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.

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.

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.

GABP Transcription Factor Is Required for Myeloid Cell Development In Vivo.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 374-374 ◽  
Author(s):  
Zhong-fa Yang ◽  
Karen Drumea ◽  
Alan G. Rosmarin
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
T Cell ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Cell Development ◽  
Wild Type ◽  
Ets Domain

Abstract GABP is an ets transcription factor that regulates genes that are required for innate immunity, including CD18 (β2 leukocyte integrin), lysozyme, and neutrophil elastase. GABP consists of two distinct and unrelated proteins. GABPα binds to DNA through its ets domain and recruits GABPβ, which contains the transactivation domain; together, they form a functional tetrameric transcription factor complex. We recently showed that GABP is required for entry into S phase of the cell cycle through its regulation of genes that are required for DNA synthesis and cyclin dependent kinase inhibitors (Yang, et al. Nature Cell Biol9:339, 2007). Furthermore, GABP is an essential component of a retinoic acid responsive myeloid enhanceosome (Resendes and Rosmarin Mol Cell Biol26:3060, 2006). We cloned Gabpa (the gene that encodes mouse Gabpα) from a mouse genomic BAC library and prepared a targeting vector in which the ets domain is flanked by loxP recombination sites (floxed allele). Deletion of both floxed Gabpa alleles causes an early embryonic lethal defect. In order to define the role of Gabpα in myelopoiesis, we bred floxed Gabpa mice to mice that bear the Mx1-Cre transgene, which drives expression of Cre recombinase in response to injection of the synthetic polynucleotide, poly I-C. Deletion of Gabpa dramatically reduced granulocytes and monocytes in the peripheral blood, spleen, and bone marrow, but myeloid cells recovered within weeks. In vitro colony forming assays indicated that myeloid cells in these mice were derived only from Gabpa replete myeloid precursors (that failed to delete both Gabpa alleles), suggesting strong pressure to retain Gabpα in vivo. We used a novel competitive bone marrow transplantation approach to determine if Gabp is required for myeloid cell development in vivo. Sub-lethally irradiated wild-type recipient mice bearing leukocyte marker CD45.1 received equal proportions of bone marrow from wild type CD45.1 donor mice and floxed-Mx1-Cre donor mice that bear CD45.2. Both the CD45.2 (floxed-Mx1-Cre) and CD45.1 (wild type) bone marrow engrafted well. Mice were then injected with pI-pC to induce Cre-mediated deletion of floxed Gabpa. The mature myeloid and T cell compartments were derived almost entirely from wild type CD45.1 cells. This indicates that the proliferation and/or differentiation of myeloid and T cell lineages requires Gabp. In contrast, B cell development was not impaired. We conclude that Gabpa disruption causes a striking loss of myeloid cells in vivo and corroborates prior in vitro data that GABP plays a crucial role in proliferation of myeloid progenitor cells.

A Conserved Mechanism of Transcription Factor Competition Controls Hematopoietic Progenitor Self-Renewal.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 91-91
Author(s):  
Shane R. Horman ◽  
Chinamenveni S. Velu ◽  
Tristan Bourdeau ◽  
Avinash Baktula ◽  
Jinfang Zhu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Binding Sites ◽  
Bone Marrow Cells ◽  
Dominant Negative ◽  
Wild Type ◽  
Hematopoietic Progenitor ◽  
Self Renewal ◽  
Conditional Deletion

Abstract An intrinsic mechanism of self-renewal is critical for the maintenance of hematopoietic stem cells (HSC), but this HSC function is extinguished during differentiation of progenitors. Here we show that the self-renewal capacity of hematopoietic progenitor cells is regulated through physical competition for occupancy of select DNA binding sites. Initially, we found that conditional deletion of the Growth factor independent-1 (Gfi1) gene results in the accumulation of abnormally persistent myeloid progenitors in vivo. Specifically, while germline Gfi1 deletion induces defective HSC self renewal and a block to granulopoiesis, we find that conditional deletion of Gfi1 induces a severe but transient block to neutrophil development with repopulation of the bone marrow by the remaining wild type HSC within 8 weeks post deletion. However, even though normal levels of granulocyte colony forming units (G-CFU) returned by 8 weeks post deletion, an abnormal Gfi1−/− myeloid progenitor remained in the bone marrow in vivo. Subsequently, we find in vitro that both wild-type bone marrow cells expressing Gfi1-dominant-negative mutants, and Gfi1−/− Lin- bone marrow contain cells that replate indefinitely. We hypothesized that Gfi1 is critical to extinguish self renewal in hematopoietic progenitors. In seemingly unrelated work, we discovered antagonism between the drosophila orthologs of Gfi1 and the Hoxa9/Pbx1/Meis1 transcription factor complex during drosophila embryo segmentation. We extended our drosophila findings to discover that a subset of mammalian DNA regulatory sequences encode DNA binding sites for both Gfi1 and Hoxa9/Pbx1/Meis1. These DNA sequences are able to bind either factor, and function as a molecular switch. Interestingly, composite Gfi1/ Hoxa9/Pbx1/Meis1 binding sites are present in the regulatory regions of the gene encoding Hoxa9. We note that Gfi1 expression is normally induced, while Hoxa9 expression is down-regulated, during the transition from common myeloid progenitor (CMP) to the granulocyte-monocyte progenitor (GMP). CMP have greater self renewal potential than GMP. Conditional deletion of Gfi1 in sorted CMP or GMP both increases Hoxa9 expression and generates progenitors capable of replating indefinitely in vitro. Thus, Gfi1 is critical to limit self renewal in these progenitors. Deregulated Hoxa9 expression or activity appears pivotal to this new Gfi1-null phenotype, because Gfi1 dominant-negative mutants immortalize wild-type (or Hoxa7−/−) but not Hoxa9−/− bone marrow cells in vitro. An abnormal gain of self-renewal can unleash the leukemic potential of progenitor cells. We find that both limiting Gfi1 gene dosage and expression of Gfi1 dominant-negative mutants significantly increases Nup98-Hoxa9-mediated colony formation. In contrast, forced expression of Gfi1 prevents Nup98-Hoxa9 immortalization. Notably, the expression of Hoxa9 (independent of cases with Nup98-Hoxa9 fusions) has been reported to be of significant prognostic value in human acute myeloid leukemia. In conclusion, Gfi1 and the Hoxa9/Pbx1/Meis1 complex compete to control the expression of genes (such as Hoxa9) which are critical to extinguish self renewal and limit the leukemogenic potential of hematopoietic progenitors. The antagonism between these transcription factor complexes is conserved from drosophila segment formation to mammalian hematopoietic progenitor biology.

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.

Myeloproliferative Disease or Myeloid Leukemia Caused by Cbl Mutants in a Mouse Transplantation Model.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3973-3973
Author(s):  
◽  
Srinivasa Rao Bandi ◽  
Marion Rensinghoff ◽  
Rebekka Grundler ◽  
Lara Tickenbrock ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Myeloproliferative Disease ◽  
Murine Bone Marrow ◽  
Hematologic Disorder ◽  
Almost All ◽  
Marrow Cells

Abstract Abstract 3973 Poster Board III-909 Purpose Somatic mutations of Kit have been found in leukemias and gastrointestinal stromal tumors. The proto-oncogene c-Cbl negatively regulates Kit and Flt3 by its E3 ligase activity and acts as a scaffold for several signaling adaptor molecules. We recently identified the first c-Cbl mutation in human disease in an AML patient, called Cbl-R420Q. Results We transduced primary murine bone marrow retrovirally with c-Cbl mutants and transplanted it into lethally irradiated mice. Almost all recipients of bone marrow cells transduced with Cbl mutants developed a lethal hematologic disorder with a mean latency of 341 days in the Cbl-R420Q group and 395 days in the Cbl-70Z group. Eleven out of 13 mice and 8 out of 11 mice died in the Cbl-R420Q group and Cbl-70Z group, respectively. Two animals succumbed to a myeloid leukemia, the other mice developed a myeloproliferative disease. The leukemic mice showed a leukocytosis of up to 140.000/μL. They developed a splenomegaly with massive expansion of myeloid cells in liver and spleen. Histology sections of spleen, liver and bone marrow and FACS analyses of spleen, bone marrow and peripheral blood showed extensive infiltration of myeloid cells. Conclusion Thus, transplantation of bone marrow cells expressing Cbl mutants leads to a myeloid leukemia or to a myeloproliferative disease with long latency and high penetrance. Disclosures: No relevant conflicts of interest to declare.

PRKD2 Serine-Threonine Kinase, a Downstream Effector Of GABP, Plays Essential Roles For The Maintenance Of HSCs

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2430-2430
Author(s):  
Zhong-Fa Yang ◽  
Wang Junling ◽  
Alan G. Rosmarin
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Protein Kinase ◽  
Bone Marrow Cells ◽  
Specific Cell ◽  
Wild Type ◽  
Serine Threonine Kinase ◽  
Hematopoietic Stem ◽  
Threonine Kinase ◽  
Marrow Cells

Abstract Hematopoietic stem cells (HSCs) are the source of all blood lineages, and HSCs must balance quiescence, self-renewal, and differentiation to meet lifelong needs for blood cell development. GABP is an ets-related transcription factor that controls critical genes in myeloid and lymphoid development, and has been implicated in control of HSC growth. GABP is an obligate multimeric transcription factor that includes the DNA-binding ets component, GABPa, along with various GABPb partner proteins. We conditionally deleted Gabpa in mouse bone marrow and found that Gabpa cells have a profound growth disadvantage due to cell cycle arrest in HSCs. We identified Protein Kinase D2 (PRKD2) as a candidate effector of GABP. PRKD2 is a diacyl glycerol- and Protein Kinase C-activated serine-threonine kinase, because deletion of Gabpa markedly reduced PRKD2 expression in normal HSCs and progenitor cells. In a Prkd2ki/ki mouse model, in which two functionally essential phosphorylation serines were inactivated genetically, their bone marrow long term HSCs reduced dramatically and the short term HSCs increased accordingly. Mice transplanted with a 1:1 mixture of Prkd2ki/ki and wild type bone marrow cells demonstrated the decreased proportion of the Prkd2ki/ki bone marrow cells with the corresponding increase of the wild type cells. Although ectopic expression of the human Chronic Myeloid Leukemia (CML) fusion oncogene BCR-ABL in wild type bone marrow cells induced rapid CML development, expression of BCR-ABL in Prkd2ki/ki bone marrow cells failed to develop CML in transplanted recipient mice. Analysis of the peripheral blood, bone marrow and spleen of these mice revealed that the BCR-ABL+, Prkd2ki/ki cells did not express myeloid or lymphoid specific cell surface antigens CD11b, Gr1, B220, or CD3e. They demonstrated an immature blast-like microscopic morphology, and recipient mice transplanted with these cells died before the onset of CML development. We conclude that the phosphorylation activated Prkd2 is required for the maintenance of HSC pool and the development of mature hematopoietic lineages from HSCs. These findings suggest that PRKD2 kinase mediate key downstream events of both PKC and transcription factor GABP, and that PRKD2 may serve as a novel therapeutic target in leukemia. Disclosures: No relevant conflicts of interest to declare.

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.

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