C/EBPα but not that of the related myeloid-specific C/EBPε gene, was significantly decreased in NPM-1+/− cells as compared to their wild type counterparts (NPM-1+/+). While neutrophils derived from NPM1+/− cells appear morphologically normal, the expression of both neutrophil primary granule protein genes (defensins) and secondary granule protein genes (lactoferrin, MMP8 and MMP9) was significantly decreased. The relationship between NPM-1 and C/EBPα is currently being investigated. Additionally, the function of NPM-1+/− derived neutrophils was compromised as assessed by ROS production as well as by chemotaxis assays. The defective oxidative burst may be the result of decreased expression of a key component of the neutrophil NADPH oxidase complex i.e. gp91phox, which is critical for ROS production. We confirmed that these neutrophil-associated defects were also seen in primary neutrophils from the NPM-1+/− mice. We are currently examining the effect of knocking down RPS14 in our factor-dependent NPM-1+/− cells to determine if the two genes cooperate to render the cells factor independent. This cell line will help to delineate the functions of NPM-1 and provide a platform for examining the potential role of haploinsufficiency of NPM-1 in the propensity for 5q- MDS to progress to AML.

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