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.

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.

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.

Novel Function of the TAL1/SCL Transcription Factor in Differentiation of Murine Bone Marrow Monocytes.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1272-1272
Author(s):  
Soumyadeep Dey ◽  
Yubin Shi ◽  
Stephen J. Brandt
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Dna Binding ◽  
Vector Control ◽  
Colony Stimulating Factor ◽  
Wild Type ◽  
Macrophage Colony Stimulating Factor ◽  
Macrophage Colony ◽  
Stimulating Factor

Abstract The TAL1 (or SCL) gene encodes a basic helix-loop-helix transcription factor that can activate or repress gene expression, depending on its interaction partners. TAL1 proteins are expressed in the vascular and hematopoietic systems and in erythroid, megakaryocytic, and mast cell precursors. TAL1 is essential for hematopoietic commitment and vascular remodeling during embryogenesis and is also important to the differentiation of erythroid and megakaryocytic progenitors postnatally. Although a mouse Tal1 cDNA was cloned from a bone marrow (BM) macrophage cDNA library and we reported expression of Tal1 protein in mouse bone marrow mononuclear cells (Blood83:1200, 1994), no studies have examined the function of this transcription factor in the monocyte-macrophage lineage. To address this issue, we characterized Tal1 expression in primary mouse BM monocytes and M1 monocytic leukemia cells induced to differentiate in vitro. We isolated BM cells from wild-type C57BL6/J mice and purified a mononuclear cell population by pronase treatment and density gradient centrifugation on horse serum. Monocytes were then induced to differentiate in culture, initially in the presence of both interleukin-3 (IL-3) and macrophage colony-stimulating factor (M-CSF) and subsequently in the presence of L-cell conditioned medium. Tal1 expression was maintained in these cultures, declining by only 14% over the full culture period, while calcitonin receptor expression, a marker of osteoclastic differentiation, was extinguished. In parallel, interleukin-6 receptor (IL-6R) and macrophage colony-stimulating factor receptor (M-CSFR) mRNA increased 3.5- and 114-fold, respectively. In M1 cells induced to differentiate with recombinant murine IL-6, Tal1 protein abundance decreased similarly with differentiation. To assess the function of Tal1 in this cell lineage, a full-length Tal1 cDNA was introduced into both primary BM monocytes and M1 cells via retroviral gene transfer. Puurified monocytes were transduced 24 hr after isolation with wild-type Tal1 or a DNA-binding-defective Tal1 mutant, T192P, in the MSCV-GFP vector. GFP-expressing cells were then sorted and induced to differentiate. Gene expression analysis using semi-quantitative PCR carried out after 8 days of culture showed increased expression of Tal1 mRNA in cells transduced with the wild-type Tal1 cDNA compared to the vector control. This was accompanied by upregulation of IL-6R and M-CSFR expression relative to both parental vector- and mutant-transduced cells. Northern blot analysis of M1 cells transduced with the Tal1 cDNA showed similar upregulation of M-CSFR mRNA relative to vector control cells. In summary, these studies show that the TAL1 transcription factor is expressed by murine monocytes during differentiation to macrophages and suggest a role for TAL1 in both gene expression and differentiation of this lineage. Further, they suggest a requirement for direct TAL1 DNA binding.

The Sal-Like 1 (SALL1) Protein is Highly Expressed in Primary Bone Marrow Cells Derived From Patients with Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1285-1285
Author(s):  
Heiko Konig ◽  
Susan Kiefer ◽  
Lynn Robbins ◽  
Michael Rauchman ◽  
Huda S. Salman
Keyword(s):  
Bone Marrow ◽  
Acute Myeloid Leukemia ◽  
Transcription Factor ◽  
Cell Lines ◽  
Western Blotting ◽  
Protein Level ◽  
Myeloid Leukemia ◽  
Mrna Levels ◽  
Healthy Controls ◽  
Acute Myeloid

Abstract Abstract 1285 Poster Board I-307 Background SALL1 is a zinc finger transcription factor that maps to chromosome 16q12.1 in humans and chromosome 8D in mice. It is a transcription factor required for kidney development, and is mutated in patients with Townes-Brocks syndrome (TBS). We have created a mouse model of TBS that expresses a truncated SALL1 protein and observed that these mice displayed a syndrome consistent with myelodysplastic syndrome (MDS) that accelerates to acute myeloid leukemia (AML). The purpose of this study was to investigate whether SALL1 is expressed in AML patients. Materials and methods 50 bone marrow (BM) samples obtained from AML patients and 10 BM samples obtained from healthy donors were studied. BM samples were processed immediately upon receipt. Immunohistochemistry was performed on paraffin tissue sections of human BM using an anti-SALL1 antibody. RNA was extracted using the RNeasy Mini Kit (Qiagen). Quantitative RT-PCR was performed using the SYBR Green PCR Master Mix and SALL1 specific primers. SALL1 mRNA expression was normalized to RPL19, a ribosomal housekeeping gene. Western blotting was performed to confirm the presence of SALL1 at the protein level. Two AML cell lines (Kasumi-1 and -3) were also screened for SALL1 expression. Results 44/50 human BM AML samples displayed positive nuclear staining for SALL1. Samples taken from healthy controls showed no evidence of staining. 9/ 9 human BM AML samples showed increased SALL1 mRNA levels as compared to healthy controls with upregulation of SALL1 mRNA levels ranging from 3-fold to 1630-fold. High interpatient variability was observed. On average, SALL1 expression of AML patients was 225.1-fold greater than that of healthy controls (n=9, p=0.004). Western blotting confirmed upregulated SALL1 protein expression in AML BM with no expression seen in healthy controls. No SALL1 mRNA was detected in Kasumi-1 and -3 AML cell lines. i.) The majority of patient samples examined in this study demonstrated positive staining for SALL1. ii.) Consistent with its role as a transcription factor SALL1 histochemical staining was nuclear. iii.) SALL1 is highly expressed in human AML BM on the mRNA as well as on the protein level. iv.) No SALL1 mRNA was detected in AML cell lines. Conclusion Our data in mice and humans link the overexpression of SALL1 to AML. The absence of SALL1 in cell lines and its presence in the BM may indicate that SALL1 is confined to a relatively primitive cell. It is to be speculated that SALL1 has functional significance for the molecular pathogenesis of AML. Further studies to elaborate on its molecular functions and interactions are therefore clearly warranted and being conducted. Disclosures No relevant conflicts of interest to declare.

Preventing chemotherapy-induced myelosuppression by repurposing the FLT3 inhibitor quizartinib

2017 ◽  
Vol 9 (402) ◽  
pp. eaam8060 ◽  
Author(s):  
Samuel J. Taylor ◽  
Johanna M. Duyvestyn ◽  
Samantha A. Dagger ◽  
Emma J. Dishington ◽  
Catherine A. Rinaldi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Myeloid Leukemia ◽  
Leukemic Cells ◽  
Wild Type ◽  
Short Term ◽  
Treatment Regimens ◽  
Multipotent Progenitors ◽  
Flt3 Inhibitor ◽  
Short Term Exposure

We describe an approach to inhibit chemotherapy-induced myelosuppression. We found that short-term exposure of mice to the FLT3 inhibitor quizartinib induced the transient quiescence of multipotent progenitors (MPPs). This property of quizartinib conferred marked protection to MPPs in mice receiving fluorouracil or gemcitabine. The protection resulted in the rapid recovery of bone marrow and blood cellularity, thus preventing otherwise lethal myelosuppression. A treatment strategy involving quizartinib priming that protected wild-type bone marrow progenitors, but not leukemic cells, from fluorouracil provided a more effective treatment than conventional induction therapy in mouse models of acute myeloid leukemia. This strategy has the potential to be extended for use in other cancers where FLT3 inhibition does not adversely affect the effectiveness of chemotherapy. Thus, the addition of quizartinib to cancer treatment regimens could markedly improve cancer patient survival and quality of life.

GA Binding Protein (GABP) Is Required for Development and Maturation of Myeloid Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 776-776
Author(s):  
Zhongfa Yang ◽  
Alan G. Rosmarin
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Target Genes ◽  
Embryonic Stem ◽  
Myeloid Cells ◽  
Transactivation Domain ◽  
Wild Type ◽  
Floxed Allele

Abstract GABP is an ets transcription factor that regulates transcription of key myeloid genes, including CD18 (beta2 leukocyte integrin), neutrophil elastase, lysozyme, and other key mediators of the inflammatory response; it is also known to regulate important cell cycle control genes. GABP consists of two distinct and unrelated proteins that, together, form a functional transcription factor complex. GABPalpha (GABPa) is an ets protein that binds to DNA; it forms a tetrameric complex by recruiting its partner, GABPbeta (GABPb), which contains the transactivation domain. GABPa is a single copy gene in both the human and murine genomes and it is the only protein that can recruit GABPb to DNA. We cloned GABPa from a murine genomic BAC library and prepared a targeting vector in which exon 9 (which encodes the GABPa ets domain) was flanked by loxP (floxed) recombination sites. The targeting construct was electroporated into embryonic stem cells, homologous recombinants were implanted into pseudopregnant mice, heterozygous floxed GABPa mice were identified, and intercrossing yielded expected Mendelian ratios of wild type, heterozygous, and homozygous floxed GABPa mice. Breeding of heterozygous floxed GABPa mice to CMV-Cre mice (which express Cre recombinase in all tissues) yielded expected numbers of hemizygous mice (only one intact GABPa allele), but no nullizygous (GABPa−/−) mice among 64 pups; we conclude that homozygous deletion of GABPa causes an embryonic lethal defect. To determine the effect of GABPa deletion on myeloid cell development, we bred heterozygous and homozygous floxed mice to LysMCre mice, which express Cre only in myeloid cells. These mice had a normal complement of myeloid cells but, unexpectedly, PCR indicated that their Gr1+ myeloid cells retained an intact (undeleted) floxed GABPa allele. We detected similar numbers of in vitro myeloid colonies from bone marrow of wild type, heterozygous floxed, and homozygous floxed progeny of LysMCre matings. However, PCR of twenty individual in vitro colonies from homozygous floxed mice indicated that they all retained an intact floxed allele. Breeding of floxed GABPa/LysMCre mice with hemizygous mice indicated that retention of a floxed allele was not due to incomplete deletion by LysMCre; rather, it appears that only myeloid cells that retain an intact GABPa allele can survive to mature in vitro or in vivo. We prepared murine embryonic fibroblasts from homozygous floxed mice and efficiently deleted GABPa in vitro. We found striking abnormalities in proliferation and G1/S phase arrest. We used quantitative RT-PCR to identify mechanisms that account for the altered growth of GABPa null cells. We found dramatically reduced expression of known GABP target genes that regulate DNA synthesis and cell cycle that appear to account for the proliferative defect. We conclude that GABPa is required for growth and maturation of myeloid cells and we identified downstream targets that may account for their failure to proliferate and mature in vitro and in vivo.

Molecular Signature for Parthenolide-Induced Apoptosis in AML CD34+ Cells Reveals Targets for Improving Induction of Leukemia-Specific Cell Death.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2592-2592 ◽  
Author(s):  
Duane C. Hassane ◽  
Monica L. Guzman ◽  
Xiaojie Li ◽  
Cheryl Corbett ◽  
Craig T. Jordan
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Biological Effects ◽  
Molecular Signature ◽  
Initiation Factor ◽  
Binding Motif ◽  
Specific Cell ◽  
Normal Bone Marrow ◽  
Normal Bone ◽  
Induced Apoptosis

Abstract Previous studies have demonstrated that the plant-derived compound parthenolide (PTL) induces AML stem cell specific apoptosis while sparing normal counterparts. We have observed that PTL strongly inhibits NF-kappaB activity. Moreover, anti-oxidants such as N-acetylcysteine block the apoptotic process. However, the molecular events involved in the process are not completely understood. Given the demonstrated efficacy of PTL at targeting AML stem cells (AML-SCs), we hypothesized that a molecular signature that captures events invoked during PTL-induced apoptosis will provide a powerful tool for improving AML-SC targeting. To this end, we have conducted pharmacogenomic studies using primary CD34-enriched cells obtained from 12 randomly selected AML specimens. To capture dynamics of the PTL response, primary CD34+ AML were cultured in vitro and exposed to 5 micromolar PTL for either 1 hour or 6 hours. Labeled mRNA was hybridized to Affymetrix HG-U133plus2 chips. Differential expression of individual genes was assessed using moderated and permutation-based paired tests with false discovery rate control. Additionally, to capture pathway-level events possibly representing drugable targets, we employed functional, network, and robust geneset-based approaches to mine the data for coordinated changes in the expression of groups of biologically related genes, e.g., those controlled by a common transcription factor. By 1 hour post-treatment, we were able to detect the beginning stages of a cascade of responses leading to activation of antioxidant enzymes, cytoskeletal reorganization and downregulation of adhesion molecules, modulation of protein synthesis, proteasome remodeling, and modulation of cell cycle progression genes (q < 0.05). Transcription factor (TF) binding motif enrichment studies show that several of these processes are governed by the involvement of transcription factors such as Nrf2, NFkappaB, and interferon regulatory factor-1 (IRF-1) and their respective upstream controllers. The biological relevance of the array findings was directly validated in independent studies demonstrating heightened oxidative state and the associated nuclear translocation of Nrf2 and upregulation of its target heme oxygenase I. We further hypothesized that a number of genes activated immediately following PTL exposure are important for promoting AML survival and/or drug resistance and that such genes would represent potentially useful drug targets. This rationale resulted in our selection of the target, eukaryotic initiation factor 5 (eIF5). Our array-based analyses revealed that the eIF5 gene is upregulated within 1 h of PTL exposure and is further upregulated at 6 h. eIF5 activation is regulated by hypusination and can be inhibited by the anti-fungal agent, ciclopirox. Therefore, we tested the biological effects of 2.5 micromolar PTL alone or in combination with ciclopirox on independent primary AML specimens. Exposure to either drug alone had a negligible effect on primary normal bone marrow or AML cells after 16 h growth in suspension culture. However, the combination of both resulted in ~20% viability for AML relative to ~80% viability for normal bone marrow. Importantly, the combination of PTL and ciclopirox completely eradicated the colony forming ability of AML cells while only modestly affecting normal bone marrow. These studies support the utility of high-throughput genomic studies as a conduit for drug discovery.

Requirement of the E2F3 Transcription Factor for BCR/ABL Leukemogenesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 33-33
Author(s):  
Anna M. Eiring ◽  
Paolo Neviani ◽  
Ramasamy Santhanam ◽  
Joshua J. Oaks ◽  
Ji Suk Chang ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transcription Factor ◽  
Blast Crisis ◽  
Scid Mice ◽  
Myelogenous Leukemia ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Hnrnp A1 ◽  
Protein Levels ◽  
Abl Kinase

Abstract Several RNA binding proteins (RBPs) have been implicated in the progression of chronic myelogenous leukemia (CML) from the indolent chronic phase to the aggressively fatal blast crisis. In the latter phase, expression and function of specific RBPs are altered at transcriptional or post-translational levels by the increased constitutive kinase activity of the BCR/ABL oncoprotein, resulting in enhanced resistance to apoptotic stimuli, growth advantage and differentiation arrest of CD34+ CML blast crisis (CML-BC) progenitors. In the current study, we identified by RIP (RNA immunoprecipitation)-mediated microarray analysis that mRNA encoding the E2F3 transcription factor associates to the BCR/ABL-regulated RBP hnRNP A1. Moreover, RNA electrophoretic mobility shift and UV-crosslinking assays revealed that hnRNP A1 interacts with E2F3 mRNA through a binding site located in the 3’UTR of both human and mouse E2F3 mRNA. Accordingly, E2F3 protein levels were upregulated in BCR/ABL-transformed myeloid precursor cell lines compared to parental cells in a BCR/ABL-kinase- and hnRNP A1 shuttling-dependent manner. In fact, treatment of BCR/ABL-expressing myeloid precursors with the kinase inhibitor Imatinib (2mM, 24 hr) or introduction of a dominant-negative shuttling-deficient hnRNP A1 protein (NLS-A1) markedly reduced E2F3 protein and mRNA levels. Similarly, upregulation of BCR/ABL expression/activity in the doxycycline inducible TonB2.10 cell line resulted in increased E2F3 protein expression. BCR/ABL kinase-dependent induction of E2F3 protein levels was also detected in CML-BCCD34+ compared to CML-CPCD34+ progenitors from paired patient samples and to normal CD34+ bone marrow samples. Importantly, the in vitro clonogenic potential of primary mouse BCR/ABL+ lineage negative (Lin−) progenitors was markedly impaired in BCR/ABL+ E2F3−/− compared to BCR/ABL-transduced E2F3+/+ myeloid progenitors and upon shRNA-mediated downregulation of E2F3 expression (90% inhibition, P<0.001). Furthermore, subcutaneous injection of shE2F3-expressing BCR/ABL+ cells into SCID mice markedly impaired in vivo tumorigenesis (>80% reduction in tumor burden, P<0.01). Accordingly, BCR/ABL leukemogenesis was strongly inhibited in SCID mice intravenously injected with E2F3 shRNA-expressing 32D-BCR/ABL cells and in mice transplanted with BCR/ABL-transduced Lin− bone marrow cells from E2F3−/− mice. Specifically, we demonstrate that reduced or absent levels of E2F3 resulted in dramatically decreased numbers of circulating BCR/ABL+ cells as determined by nested RT-PCR at 4 weeks post-injection (P=0.0001), normal splenic architecture and bone marrow cellularity and the absence of infiltrating myeloid blasts into non-hematopoietic compartments (i.e. liver). By contrast, SCID mice transplanted with vector-transduced 32D-BCR/ABL cells or BCR/ABL+ E2F3+/+ Lin− BM progenitors showed signs of an overt acute leukemia-like process with blast infiltration of hematopoietic and non-hematopoietic organs. Altogether, these data outline the importance of E2F3 expression for BCR/ABL leukemogenesis and characterize a new potential therapeutic target for the treatment of patients with advanced phase CML.

Sign in / Sign up

Export Citation Format

Share Document