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Beatrice U. Mueller - One of the best experts on this subject based on the ideXlab platform.
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Inactivation of the p53-KLF4-CEBPA Axis in Acute Myeloid Leukemia.
Clinical cancer research : an official journal of the American Association for Cancer Research, 2015Co-Authors: Katja Seipel, Beatrice U. Mueller, Miguel A.t. Marques, Marie-ange Bozzini, Christina Meinken, Thomas PabstAbstract:Purpose: In acute myeloid leukemia (AML), the transcription factors CEBPA and KLF4 as well as the universal tumor suppressor p53 are frequently deregulated. Here, we investigated the extent of dysregulation, the molecular interactions and the mechanisms involved. Experimental Design: 110 AML patient samples were analyzed for protein levels of CEBPA, KLF4, p53 and p53 modulators. Regulation of CEBPA gene expression by KLF4 and p53 or by chemical p53 activators was characterized in AML cell lines. Results: We found that CEBPA gene transcription can be directly activated by p53 and KLF4 suggesting a p53-KLF4-CEBPA axis. In AML patient cells, we observed a prominent loss of p53 function and concomitant reduction of KLF4 and CEBPA protein levels. Assessment of cellular p53 modulator proteins indicated that p53 inactivation in leukemic cells correlated with elevated levels of the nuclear export protein XPO1/CRM1 and increase of the p53 inhibitors MDM2 and CUL9/PARC in the cytoplasm. Finally, restoring p53 function following treatment with cytotoxic chemotherapy compounds and p53 restoring non-genotoxic agents induced CEBPA gene expression, myeloid differentiation and cell cycle arrest in AML cells. Conclusions: The p53-KLF4-CEBPA axis is deregulated in AML, but can be functionally restored by conventional chemotherapy and novel p53 activating treatments.
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Protein disulfide isomerase blocks CEBPA translation and is up-regulated during the unfolded protein response in AML
Blood, 2011Co-Authors: Simon Haefliger, Beatrice U. Mueller, Julian Schardt, Nikolai A. Timchenko, Christiane Klebig, Kerstin Schaubitzer, Thomas PabstAbstract:Deregulation of the myeloid key transcription factor CEBPA is a common event in acute myeloid leukemia (AML). We previously reported that the chaperone calreticulin is activated in subgroups of AML patients and that calreticulin binds to the stem loop region of the CEBPA mRNA, thereby blocking CEBPA translation. In this study, we screened for additional CEBPA mRNA binding proteins and we identified protein disulfide isomerase (PDI), an endoplasmic reticulum (ER) resident protein, to bind to the CEBPA mRNA stem loop region. We found that forced PDI expression in myeloid leukemic cells in fact blocked CEBPA translation, but not transcription, whereas abolishing PDI function restored CEBPA protein. In addition, PDI protein displayed direct physical interaction with calreticulin. Induction of ER stress in leukemic HL60 and U937 cells activated PDI expression, thereby decreasing CEBPA protein levels. Finally, leukemic cells from 25.4% of all AML patients displayed activation of the unfolded protein response as a marker for ER stress, and these patients also expressed significantly higher PDI levels. Our results indicate a novel role of PDI as a member of the ER stress–associated complex mediating blocked CEBPA translation and thereby suppressing myeloid differentiation in AML patients with activated unfolded protein response (UPR).
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Deficient CEBPA DNA binding function in normal karyotype AML patients is associated with favorable prognosis
Blood, 2011Co-Authors: José Fos, Thomas Pabst, Vibor Petkovic, Daniel Ratschiller, Beatrice U. MuellerAbstract:CCAAT/enhancer binding protein-α (CEBPA) mutations in acute myeloid leukemia (AML) patients with a normal karyotype (NK) confer favorable prognosis, whereas NK-AML patients per se are of intermediate risk. This suggests that blocked CEBPA function characterizes NK-AML with favorable outcome. We determined the prognostic significance of CEBPA DNA binding function by enzyme-linked immunosorbent assay in 105 NK-AML patients. Suppressed CEBPA DNA binding was defined by 21 good-risk AML patients with inv(16) or t(8;21) (both abnormalities targeting CEBPA) and 8 NK-AML patients with dominant-negative CEBPA mutations. NK-AML patients with suppressed CEBPA function showed a better overall survival (P = .0231) and disease-free survival (P = .0069) than patients with conserved CEBPA function. Suppressed CEBPA DNA binding was an independent marker for better overall survival and disease-free survival in a multivariable analysis that included FLT3-ITD, NPM1 and CEBPA mutation status, white blood cell count, age and lactate dehydrogenase. These data indicate that suppressed CEBPA function is associated with favorable prognosis in NK-AML patients.
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The tumour-suppressive miR-29a/b1 cluster is regulated by CEBPA and blocked in human AML.
British journal of cancer, 2010Co-Authors: Marianne Eyholzer, Beatrice U. Mueller, Sabine Schmid, L Wilkens, Thomas PabstAbstract:CCAAT/enhancer-binding protein-α (CEBPA) is crucial for normal granulopoiesis and is frequently disrupted in acute myeloid leukaemia (AML). Increasing evidence suggests that CEBPA exerts its effects, in parts, by regulating specific microRNAs (miRNAs), as previously shown for miR-223. The aim of this study was to investigate the genome-wide pattern of miRNAs regulated by CEBPA in myeloid cells. In Kasumi-1 cells, conditionally expressing CEBPA, we assessed the expression of 470 human miRNAs by microarray analysis. We further investigated the microarray results by qRT-PCR, luciferase reporter assays, and chromatin immunoprecipitation assays. In all, 18 miRNAs were more than two-fold suppressed or induced after CEBPA restoration. Among these 18 miRNAs, we focused on CEBPA-mediated regulation of the tumour-suppressive miR-29b. We observed that miR-29b is suppressed in AML patients with impaired CEBPA function or loss of chromosome 7q. We found that CEBPA selectively regulates miR-29b expression on its miR-29a/b1 locus on chromosome 7q32.3, whereas miR-29b2/c on chromosome 1q32.2 is not affected. This study reports the activation of the tumour-suppressive miR-29b by the haematopoietic key transcription factor CEBPA. Our data provide a rationale for miR-29b suppression in AML patients with loss of chromosome 7q or CEBPA deficiency.
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the tumour suppressive mir 29a b1 cluster is regulated by CEBPA and blocked in human aml
British Journal of Cancer, 2010Co-Authors: Marianne Eyholzer, Beatrice U. Mueller, Sabine Schmid, L Wilkens, Thomas PabstAbstract:CCAAT/enhancer-binding protein-α (CEBPA) is crucial for normal granulopoiesis and is frequently disrupted in acute myeloid leukaemia (AML). Increasing evidence suggests that CEBPA exerts its effects, in parts, by regulating specific microRNAs (miRNAs), as previously shown for miR-223. The aim of this study was to investigate the genome-wide pattern of miRNAs regulated by CEBPA in myeloid cells. In Kasumi-1 cells, conditionally expressing CEBPA, we assessed the expression of 470 human miRNAs by microarray analysis. We further investigated the microarray results by qRT-PCR, luciferase reporter assays, and chromatin immunoprecipitation assays. In all, 18 miRNAs were more than two-fold suppressed or induced after CEBPA restoration. Among these 18 miRNAs, we focused on CEBPA-mediated regulation of the tumour-suppressive miR-29b. We observed that miR-29b is suppressed in AML patients with impaired CEBPA function or loss of chromosome 7q. We found that CEBPA selectively regulates miR-29b expression on its miR-29a/b1 locus on chromosome 7q32.3, whereas miR-29b2/c on chromosome 1q32.2 is not affected. This study reports the activation of the tumour-suppressive miR-29b by the haematopoietic key transcription factor CEBPA. Our data provide a rationale for miR-29b suppression in AML patients with loss of chromosome 7q or CEBPA deficiency.
Thomas Pabst - One of the best experts on this subject based on the ideXlab platform.
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Inactivation of the p53-KLF4-CEBPA Axis in Acute Myeloid Leukemia.
Clinical cancer research : an official journal of the American Association for Cancer Research, 2015Co-Authors: Katja Seipel, Beatrice U. Mueller, Miguel A.t. Marques, Marie-ange Bozzini, Christina Meinken, Thomas PabstAbstract:Purpose: In acute myeloid leukemia (AML), the transcription factors CEBPA and KLF4 as well as the universal tumor suppressor p53 are frequently deregulated. Here, we investigated the extent of dysregulation, the molecular interactions and the mechanisms involved. Experimental Design: 110 AML patient samples were analyzed for protein levels of CEBPA, KLF4, p53 and p53 modulators. Regulation of CEBPA gene expression by KLF4 and p53 or by chemical p53 activators was characterized in AML cell lines. Results: We found that CEBPA gene transcription can be directly activated by p53 and KLF4 suggesting a p53-KLF4-CEBPA axis. In AML patient cells, we observed a prominent loss of p53 function and concomitant reduction of KLF4 and CEBPA protein levels. Assessment of cellular p53 modulator proteins indicated that p53 inactivation in leukemic cells correlated with elevated levels of the nuclear export protein XPO1/CRM1 and increase of the p53 inhibitors MDM2 and CUL9/PARC in the cytoplasm. Finally, restoring p53 function following treatment with cytotoxic chemotherapy compounds and p53 restoring non-genotoxic agents induced CEBPA gene expression, myeloid differentiation and cell cycle arrest in AML cells. Conclusions: The p53-KLF4-CEBPA axis is deregulated in AML, but can be functionally restored by conventional chemotherapy and novel p53 activating treatments.
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Protein disulfide isomerase blocks CEBPA translation and is up-regulated during the unfolded protein response in AML
Blood, 2011Co-Authors: Simon Haefliger, Beatrice U. Mueller, Julian Schardt, Nikolai A. Timchenko, Christiane Klebig, Kerstin Schaubitzer, Thomas PabstAbstract:Deregulation of the myeloid key transcription factor CEBPA is a common event in acute myeloid leukemia (AML). We previously reported that the chaperone calreticulin is activated in subgroups of AML patients and that calreticulin binds to the stem loop region of the CEBPA mRNA, thereby blocking CEBPA translation. In this study, we screened for additional CEBPA mRNA binding proteins and we identified protein disulfide isomerase (PDI), an endoplasmic reticulum (ER) resident protein, to bind to the CEBPA mRNA stem loop region. We found that forced PDI expression in myeloid leukemic cells in fact blocked CEBPA translation, but not transcription, whereas abolishing PDI function restored CEBPA protein. In addition, PDI protein displayed direct physical interaction with calreticulin. Induction of ER stress in leukemic HL60 and U937 cells activated PDI expression, thereby decreasing CEBPA protein levels. Finally, leukemic cells from 25.4% of all AML patients displayed activation of the unfolded protein response as a marker for ER stress, and these patients also expressed significantly higher PDI levels. Our results indicate a novel role of PDI as a member of the ER stress–associated complex mediating blocked CEBPA translation and thereby suppressing myeloid differentiation in AML patients with activated unfolded protein response (UPR).
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Deficient CEBPA DNA binding function in normal karyotype AML patients is associated with favorable prognosis
Blood, 2011Co-Authors: José Fos, Thomas Pabst, Vibor Petkovic, Daniel Ratschiller, Beatrice U. MuellerAbstract:CCAAT/enhancer binding protein-α (CEBPA) mutations in acute myeloid leukemia (AML) patients with a normal karyotype (NK) confer favorable prognosis, whereas NK-AML patients per se are of intermediate risk. This suggests that blocked CEBPA function characterizes NK-AML with favorable outcome. We determined the prognostic significance of CEBPA DNA binding function by enzyme-linked immunosorbent assay in 105 NK-AML patients. Suppressed CEBPA DNA binding was defined by 21 good-risk AML patients with inv(16) or t(8;21) (both abnormalities targeting CEBPA) and 8 NK-AML patients with dominant-negative CEBPA mutations. NK-AML patients with suppressed CEBPA function showed a better overall survival (P = .0231) and disease-free survival (P = .0069) than patients with conserved CEBPA function. Suppressed CEBPA DNA binding was an independent marker for better overall survival and disease-free survival in a multivariable analysis that included FLT3-ITD, NPM1 and CEBPA mutation status, white blood cell count, age and lactate dehydrogenase. These data indicate that suppressed CEBPA function is associated with favorable prognosis in NK-AML patients.
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The tumour-suppressive miR-29a/b1 cluster is regulated by CEBPA and blocked in human AML.
British journal of cancer, 2010Co-Authors: Marianne Eyholzer, Beatrice U. Mueller, Sabine Schmid, L Wilkens, Thomas PabstAbstract:CCAAT/enhancer-binding protein-α (CEBPA) is crucial for normal granulopoiesis and is frequently disrupted in acute myeloid leukaemia (AML). Increasing evidence suggests that CEBPA exerts its effects, in parts, by regulating specific microRNAs (miRNAs), as previously shown for miR-223. The aim of this study was to investigate the genome-wide pattern of miRNAs regulated by CEBPA in myeloid cells. In Kasumi-1 cells, conditionally expressing CEBPA, we assessed the expression of 470 human miRNAs by microarray analysis. We further investigated the microarray results by qRT-PCR, luciferase reporter assays, and chromatin immunoprecipitation assays. In all, 18 miRNAs were more than two-fold suppressed or induced after CEBPA restoration. Among these 18 miRNAs, we focused on CEBPA-mediated regulation of the tumour-suppressive miR-29b. We observed that miR-29b is suppressed in AML patients with impaired CEBPA function or loss of chromosome 7q. We found that CEBPA selectively regulates miR-29b expression on its miR-29a/b1 locus on chromosome 7q32.3, whereas miR-29b2/c on chromosome 1q32.2 is not affected. This study reports the activation of the tumour-suppressive miR-29b by the haematopoietic key transcription factor CEBPA. Our data provide a rationale for miR-29b suppression in AML patients with loss of chromosome 7q or CEBPA deficiency.
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the tumour suppressive mir 29a b1 cluster is regulated by CEBPA and blocked in human aml
British Journal of Cancer, 2010Co-Authors: Marianne Eyholzer, Beatrice U. Mueller, Sabine Schmid, L Wilkens, Thomas PabstAbstract:CCAAT/enhancer-binding protein-α (CEBPA) is crucial for normal granulopoiesis and is frequently disrupted in acute myeloid leukaemia (AML). Increasing evidence suggests that CEBPA exerts its effects, in parts, by regulating specific microRNAs (miRNAs), as previously shown for miR-223. The aim of this study was to investigate the genome-wide pattern of miRNAs regulated by CEBPA in myeloid cells. In Kasumi-1 cells, conditionally expressing CEBPA, we assessed the expression of 470 human miRNAs by microarray analysis. We further investigated the microarray results by qRT-PCR, luciferase reporter assays, and chromatin immunoprecipitation assays. In all, 18 miRNAs were more than two-fold suppressed or induced after CEBPA restoration. Among these 18 miRNAs, we focused on CEBPA-mediated regulation of the tumour-suppressive miR-29b. We observed that miR-29b is suppressed in AML patients with impaired CEBPA function or loss of chromosome 7q. We found that CEBPA selectively regulates miR-29b expression on its miR-29a/b1 locus on chromosome 7q32.3, whereas miR-29b2/c on chromosome 1q32.2 is not affected. This study reports the activation of the tumour-suppressive miR-29b by the haematopoietic key transcription factor CEBPA. Our data provide a rationale for miR-29b suppression in AML patients with loss of chromosome 7q or CEBPA deficiency.
Masao Matsuoka - One of the best experts on this subject based on the ideXlab platform.
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A regulatory element in the 3′‐untranslated region of CEBPA is associated with myeloid/NK/T‐cell leukemia
European journal of haematology, 2020Co-Authors: Yukiko Kimura, Kenji Tokunaga, Eisaku Iwanaga, Kouta Iwanaga, Shinya Endo, Yoshitaka Inoue, Yousuke Nagahata, Kyoko Masuda, Hiroshi Kawamoto, Masao MatsuokaAbstract:OBJECTIVES CCAAT/enhancer-binding protein α (CEBPA) is an essential transcription factor for myeloid differentiation. Not only mutation of the CEBPA gene, but also promoter methylation, which results in silencing of CEBPA, contributes to the pathogenesis of acute myeloid leukemia (AML). We sought for another differentially methylated region (DMR) that associates with the CEBPA silencing and disease phenotype. METHODS Using databases, we identified a conserved DMR in the CEBPA 3'-untranslated region (UTR). RESULTS Methylation-specific PCR analysis of 231 AML cases showed that hypermethylation of the 3'-UTR was associated with AML that had a myeloid/NK/T-cell phenotype and downregulated CEBPA. Most of these cases were of an immature phenotype with CD7/CD56 positivity. These cases were significantly associated with lower hemoglobin levels than the others. Furthermore, we discovered that the CEBPA 3'-UTR DMR can enhance transcription from the CEBPA native promoter. In vitro experiments identified IKZF1-binding sites in the 3'-UTR that are responsible for this increased transcription of CEBPA. CONCLUSIONS These results indicate that the CEBPA 3'-UTR DMR is a novel regulatory element of CEBPA related to myeloid/NK/T-cell lineage leukemogenesis. Transcriptional regulation of CEBPA by IKZF1 may provide a clue for understanding the fate determination of myeloid vs. NK/T-lymphoid progenitors.
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a regulatory element in the 3 untranslated region of CEBPA is associated with myeloid nk t cell leukemia
European Journal of Haematology, 2020Co-Authors: Yukiko Kimura, Kenji Tokunaga, Eisaku Iwanaga, Kouta Iwanaga, Shinya Endo, Yoshitaka Inoue, Yousuke Nagahata, Kyoko Masuda, Hiroshi Kawamoto, Masao MatsuokaAbstract:OBJECTIVES CCAAT/enhancer-binding protein α (CEBPA) is an essential transcription factor for myeloid differentiation. Not only mutation of the CEBPA gene, but also promoter methylation, which results in silencing of CEBPA, contributes to the pathogenesis of acute myeloid leukemia (AML). We sought for another differentially methylated region (DMR) that associates with the CEBPA silencing and disease phenotype. METHODS Using databases, we identified a conserved DMR in the CEBPA 3'-untranslated region (UTR). RESULTS Methylation-specific PCR analysis of 231 AML cases showed that hypermethylation of the 3'-UTR was associated with AML that had a myeloid/NK/T-cell phenotype and downregulated CEBPA. Most of these cases were of an immature phenotype with CD7/CD56 positivity. These cases were significantly associated with lower hemoglobin levels than the others. Furthermore, we discovered that the CEBPA 3'-UTR DMR can enhance transcription from the CEBPA native promoter. In vitro experiments identified IKZF1-binding sites in the 3'-UTR that are responsible for this increased transcription of CEBPA. CONCLUSIONS These results indicate that the CEBPA 3'-UTR DMR is a novel regulatory element of CEBPA related to myeloid/NK/T-cell lineage leukemogenesis. Transcriptional regulation of CEBPA by IKZF1 may provide a clue for understanding the fate determination of myeloid vs. NK/T-lymphoid progenitors.
Alan D Friedman - One of the best experts on this subject based on the ideXlab platform.
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pathways relevant to aml pathogenesis targeting the hematopoietic specific CEBPA 37 kb enhancer
Blood, 2016Co-Authors: Stacy Cooper, Hong Guo, Justin Thomas, Jay L. Hess, Peter D. Aplan, Donald Small, Alan D FriedmanAbstract:C/EBPα mediates myeloid differentiation, and its reduced activity is central to myeloid transformation (Friedman Int. J. Hematol. 2015). The murine CEBPA gene contains a 450 bp, +37 kb element that acquires the enhancer-specific H3K4me1 and H3K27Ac histone marks as LT-HSC progress to GMP and directs hCD4 transgene expression to GMP and myeloid CFUs (Guo et al. Blood 2012; Guo et al. J. Leuk. Biol . 2014). Moreover, CRISPR/Cas9 mediated, biallelic replacement of the enhancer with a variant harboring mutations in its seven Ets sites reduces CEBPA RNA >10-fold in 32Dcl3 cells (Cooper et al. PLoS One 2015), and germline or Cre-mediated enhancer deletion leads to marked reduction in CEBPA RNA in marrow LSK, CMP, and GMP, with 3-fold reduction in GMP, CFU-G, and neutrophils and indefinite myeloid colony replating in IL-3, a preleukemic phenotype (Guo et al. PLoS One 2016; Avellino et al. Blood 2016). The enhancer contains four conserved RUNX1 cis elements that bind RUNX1 in gel shift or ChIP assays, Runx1 gene deletion reduces CEBPA RNA 5-fold in Lin - and 2-fold in GMP marrow cells, Runx1 cis element mutation reduces luciferase reporter activity 6-fold in 32Dcl3 myeloid cells (Guo et al. Blood 2012), and in AMLs with t(8;21) RUNX1-ETO interacts with the human CEBPA locus at the homologous +42 kb enhancer (Ptasinska et al. Leukemia 2012). Mutation of the CEBPA enhancer has not been seen in human AML cases, perhaps reflecting preference for upstream pathway alteration to suppress both alleles and additional genes. As a further example of a leukemic alteration affecting enhancer activity, elevated EVI1 is a high-risk feature in AML, and EVI1 binds and represses the +37 kb CEBPA enhancer (Wilson et al. J. Biol. Chem. 2016). We have characterized additional pathways whose modulation in AML may reduce CEBPA enhancer activity. Deletion of the Pu.1 -14 kb enhancer leads to AML in mice, and PU.1 binds the CEBPA enhancer in ChIP and gel shift; we now find that mutation of the one CEBPA enhancer Ets site that binds PU.1 reduces reporter activity 4-fold in 32Dcl3 cells, providing the first functional evidence that PU.1 regulates the enhancer. ~10% of human AMLs harbor mutant C/EBPα proteins, CEBPα binds the enhancer in ChIP and gel shift, and mutation of the two enhancer C/EBP elements reduces reporter activity; we have now used CRISPR/Cas9 to generate a 32Dcl3 cell line with the two enhancer C/EBPsites mutated in one allele and find 60% reduction in CEBPA RNA expression. FLT3/ITD is a constitutively activated mutant form of the receptor tyrosine kinase found in ~30% of AML cases and confers high risk. While FLT3/ITD predominantly provides proliferative signals, it may also contribute to impaired differentiation, as FLT3/ITD inhibition in AML cases leads to a neutrophilic differentiation syndrome. We previously demonstrated that FLT3/ITD reduces CEBPA RNA in 32Dcl3 cells and patient leukemic blasts and that the first generation FLT3/ITD inhibitor lestaurtinib increases CEBPA ; we now find that the effect on CEBPA RNA is also reversed in 32Dcl3 cells by the next generation inhibitor crenolanib, that FLT3/ITD reduces CEBPA enhancer H3K4me1 and H3K27Ac histone marks several-fold, and that both of these activating marks are also restored by crenolanib. FLT3/ITD signaling leads to C/EBPα serine phosphorylation to reduce its activity, which may account in part for the inhibitory effect of FLT3/ITD on CEBPA enhancer activity. Finally, NUP98-HOX fusion proteins contribute to a small percent of AML cases. Vav-NUP98/HOXD13 (NHD13) mice develop MDS and can progress to AML; we now find that CEBPA RNA is reduced 5-fold in Vav-NHD13 CMP and LSK, with 5-fold increased HoxA9 RNA, that Vav-NHD13:: CEBPA Enh-hCD4 compound heterozygous mice have reduced hCD4 expression in GMP and CMP, implicating a direct effect on enhancer activity, and that expression of NHD13 in 32Dcl3 subclones prevents G-CSF induction of CEBPA , MPO, and morphologic differentiation. These 32Dcl3 lines retain levels of Gcsfr equal to empty vector-transduced cells in IL-3 and manifest increased HoxA9 in response to G-CSF. Moreover, HoxA9 binds the CEBPA enhancer in ChIP using marrow-derived HoxA9/Meis1 myeloid lines. Potentially, NHD13 induces HoxA9, which then binds the CEBPA enhancer directly or via other factors to repress transcription. Identifying mechanisms that repress CEBPA hematopoietic enhancer activity in AML may identify approaches to induce differentiation. Disclosures No relevant conflicts of interest to declare.
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Pathways Relevant to AML Pathogenesis Targeting the Hematopoietic-Specific CEBPA +37 Kb Enhancer
Blood, 2016Co-Authors: Stacy Cooper, Hong Guo, Justin Thomas, Jay L. Hess, Peter D. Aplan, Donald Small, Alan D FriedmanAbstract:C/EBPα mediates myeloid differentiation, and its reduced activity is central to myeloid transformation (Friedman Int. J. Hematol. 2015). The murine CEBPA gene contains a 450 bp, +37 kb element that acquires the enhancer-specific H3K4me1 and H3K27Ac histone marks as LT-HSC progress to GMP and directs hCD4 transgene expression to GMP and myeloid CFUs (Guo et al. Blood 2012; Guo et al. J. Leuk. Biol . 2014). Moreover, CRISPR/Cas9 mediated, biallelic replacement of the enhancer with a variant harboring mutations in its seven Ets sites reduces CEBPA RNA >10-fold in 32Dcl3 cells (Cooper et al. PLoS One 2015), and germline or Cre-mediated enhancer deletion leads to marked reduction in CEBPA RNA in marrow LSK, CMP, and GMP, with 3-fold reduction in GMP, CFU-G, and neutrophils and indefinite myeloid colony replating in IL-3, a preleukemic phenotype (Guo et al. PLoS One 2016; Avellino et al. Blood 2016). The enhancer contains four conserved RUNX1 cis elements that bind RUNX1 in gel shift or ChIP assays, Runx1 gene deletion reduces CEBPA RNA 5-fold in Lin - and 2-fold in GMP marrow cells, Runx1 cis element mutation reduces luciferase reporter activity 6-fold in 32Dcl3 myeloid cells (Guo et al. Blood 2012), and in AMLs with t(8;21) RUNX1-ETO interacts with the human CEBPA locus at the homologous +42 kb enhancer (Ptasinska et al. Leukemia 2012). Mutation of the CEBPA enhancer has not been seen in human AML cases, perhaps reflecting preference for upstream pathway alteration to suppress both alleles and additional genes. As a further example of a leukemic alteration affecting enhancer activity, elevated EVI1 is a high-risk feature in AML, and EVI1 binds and represses the +37 kb CEBPA enhancer (Wilson et al. J. Biol. Chem. 2016). We have characterized additional pathways whose modulation in AML may reduce CEBPA enhancer activity. Deletion of the Pu.1 -14 kb enhancer leads to AML in mice, and PU.1 binds the CEBPA enhancer in ChIP and gel shift; we now find that mutation of the one CEBPA enhancer Ets site that binds PU.1 reduces reporter activity 4-fold in 32Dcl3 cells, providing the first functional evidence that PU.1 regulates the enhancer. ~10% of human AMLs harbor mutant C/EBPα proteins, CEBPα binds the enhancer in ChIP and gel shift, and mutation of the two enhancer C/EBP elements reduces reporter activity; we have now used CRISPR/Cas9 to generate a 32Dcl3 cell line with the two enhancer C/EBPsites mutated in one allele and find 60% reduction in CEBPA RNA expression. FLT3/ITD is a constitutively activated mutant form of the receptor tyrosine kinase found in ~30% of AML cases and confers high risk. While FLT3/ITD predominantly provides proliferative signals, it may also contribute to impaired differentiation, as FLT3/ITD inhibition in AML cases leads to a neutrophilic differentiation syndrome. We previously demonstrated that FLT3/ITD reduces CEBPA RNA in 32Dcl3 cells and patient leukemic blasts and that the first generation FLT3/ITD inhibitor lestaurtinib increases CEBPA ; we now find that the effect on CEBPA RNA is also reversed in 32Dcl3 cells by the next generation inhibitor crenolanib, that FLT3/ITD reduces CEBPA enhancer H3K4me1 and H3K27Ac histone marks several-fold, and that both of these activating marks are also restored by crenolanib. FLT3/ITD signaling leads to C/EBPα serine phosphorylation to reduce its activity, which may account in part for the inhibitory effect of FLT3/ITD on CEBPA enhancer activity. Finally, NUP98-HOX fusion proteins contribute to a small percent of AML cases. Vav-NUP98/HOXD13 (NHD13) mice develop MDS and can progress to AML; we now find that CEBPA RNA is reduced 5-fold in Vav-NHD13 CMP and LSK, with 5-fold increased HoxA9 RNA, that Vav-NHD13:: CEBPA Enh-hCD4 compound heterozygous mice have reduced hCD4 expression in GMP and CMP, implicating a direct effect on enhancer activity, and that expression of NHD13 in 32Dcl3 subclones prevents G-CSF induction of CEBPA , MPO, and morphologic differentiation. These 32Dcl3 lines retain levels of Gcsfr equal to empty vector-transduced cells in IL-3 and manifest increased HoxA9 in response to G-CSF. Moreover, HoxA9 binds the CEBPA enhancer in ChIP using marrow-derived HoxA9/Meis1 myeloid lines. Potentially, NHD13 induces HoxA9, which then binds the CEBPA enhancer directly or via other factors to repress transcription. Identifying mechanisms that repress CEBPA hematopoietic enhancer activity in AML may identify approaches to induce differentiation. Disclosures No relevant conflicts of interest to declare.
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granulopoiesis requires increased c ebpα compared to monopoiesis correlated with elevated CEBPA in immature g csf receptor versus m csf receptor expressing cells
PLOS ONE, 2014Co-Authors: Sun Hwa Hong, Hong Guo, Gabriel Ghiaur, Alan D FriedmanAbstract:C/EBPα is required for the formation of granulocyte-monocyte progenitors; however, its role in subsequent myeloid lineage specification remains uncertain. Transduction of murine marrow with either of two CEBPA shRNAs markedly increases monocyte and reduces granulocyte colonies in methylcellulose or the monocyte to neutrophil ratio in liquid culture. Similar findings were found after marrow shRNA transduction and transplantation and with CEBPA knockdown in human marrow CD34+ cells. These results apparently reflect altered myeloid lineage specification, as similar knockdown allowed nearly complete 32Dcl3 granulocytic maturation. CEBPA knockdown also generated lineage-negative blasts with increased colony replating capacity but unchanged cell cycle parameters, likely reflecting complete differentiation block. The shRNA having the greatest effect on lineage skewing reduced CEBPA 3-fold in differentiating cells but 6-fold in accumulating blasts. Indicating that CEBPA is the relevant shRNA target, shRNA-resistant C/EBPα-ER rescued marrow myelopoiesis. CEBPA knockdown in murine marrow cells also increased in vitro erythropoiesis, perhaps reflecting 1.6-fold reduction in PU.1 leading to GATA-1 derepression. Global gene expression analysis of lineage-negative blasts that accumulate after CEBPA knockdown demonstrated reduction in Cebpe and Gfi1, known transcriptional regulators of granulopoiesis, and also reduced Ets1 and Klf5. Populations enriched for immature granulocyte or monocyte progenitor/precursors were isolated by sorting Lin−Sca-1−c-Kit+ cells into GCSFR+MCSFR− or GCSFR−MCSFR+ subsets. CEBPA, Cebpe, Gfi1, Ets1, and Klf5 RNAs were increased in the c-Kit+GCSFR+ and Klf4 and Irf8 in the c-Kit+MCSFR+ populations, with PU.1 levels similar in both. In summary, higher levels of C/EBPα are required for granulocyte and lower levels for monocyte lineage specification, and this myeloid bifurcation may be facilitated by increased CEBPA gene expression in granulocyte compared with monocyte progenitors.
Yukiko Kimura - One of the best experts on this subject based on the ideXlab platform.
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A regulatory element in the 3′‐untranslated region of CEBPA is associated with myeloid/NK/T‐cell leukemia
European journal of haematology, 2020Co-Authors: Yukiko Kimura, Kenji Tokunaga, Eisaku Iwanaga, Kouta Iwanaga, Shinya Endo, Yoshitaka Inoue, Yousuke Nagahata, Kyoko Masuda, Hiroshi Kawamoto, Masao MatsuokaAbstract:OBJECTIVES CCAAT/enhancer-binding protein α (CEBPA) is an essential transcription factor for myeloid differentiation. Not only mutation of the CEBPA gene, but also promoter methylation, which results in silencing of CEBPA, contributes to the pathogenesis of acute myeloid leukemia (AML). We sought for another differentially methylated region (DMR) that associates with the CEBPA silencing and disease phenotype. METHODS Using databases, we identified a conserved DMR in the CEBPA 3'-untranslated region (UTR). RESULTS Methylation-specific PCR analysis of 231 AML cases showed that hypermethylation of the 3'-UTR was associated with AML that had a myeloid/NK/T-cell phenotype and downregulated CEBPA. Most of these cases were of an immature phenotype with CD7/CD56 positivity. These cases were significantly associated with lower hemoglobin levels than the others. Furthermore, we discovered that the CEBPA 3'-UTR DMR can enhance transcription from the CEBPA native promoter. In vitro experiments identified IKZF1-binding sites in the 3'-UTR that are responsible for this increased transcription of CEBPA. CONCLUSIONS These results indicate that the CEBPA 3'-UTR DMR is a novel regulatory element of CEBPA related to myeloid/NK/T-cell lineage leukemogenesis. Transcriptional regulation of CEBPA by IKZF1 may provide a clue for understanding the fate determination of myeloid vs. NK/T-lymphoid progenitors.
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a regulatory element in the 3 untranslated region of CEBPA is associated with myeloid nk t cell leukemia
European Journal of Haematology, 2020Co-Authors: Yukiko Kimura, Kenji Tokunaga, Eisaku Iwanaga, Kouta Iwanaga, Shinya Endo, Yoshitaka Inoue, Yousuke Nagahata, Kyoko Masuda, Hiroshi Kawamoto, Masao MatsuokaAbstract:OBJECTIVES CCAAT/enhancer-binding protein α (CEBPA) is an essential transcription factor for myeloid differentiation. Not only mutation of the CEBPA gene, but also promoter methylation, which results in silencing of CEBPA, contributes to the pathogenesis of acute myeloid leukemia (AML). We sought for another differentially methylated region (DMR) that associates with the CEBPA silencing and disease phenotype. METHODS Using databases, we identified a conserved DMR in the CEBPA 3'-untranslated region (UTR). RESULTS Methylation-specific PCR analysis of 231 AML cases showed that hypermethylation of the 3'-UTR was associated with AML that had a myeloid/NK/T-cell phenotype and downregulated CEBPA. Most of these cases were of an immature phenotype with CD7/CD56 positivity. These cases were significantly associated with lower hemoglobin levels than the others. Furthermore, we discovered that the CEBPA 3'-UTR DMR can enhance transcription from the CEBPA native promoter. In vitro experiments identified IKZF1-binding sites in the 3'-UTR that are responsible for this increased transcription of CEBPA. CONCLUSIONS These results indicate that the CEBPA 3'-UTR DMR is a novel regulatory element of CEBPA related to myeloid/NK/T-cell lineage leukemogenesis. Transcriptional regulation of CEBPA by IKZF1 may provide a clue for understanding the fate determination of myeloid vs. NK/T-lymphoid progenitors.