PU.1 Is a Downstream Target of SOX4 in Myeloid Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2217-2217
Author(s):  
Georg Aue ◽  
Yang Du ◽  
Cynthia E. Dunbar ◽  
Nancy A. Jenkins ◽  
Neal G. Copeland
Keyword(s):  
Bone Marrow ◽  
Vector Control ◽  
De Novo ◽  
Bone Marrow Cells ◽  
Regulatory Element ◽  
Myeloid Cells ◽  
P Value ◽  
Mrna Levels ◽  
Wild Type ◽  
Early Results

Abstract Mice that express 20% the normal levels of the Ets transcription factor PU.1 develop AML, unlike mice that express 50% or 80% the normal levels, indicating that PU.1 is a dosage-sensitive tumor suppressor gene. In addition, 3 of 13 AMLs induced by transplanting mice with cells transduced with a Sox4 oncogene-containing retrovirus were found to carry a Sox4 retroviral integration in one PU.1 allele, suggesting that downregulation of PU.1 may cooperate with Sox4 in AML induction. Since the other PU.1 allele remains intact in these AMLs and a 50% decrease in PU.1 expression is not sufficient to induce AML, we hypothesized that Sox4 might further downregulate PU.1 expression in these AMLs. To test this hypothesis, we transfected HL60 promyelocytes with an expression vector carrying both GFP and Sox4 cDNAs or a GFP vector control. Transfected GFP+ cells were purified by flow cytometry and PU.1 mRNA levels were analyzed by real-time RT-PCR. PU.1 mRNA levels were consistently downregulated 4 to 10 fold in cells transfected with Sox4 cDNA compared to cells transfected with the vector control, while β-actin mRNA levels were maintained constant, confirming that overexpression of Sox4 downregulates PU.1 expression in myeloid cells. The decrease of PU.1 mRNA was observed as early as 8 hours after Sox4 transfection, further suggesting that Sox4 may directly repress the PU.1 promoter in myeloid cells. Consistent with this, analysis of 2 published microarray databases comprising 401 de novo AML patient samples showed that SOX4 expression is significantly negatively correlated with PU.1 expression (coefficient: −0.337, P-value: 1E-07). Interestingly, AML FAB M1 and M2 subtypes were associated with statistically significant higher SOX4 expression levels compared to AML FAB M3, M4, M5. In order to confirm that downregulation of PU.1 cooperates with Sox4 in AML induction, we infected wild type or PU.1 heterozygous knockout bone marrow cells with the Sox4 retrovirus and then monitored the time of AML development in transplanted mice. Early results show accelerated leukemogenesis in mice transplanted with Sox4-infected PU +/− bone marrow (115 days) compared to mice receiving Sox4-infected wild type marrow (160 days). We are currently trying to identify Sox4 binding sites in the PU.1 promoter, or in an upper regulatory element that may be responsible for mediating the repression of PU.1.

PU.1 Cooperates with SOX4 in Myeloid Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2633-2633
Author(s):  
Georg Aue ◽  
Yang Du ◽  
Nancy A. Jenkins ◽  
Cynthia E. Dunbar ◽  
Neal G. Copeland
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Vector Control ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Bone Marrow Cells ◽  
Myeloid Cells ◽  
P Value ◽  
Mrna Levels ◽  
Wild Type

Abstract Mice that express 20% the normal levels of the Ets transcription factor PU.1 develop AML, unlike mice that express 50% or 80% the normal levels, indicating that PU.1 is a dosage-sensitive tumor suppressor gene. In addition, 3 of 13 AMLs induced by transplanting mice with cells transduced with a Sox4 oncogene-containing retrovirus were found to carry a Sox4 retroviral integration in one PU.1 allele, suggesting that downregulation of PU.1 may cooperate with Sox4 in AML induction. Since the other PU.1 allele remains intact in these AMLs and a 50% decrease in PU.1 expression is not sufficient to induce AML, we hypothesized that Sox4 might further downregulate PU.1 expression in these AMLs. To test this hypothesis, we transfected HL60 cells with an expression vector carrying GFP and Sox4 cDNA or a GFP vector control alone. PU.1 mRNA levels were consistently downregulated 4 to 10 fold in cells transfected with Sox4 cDNA compared to cells transfected with the vector control, confirming that overexpression of Sox4 downregulates PU.1 expression in myeloid cells. The decrease of PU.1 mRNA was observed as early as 8 hours after Sox4 transfection, further suggesting that Sox4 may directly interact with PU.1 in myeloid cells. Consistent with this, analysis of 2 published microarray databases comprising 401 de novo AML patient samples showed that SOX4 expression is significantly negatively correlated with PU.1 expression (coefficient: −0.337, P-value: 1E-07). In order to confirm that downregulation of PU.1 cooperates with Sox4 in AML induction, we infected wild type or PU.1 heterozygous knockout bone marrow cells with the Sox4 retrovirus and then monitored the time of AML development in transplanted mice. Results showed increased penetrance (95%) of myeloid leukemia in mice transplanted with Sox4-infected PU +/– bone marrow compared to mice receiving Sox4-infected wild type marrow (60%). Myeloid leukemia was confirmed by histology in all animals of the Sox4-infected PU +/ cohort while T cell lymphoma was diagnosed in 3 animals of the Sox4 wild type cohort. Together, all experiments support the hypothesis that Sox4 cooperates with the transcription factor PU.1.

Sox4 Downregulates Pu.1 Gene Expression by Binding to An Upper Regulatory Element of Pu.1, a Mechanism Contributing to Leukemogenesis.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3979-3979
Author(s):  
Georg Aue ◽  
Yang Du ◽  
Susan Cleveland ◽  
Stephen Smith ◽  
Utpal P. Dave ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Vector Control ◽  
Myeloid Leukemia ◽  
Integration Site ◽  
Regulatory Element ◽  
Myeloid Cells ◽  
Binding Motif ◽  
Mrna Levels ◽  
Wild Type

Abstract Abstract 3979 Poster Board III-915 Mice that express 20% the normal levels of the Ets transcription factor Pu.1 develop AML, unlike mice that express 50% to 90% the normal levels, indicating that Pu.1 is a dosage-sensitive tumor suppressor gene. Furthermore, 3 of 13 AMLs induced by transplanting mice with cells transduced with a Sox4 oncogene-containing retrovirus were found to carry a Sox4 retroviral integration in one Pu.1 allele, suggesting that downregulation of Pu.1 may cooperate with Sox4 in AML induction. Since the other Pu.1 allele remains intact in these AMLs and a 50% decrease in Pu.1 expression is not sufficient to induce AML, we hypothesized that Sox4 might further downregulate Pu.1 expression in these AMLs. To test this hypothesis, we transfected HL60 promyelocytes with an expression vector carrying both GFP and Sox4 cDNAs or a GFP vector control. Transfected GFP+ cells were purified by flow cytometry and Pu.1 mRNA levels were analyzed by real-time RT-PCR. Pu.1 mRNA levels were consistently downregulated 4 to 10 fold in cells transfected with Sox4 cDNA compared to cells transfected with the vector control, while Beta-actin mRNA levels were maintained constant, confirming that overexpression of Sox4 downregulates Pu.1 expression in myeloid cells. The decrease of Pu.1 mRNA was observed as early as 8 hours after Sox4 transfection, further suggesting that Sox4 may directly repress the Pu.1 promoter in myeloid cells. Consistent with this, analysis of a published microarray databases comprising 285 de novo AML patient samples showed that SOX4 expression is significantly negatively correlated with Pu.1 expression (r= -0.337, p-value<0.001). In order to confirm that downregulation of Pu.1 cooperates with Sox4 in AML induction, we infected Pu.1 heterozygous knockout or wild type bone marrow cells with the Sox4 retrovirus and then monitored the time of AML development in transplanted mice. An increased penetrance of myeloid leukemia was observed in mice transplanted with Sox4-infected Pu.1 +/- bone marrow (95%) compared to mice receiving Sox4-infected wild type marrow (60%, p<0.001). Myeloid leukemia was confirmed by histology in all animals (100%) of the Sox4-infected Pu.1 +/ cohort. A Southern blot with a Sox4 probe confirmed clonal integrations. Consistent with our hypothesis, integration site analysis of the Sox4-infected Pu.1 +/- cohort tumor spleen DNA could not detect a Pu.1 integration site. Binding motif analysis found a Sox4 binding site in an upper regulatory element (URE) 14 kb upstream of the Pu.1 gene. Chromatin immunohybridization (ChIP) with a Sox4 antibody performed in 32D clone 3 lymphoblasts confirmed binding in a highly conserved area of the Pu.1 upstream control region. An electromobility shift assay (EMSA) is currently pursued. In summary, these results elucidate how the transcription factor Pu.1 is regulated by Sox4 though an upper regulatory element and can play a role in leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Sox4 cooperates with PU.1 haploinsufficiency in murine myeloid leukemia

Blood ◽  
2011 ◽  
Vol 118 (17) ◽  
pp. 4674-4681 ◽  
Author(s):  
Georg Aue ◽  
Yang Du ◽  
Susan M. Cleveland ◽  
Stephen B. Smith ◽  
Utpal P. Davé ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
De Novo ◽  
Bone Marrow Cells ◽  
Regulatory Element ◽  
Mrna Levels ◽  
Wild Type ◽  
Myeloid Leukemias ◽  
Acute Myeloid

Abstract Cooperation of multiple mutations is thought to be required for cancer development. In previous studies, murine myeloid leukemias induced by transducing wild-type bone marrow progenitors with a SRY sex determining region Y-box 4 (Sox4)–expressing retrovirus frequently carried proviral insertions at Sfpi1, decreasing its mRNA levels, suggesting that reduced Sfpi1 expression cooperates with Sox4 in myeloid leukemia induction. In support of this hypothesis, we show here that mice receiving Sox4 virus-infected Sfpi1ko/+ bone marrow progenitors developed myeloid leukemia with increased penetrance and shortened latency. Interestingly, Sox4 expression further decreased Sfpi1 transcription. Ectopic SOX4 expression reduced endogenous PU.1 mRNA levels in HL60 promyelocytes, and decreased Sfpi1 mRNA levels were also observed in the spleens of leukemic and preleukemic mice receiving Sox4 virus-infected wild-type bone marrow cells. In addition, Sox4 protein bound to a critical upstream regulatory element of Sfpi1 in ChIP assays. Such cooperation probably occurs in de novo human acute myeloid leukemias, as an analysis of 285 acute myeloid leukemia patient samples found a significant negative correlation between SOX4 and PU.1 expression. Our results establish a novel cooperation between Sox4 and reduced Sfpi1 expression in myeloid leukemia development and suggest that SOX4 could be an important new therapeutic target in human acute myeloid leukemia.

Stat3β Promotes Basal Granulopoiesis and Inhibits Emergency Granulopoiesis, While Stat3α Inhibits Basal Granulopoiesis and Promotes Emergency Granulopoiesis

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3871-3871
Author(s):  
Michele Redell ◽  
S. Wen-Wen Chen ◽  
Marcos J. Ruiz ◽  
David J. Tweardy
Keyword(s):  
Bone Marrow ◽  
B Lymphocyte ◽  
Inflammatory Responses ◽  
Bone Marrow Cells ◽  
Myeloid Cells ◽  
Lymphoid Cells ◽  
Transactivation Domain ◽  
Wild Type ◽  
Alternative Mrna Splicing ◽  
Deficient Mice

Abstract Signal transducer and activator of transcription 3 (Stat3) is a key signaling intermediate that is activated by several cytokines that regulate hematopoiesis, including granulocyte-colony stimulating factor (G-CSF), interleukin 6, and stem cell factor (SCF). Studies using mice with Stat3 deletion targeted to hematopoietic cells have shown that Stat3 negatively regulates basal granulopoiesis but positively regulates emergency granulopoiesis. Stat3 also has been reported to promote B lymphocyte differentiation. Defining the hematopoietic functions of Stat3 is further complicated by the existence of two isoforms: full-length Stat3α (p92), and truncated Stat3β (p83). Stat3β is derived from alternative mRNA splicing resulting in replacement of the C-terminal transactivation domain with 7 unique amino acids (CT7), which have been demonstrated to confer markedly prolonged nuclear retention. Homozygous Stat3α-deficient mice are not viable, whereas Stat3β-deficient mice survive to adulthood and are fertile, but have increased inflammatory responses compared to wild-type mice. We compared basal granulopoiesis and lymphopoiesis, as well as emergency granulopoiesis, in homozygous Stat3β-deficient mice (βΔ/βΔ), which express only Stat3α, vs. their wild-type (+/+) littermates. We found that βΔ/βΔ mice were significantly leukopenic (2880 ± 1260/ml v. 4600 ± 1670/ml; p<0.05), with lower absolute neutrophil counts (ANC, 360 ± 180/ml v. 800 ± 380/ml, p<0.05) and B lymphocyte counts (780 ± 470/ml v. 1830 ± 1260/ml, p<0.05), compared to +/+ mice. Within the circulating B-lymphocyte population, the mature B220hi/IgM− cells were most dramatically reduced (170 ± 70/ml v. 480 ± 350/ml, p<0.05). Percentages of myeloid and lymphoid cells in the spleen and bone marrow were not significantly different between βΔ/βΔ and +/+ mice. Bone marrow from βΔ/βΔ mice generated significantly fewer myeloid colonies (CFU-GM) compared to wild-type marrow (28 ± 9 v. 42 ± 8 colonies per 20,000 cells, p<0.05). Additionally, βΔ/βΔ lineage-depleted bone marrow cells cultured in G-CSF and SCF produced significantly fewer CD11b+/Gr1+ myeloid cells compared to +/+ cells (52.8 ± 6.5% v. 68.3 ± 2.6%, p<0.05). In contrast, bone marrow from βΔ/βΔ and +/+ mice produced equal numbers of pro-B colonies in CFU assays containing the lymphopoietic cytokine IL-7. Finally, as a test of emergency granulopoiesis, we administered a single dose of G-CSF (250 μg/kg subcutaneously) or an equal volume of PBS, and 24 hr later measured the ANC, percentage of CD11b+/Gr1+ myeloid cells in the bone marrow, and CFU-GM generation. Mice of both genotypes responded to G-CSF stimulation with increases in ANC, percent of myeloid cells within the marrow, and CFU-GM. Bone marrow from βΔ/βΔ mice showed a larger G-CSF-induced increase in CFU-GM (PBS: 22 ± 5 v. G-CSF: 39 ± 1, p<0.05) compared to +/+ marrow (PBS: 24 ± 14 v. G-CSF: 31 ± 14, NS). Thus, Stat3β positively regulates basal granulopoiesis in the bone marrow, and may negatively regulate emergency granulopoiesis. This pattern is the opposite of that seen with deletion of both Stat3 isoforms, indicating that Stat3α’s function is to negatively regulate basal granulopoiesis and positively regulate emergency granulopoiesis. Stat3β also positively regulates circulating B lymphocyte numbers, via a mechanism other than B lymphocyte production in the bone marrow.

IMMU-05. CCR2+ MYELOID-DERIVED SUPPRESSOR CELLS FROM MURINE GLIOMAS ARE SOURCED FROM THE BONE MARROW, SUPPRESS T CELL ACTIVATION, AND MIGRATE TO CCL2

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi92-vi93
Author(s):  
Gregory Takacs ◽  
Christian Kreiger ◽  
Defang Luo ◽  
Joseph Flores-Toro ◽  
Loic Deleyrolle ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
T Cell Activation ◽  
Cell Activation ◽  
Bone Marrow Cells ◽  
Myeloid Cells ◽  
Suppressor Cells ◽  
Myeloid Derived Suppressor Cells ◽  
Wild Type ◽  
Suppressive Phenotype

Abstract INTRODUCTION Mounting evidence suggests infiltrating immune-suppressive cells contribute to immune checkpoint inhibitor resistance and poor survival in Glioblastoma (GBM) patients. We have previously shown glioma-associated monocytic-myeloid derived suppressor cells (M-MDSCs) express chemokine receptors CCR2 and CX3CR1. Genetic and pharmacologic targeting of CCR2 promoted sequestration of M-MDSCs in the bone marrow and, in combination with PD-1 blockade, slowed progression of KR158 and 005GSC murine gliomas. This combination treatment also enhanced infiltration of IFNg-producing T cells that were less exhausted. Although CCR2+/CX3CR1+ cells display surface markers indicative of bone marrow-derived M-MDSCs, additional studies are needed to formally establish the source of these cells and to determine if they exhibit an immune-suppressive phenotype as well as migrate to the CCR2 ligands, CCL2 and/or CCL7. OBJECTIVE Evaluate the source, migration, and immune suppressive function of CCR2+/CX3CR1+ myeloid cells from glioma bearing mice. METHODS To identify the source of CCR2+/CX3CR1+ myeloid cells, chimeric wild type mice harboring bone marrow cells from transgenic CCR2WT/RFP/CX3CR1WT/GFP mice were generated. CCR2+/CX3CR1+ cells were enriched from bone marrow obtained from either wild-type or CCR2WT/RFP/CX3CR1WT/GFP naïve and glioma-bearing mice in order to evaluate their immune suppressive phenotype and ability to migrate to CCL2 and CCL7. RESULTS CCR2+/CX3CR1+ cells are present in glioma isolates from chimeric mice, indicative of a bone marrow-derived cell population, and are detectable within the tumor microenvironment as early as 3 days post orthotopic implantation of KR158 cells; these cells accumulate as tumors increase in size (r=0.7605, p=0.007). CCR2+/CX3CR1+ M-MDSCs isolated from the bone marrow of tumor bearing mice suppress CD8+ T cell production of IFNg and migrate to CCL2 more efficiently than CCL7. CONCLUSION CCR2+/CX3CR1+ cells from glioma bearing mice are derived from the bone marrow and represent an immune suppressive population that migrates to CCL2.

Hematopoietic Cells from Gadd45a, Gadd45b Deficent Mice Exhibit Altered Myelopoiesis and Higher Apoptosis after Cytokine Treatment.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1697-1697
Author(s):  
Shiv K. Gupta ◽  
Mamta Gupta ◽  
Barbara Hoffman ◽  
Dan A. Liebermann
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Myeloid Cells ◽  
Methyl Cellulose ◽  
Myeloid Cell ◽  
Cell Maturation ◽  
Wild Type ◽  
Wild Type Littermate ◽  
Deficient Mice ◽  
Marrow Cells

Abstract Growth arrest and DNA damage, Gadd45 gene family members are rapidly induced by genotoxic agents as well as by apoptosis and differentiation inducing cytokines. Their role in hemetopoiesis, wherein proliferation, differentiation and apoptosis integrate to maintain cellular homeostasis, is not clear. Using bone marrow cells from gadd45a or gadd45b deficient and wild type littermate mice we have investigated the role of Gadd45 proteins in cytokine induced myeloid cell differentiation in vitro. Bone marrow cells obtained from either gadd45a or gadd45b deficient mice displayed compromised cytokines (IL3, GM-CSF, M-CSF or G-CSF) induced myelopoiesis, resulting in a quantitatively decreased population of mature myeloid cells. Immuno-phenotyping with antibodies to cell surface molecules associated with myeloid cell maturation confirmed impaired myeloid cell maturation in Gadd45a or b deficient bone marrow cells treated with the above cytokines. Analysis of apoptosis by annexin-V and PI staining followed by FACS analysis showed a substantially higher apoptosis in Gadd45a−/− as well as gadd45b−/− cells compared to wild type cells after treatment with M-CSF or G-CSF. Gadd45a−/− as well as gadd45b−/− bone marrow cells were found to be less clonogenic in methylcellulose medium. Morphologically compact and round colonies consisting of immature myeloid cells prevailed over dispersed- colonies consisting of mature myeloid cells in gadd45- deficient cells cultured in methyl cellulose containing IL-3. Furthermore, colony re-plating assay showed better self-renewal abilities in gadd45a−/− as well as gadd45b−/− progenitors, compared to wild type progenitor cells. Altered myelopoiesis in gadd45 a or b deficient mice was further confirmed in vivo by intra-peritoneal administration of sodium casienate - a known inducer of inflammatory response and myelopoiesis in mice bone marrow. Sodium casienate failed to enhance myelopoiesis in gadd45a or gadd45b deficient mice bone marrow, while wild type littermate mice showed a rapid induction of myelopoiesis. Simultaneously peritoneal exudates collected from gadd45 deficient mice consisted of 2–3 fold less myeloid cells compared to age matched wild type control mice after sodium casienate treatment. Gadd45a−/− or gadd45b−/− mice showed a slow recovery after myelo-suppressive effect of antimetabolite 5-Fluorouracil, which further confirmed that gadd45 deficiency leads to delayed myelopoiesis in mouse. Mechanistic aspects of Gadd45 deficiency, which results in impaired myelopoiesis are under investigation.

Loss of Sos1 Inhibits Oncogenic Kras-Induced Hyperactivation of Wild-Type Ras during Leukemogenesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1543-1543
Author(s):  
Xiaona You ◽  
Guangyao Kong ◽  
Erik A. Ranheim ◽  
Yun Zhou ◽  
Jing Zhang
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Myeloid Cells ◽  
Small Gtpase ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Erk Activation ◽  
Gm Csf ◽  
Marrow Cells

Abstract As members of small GTPase super family, the functional output of Ras proteins depends on their GTP binding status, which is regulated by the interactions with guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). Activating mutations in NRAS and KRAS isoforms are identified in various types of hematopoietic malignancies. Interestingly, the same oncogenic mutation (G12D) at the endogenous Kras locus displays much more potent leukemogenic activity than that at the endogenous Nras locus in vivo. Moreover, combined inhibition of MEK and ERK provides long-term disease-free survival in NrasG12D/G12D mice but had much less effect in KrasG12D/+ mice. During our investigation to understand the potent leukemogenic activity of oncogenic Kras, we found that in total bone marrow cells, oncogenic Kras, but not oncogenic Nras, induces hyperactivation of wild-type (WT) Hras and Nras. We hypothesize that the hyperactivated WT Ras significantly contributes to oncogenic Kras-mediated leukemogenesis and inhibition of this process might improve the sensitivity of oncogenic Kras cells towards combined therapy. Because Sos1, a RAS GEF, has been implicated in oncogenic Ras-mediated activation of WT Ras in human cancer cell lines, we investigated whether Sos1 plays an essential role in this process in vivo. We find that Sos1 is overexpressed in KrasG12D/+ bone marrow cells. Genetic deletion of Sos1 indeed significantly decreases the GTP-bound active form of WT Nras and Hras without affecting the activation status of oncogenic Kras. Consequently, Sos1 deficiency-mediated downregulation of ERK activation rescues oncogenic Kras mediated depletion of hematopoietic stem cells (HSCs). HSCs, multipotent progenitors (MPPs) and LSKs (Lin-Sca-1+c-Kit+) in KrasG12D/+;Sos1-/- mice are much more quiescent than those in KrasG12D/+ mice. Moreover, Sos1 deficiency significantly inhibits granulocyte-macrophage colony stimulating factor (GM-CSF) evoked ERK signaling in KrasG12D/+ myeloid progenitor and precursor cells. Consistent with these biochemical data, we show that myeloproliferative neoplasm (MPN) phenotypes are significantly alleviated in KrasG12D/+;Sos1-/- mice and these animals survived significantly longer than KrasG12D/+ mice. However, we find that in differentiated myeloid cells (e.g. neutrophils), loss of Sos1 does not affect GM-CSF-evoked ERK activation. This result is consistent with our previous finding that Ras-mediated ERK activation in differentiated myeloid cells is predominantly through Kras but not Hras or Nras. Together, our results demonstrate that Sos1 mediates oncogenic Kras-induced hyperactivation of WT Ras. Inhibition of Sos1 thus blocks this process and attenuates the leukemogenic activity of oncogenic Kras. In contrast, Sos1 deficiency does not affect the unique signaling mediated by oncogenic Kras itself. Therefore, we hypothesize that targeting Sos1 alone will not effectively treat KrasG12D-associated leukemias but it might increase the sensitivity of KrasG12D cells to other therapies, such as combined inhibition of MEK and JAK. We are currently testing this hypothesis in vivo. Disclosures No relevant conflicts of interest to declare.

scholarly journals Heterogeneous Expression of OLFM4 in Neutrophils and Plasma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2213-2213
Author(s):  
Stine N. Clemmensen ◽  
Sara Heebøll ◽  
Claus Koch ◽  
Niels Borregaard
Keyword(s):  
Bone Marrow ◽  
Blood Plasma ◽  
Bone Marrow Cells ◽  
Myeloid Cells ◽  
Human Neutrophils ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mrna Levels ◽  
Highly Sensitive ◽  
Bone Marrow Plasma ◽  
Marrow Cells

Abstract Introduction: Olfactomedin 4 (OLFM4) is a glycoprotein predominantly expressed in myeloid cells and in gastrointestinal tissues. OLFM4 is stored in specific granules of human neutrophils where it defines a subset of neutrophils ranging from 5-40% OLFM4 positive neutrophils. OLFM4 has been proposed to inhibit cathepsin C, a cysteine protease essential for activation of serine proteases. Elevated levels of OLFM4 has been seen primary myelofibrosis (PMF) patients, these patients furthermore display a significantly elevated serum OLFM4. Methods: We developed monoclonal antibodies against OLFM4 and established an Enzyme Linked Immunosorbent Assay for OLFM4 for further investigation of OLFM4 in myeloid cells. Results: We observed two populations of individuals with respect to OLFM4 levels in plasma, the majority with OLFM4 in plasma between 0 and 0.1 μg/mL, mean 0.028 μg/mL while approximately 10% had OLFM4 between 4 and 60 μg/mL, mean 15 μg/mL. The levels were constant over time. The level did not relate to the size of the OLFM4 positive neutrophil subset detected in peripheral blood. We studied the biosynthesis of OLFM4 in isolated bone marrow cells from an individual with high plasma OLFM4 and an individual with low plasma OLFM4. The levels of OLFM4 mRNA were comparable and the amounts of OLFM4 synthesized and retained in cells were similar between the two individuals. We next determined whether OLFM4 might be produced by bone marrow cells and released into bone marrow plasma. Corresponding levels of OLFM4 determined in bone marrow plasma and blood plasma from two persons with high levels of OLFM4 showed lower levels in bone marrow plasma than in blood plasma, arguing against bone marrow as the direct source of OLFM4 in plasma. To estimate the amount of OLFM4 generated daily during myelopoiesis, we quantitated the amount of OLFM4 in neutrophils from 3 sets of buffy coat neutrophils, each pooled from 4 healthy donors. The amount of OLFM4 was 1.2 μg/107 neutrophils. As the production of neutrophils is about 1 x 109 cells/kg body weight/day, this would indicate production of 10 mg OLFM4/day in an adult. To rule out the liver as a production site, mRNA was determined by Affymetrix gene array in liver biopsies from 42 patients evaluated for liver steatosis. OLFM4 mRNA levels were uniformly at the border of detection in all (data not shown). OLFM4 is secreted from PMA stimulated neutrophils in parallel with other specific granule proteins. When purified OLFM4 was added to medium from neutrophils induced to degranulate with PMA, the ability to detect OLFM4 was rapidly lost, indicating that OLFM4 is highly sensitive to proteolysis. Adding a cocktail of protease inhibitors to the material secreted from PMA activated neutrophils preserved OLFM4. As we find that OLFM4 is highly sensitive to proteolysis we suggest that the differences in OLFM4 levels in plasma may be related to individual differences in the susceptibility of OLFM4 to escape degradation when neutrophils decease as part of their normal life cycle. Production of 10 mg OLFM4/day would support a plasma level of 3-4 µg/ml plasma depending of the half-life of OLFM4 in plasma. This hypothesis is not easily tested, but if proven correct, might open for novel insight into the fate of neutrophils after exiting circulation, an issue that is still a matter of debate. Disclosures No relevant conflicts of interest to declare.

scholarly journals Src Homology 2–containing 5-Inositol Phosphatase (SHIP) Suppresses an Early Stage of Lymphoid Cell Development through Elevated Interleukin-6 Production by Myeloid Cells in Bone Marrow

2004 ◽  
Vol 199 (2) ◽  
pp. 243-254 ◽  
Author(s):  
Koji Nakamura ◽  
Taku Kouro ◽  
Paul W. Kincade ◽  
Alexander Malykhin ◽  
Kazuhiko Maeda ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Early Stage ◽  
Myeloid Cells ◽  
Cell Development ◽  
Wild Type ◽  
Src Homology ◽  
B Lineage

The Src homology (SH)2–containing inositol 5-phosphatase (SHIP) negatively regulates a variety of immune responses through inhibitory immune receptors. In SHIP−/− animals, we found that the number of early lymphoid progenitors in the bone marrow was significantly reduced and accompanied by expansion of myeloid cells. We exploited an in vitro system using hematopoietic progenitors that reproduced the in vivo phenotype of SHIP−/− mice. Lineage-negative marrow (Lin−) cells isolated from wild-type mice failed to differentiate into B cells when cocultured with those of SHIP−/− mice. Furthermore, culture supernatants of SHIP−/− Lin− cells suppressed the B lineage expansion of wild-type lineage-negative cells, suggesting the presence of a suppressive cytokine. SHIP−/− Lin− cells contained more IL-6 transcripts than wild-type Lin− cells, and neutralizing anti–IL-6 antibody rescued the B lineage expansion suppressed by the supernatants of SHIP−/− Lin− cells. Finally, we found that addition of recombinant IL-6 to cultures of wild-type Lin− bone marrow cells reproduced the phenotype of SHIP−/− bone marrow cultures: suppression of B cell development and expansion of myeloid cells. The results identify IL-6 as an important regulatory cytokine that can suppress B lineage differentiation and drive excessive myeloid development in bone marrow.

scholarly journals Bisecting GlcNAc structure is implicated in suppression of stroma-dependent haemopoiesis in transgenic mice expressing N-acetylglucosaminyltransferase III

1998 ◽  
Vol 331 (3) ◽  
pp. 733-742 ◽  
Author(s):  
Masafumi YOSHIMURA ◽  
Yoshito IHARA ◽  
Tetsuo NISHIURA ◽  
Yu OKAJIMA ◽  
Megumu OGAWA ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Stromal Cells ◽  
Bone Marrow Cells ◽  
Adherent Cell ◽  
Wild Type ◽  
Spleen Cells ◽  
Bisecting Glcnac ◽  
Marrow Cells

Several sugar structures have been reported to be necessary for haemopoiesis. We analysed the haematological phenotypes of transgenic mice expressing β-1,4 N-acetylglucosaminyltransferase III (GnT-III), which forms bisecting N-acetylglucosamine on asparagine-linked oligosaccharides. In the transgenic mice, the GnT-III activity was elevated in bone marrow, spleen and peripheral blood and in isolated mononuclear cells from these tissues, whereas no activity was found in these tissues of wild-type mice. Stromal cells after long-term cultures of transgenic-derived bone marrow and spleen cells also showed elevated GnT-III activity, compared with an undetectable activity in wild-type stromal cells. As judged by HPLC analysis, lectin blotting and lectin cytotoxicity assay, bisecting GlcNAc residues were increased on both blood cells and stromal cells from bone marrow and spleen in transgenic mice. The transgenic mice displayed spleen atrophy, hypocellular bone marrow and pancytopenia. Bone marrow cells and spleen cells from transgenic mice produced fewer haemopoietic colonies. After lethal irradiation followed by bone marrow transplantation, transgenic recipient mice showed pancytopenia compared with wild-type recipient mice. Bone marrow cells from transgenic donors gave haematological reconstitution at the same level as wild-type donor cells. In addition, non-adherent cell production was decreased in long-term bone marrow cell cultures of transgenic mice. Collectively these results indicate that the stroma-supported haemopoiesis is compromised in transgenic mice expressing GnT-III, providing the first demonstration that the N-glycans have some significant roles in stroma-dependent haemopoiesis.

Sign in / Sign up

Export Citation Format

Share Document