RUNX1/EVI1 That Blocks Myeloid Differentiation Inhibits C/EBPα Function.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4161-4161
Author(s):  
Katsuya Tokita ◽  
Kazuhiro Maki ◽  
Kinuko Mitani
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Dominant Negative ◽  
Myelogenous Leukemia ◽  
Dominant Negative Effect ◽  
Granulocytic Differentiation ◽  
Leukemic Transformation ◽  
Hematopoietic Stem ◽  
Differentiation Block ◽  
Myeloid Progenitors

Abstract RUNX1/EVI1 chimeric transcription factor produced by t(3;21) causes leukemic transformation in hematopoietic stem cell tumors such as chronic myelogenous leukemia (CML) in blastic crisis and myelodysplastic syndrome (MDS) in leukemic transformation, possibly through differentiation block of malignant myeloid progenitors. We have recently reported that Runx1/EVI1 knock-in heterozygous mice show defective hematopoiesis in the fetal liver similar to Runx1 knock-out mice, but possess dysplastic hematopoietic progenitors with high self-renewal capacity. Notably, Runx1/EVI1 knock-in chimeric mice developed acute megakaryoblastic leukemia. The molecular characterization of RUNX1/EVI1 points two major functions; one is dominant-suppressive function over wild-type RUNX1 and the other is EVI1’s own function; blockade of TGFb-mediated signal, inhibition of JNK and stimulation of AP-1 activity. C/EBPa is a key transcriptional regulator that induces the granulocytic differentiation of myeloid progenitors and several lines of evidence suggest that disturbance in C/EBPa signaling is one of the major molecular events in myeloid malignancies. In this study, we investigated whether RUNX1/EVI1 affects the expression and function of C/EBPa. We introduced RUNX1/EVI1 cDNA into LG-3 cells that differentiate along the myeloid lineage upon granulocyte colony-stimulating factor exposure, and confirmed that RUNX1/EVI1 suppressed the differentiation. To further investigate the molecular mechanisms of RUNX1/EVI1-mediated differentiation block, we analyzed RUNX1/EVI1’s effect on the functions of C/EBPa. RUNX1/EVI1 was found to associate with C/EBPa. By using the reporter assay with the CEBPA promoter, we observed a dominant-negative effect of RUNX1/EVI1 over C/EBPa-mediated transcriptional activation via the CtBP-binding site in the EVI1 portion. In the gel-shift assay, RUNX1/EVI1 down-regulated DNA-binding activity of C/EBPa. Therefore, recruitment of histone deacetylase via CtBP and disruption of DNA binding could be likely scenarios for the RUNX1/EVI1-induced dominant repression on C/EBPa. Importantly, co-expression of C/EBPa restored differentiation ability of the RUNX1/EVI1-expressing LG-3 cells. All these data argue that inhibition of C/EBPa function may be causatively related to the RUNX1/EVI1’s leukemogenic potential.

scholarly journals RUNX1 mutations enhance self-renewal and block granulocytic differentiation in human in vitro models and primary AMLs

2019 ◽  
Vol 3 (3) ◽  
pp. 320-332 ◽  
Author(s):  
Mylène Gerritsen ◽  
Guoqiang Yi ◽  
Esther Tijchon ◽  
Jorren Kuster ◽  
Jan Jacob Schuringa ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Target Genes ◽  
Proliferative Potential ◽  
Cell Of Origin ◽  
Granulocytic Differentiation ◽  
Leukemic Transformation ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract To unravel molecular mechanisms by which Runt-related transcription factor 1 (RUNX1) mutations contribute to leukemic transformation, we introduced the RUNX1-S291fs300X mutation in human CD34+ stem/progenitor cells and in human induced pluripotent stem cells (iPSCs). In both models, RUNX1mut overexpression strongly impaired myeloid commitment. Instead, self-renewal was enhanced, as shown, by increased long-term culture-initiating cell frequencies and enhanced colony-forming cell replating capacity. Long-term suspension cultures with RUNX1mut-transduced cord blood (CB) CD34+ cells continued for more than 100 days, during which the cells displayed an immature granulocyte-macrophage progenitor-like CD34+/CD123+/CD45RA+ phenotype. The CD34+/CD38− hematopoietic stem cell (HSC) population most likely acted as cell of origin, as HSCs provided the best long-term proliferative potential on overexpression of RUNX1mut. CEBPA expression was reduced in RUNX1mut cells, and reexpression of CEBPA partly restored differentiation. RNA-seq analysis on CB/iPSC systems and on primary patient samples confirmed that RUNX1 mutations induce a myeloid differentiation block, and that a common set of RUNX1mut-upregulated target genes was strongly enriched for gene ontology terms associated with nucleosome assembly and chromatin structure. Interestingly, in comparison with AML1-ETO binding in acute myeloid leukemias (AMLs), we found significantly distinct genomic distribution and differential expression for RUNX1mut of genes such as TCF4, MEIS1, and HMGA2 that may potentially contribute to the underlying difference in clinical outcomes between RUNX1mut and AML1-ETO patients. In conclusion, RUNX1mut appears to induce a specific transcriptional program that contributes to leukemic transformation.

scholarly journals Dominant Negative Mutants Implicate STAT5 in Myeloid Cell Proliferation and Neutrophil Differentiation

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4154-4166 ◽  
Author(s):  
Robert L. Ilaria ◽  
Robert G. Hawley ◽  
Richard A. Van Etten
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Dominant Negative ◽  
Dominant Negative Effect ◽  
Cytokine Signaling ◽  
Transactivation Domain ◽  
Negative Effects ◽  
Murine Bone Marrow ◽  
Negative Effect

Abstract STAT5 is a member of the signal transducers and activation of transcription (STAT) family of latent transcription factors activated in a variety of cytokine signaling pathways. We introduced alanine substitution mutations in highly conserved regions of murine STAT5A and studied the mutants for dimerization, DNA binding, transactivation, and dominant negative effects on erythropoietin-induced STAT5-dependent transcriptional activation. The mutations included two near the amino-terminus (W255KR→AAA and R290QQ→AAA), two in the DNA-binding domain (E437E→AA and V466VV→AAA), and a carboxy-terminal truncation of STAT5A (STAT5A/▵53C) analogous to a naturally occurring isoform of rat STAT5B. All of the STAT mutant proteins were tyrosine phosphorylated by JAK2 and heterodimerized with STAT5B except for the WKR mutant, suggesting an important role for this region in STAT5 for stabilizing dimerization. The WKR, EE, and VVV mutants had no detectable DNA-binding activity, and the WKR and VVV mutants, but not EE, were defective in transcriptional induction. The VVV mutant had a moderate dominant negative effect on erythropoietin-induced STAT5 transcriptional activation, which was likely due to the formation of heterodimers that are defective in DNA binding. Interestingly, the WKR mutant had a potent dominant negative effect, comparable to the transactivation domain deletion mutant, ▵53C. Stable expression of either the WKR or ▵53C STAT5 mutants in the murine myeloid cytokine-dependent cell line 32D inhibited both interleukin-3–dependent proliferation and granulocyte colony-stimulating factor (G-CSF)–dependent differentiation, without induction of apoptosis. Expression of these mutants in primary murine bone marrow inhibited G-CSF–dependent granulocyte colony formation in vitro. These results demonstrate that mutations in distinct regions of STAT5 exert dominant negative effects on cytokine signaling, likely through different mechanisms, and suggest a role for STAT5 in proliferation and differentiation of myeloid cells.

Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis

Blood ◽  
2008 ◽  
Vol 112 (4) ◽  
pp. 1056-1067 ◽  
Author(s):  
Mira T. Kassouf ◽  
Hedia Chagraoui ◽  
Paresh Vyas ◽  
Catherine Porcher
Keyword(s):  
Dna Binding ◽  
Molecular Mechanisms ◽  
Binding Activity ◽  
Hematopoietic Stem ◽  
Helix Loop Helix ◽  
Experimental Systems ◽  
Dna Binding Activity ◽  
Independent Functions

Abstract Dissecting the molecular mechanisms used by developmental regulators is essential to understand tissue specification/differentiation. SCL/TAL-1 is a basic helix-loop-helix transcription factor absolutely critical for hematopoietic stem/progenitor cell specification and lineage maturation. Using in vitro and forced expression experimental systems, we previously suggested that SCL might have DNA-binding–independent functions. Here, to assess the requirements for SCL DNA-binding activity in vivo, we examined hematopoietic development in mice carrying a germline DNA-binding mutation. Remarkably, in contrast to complete absence of hematopoiesis and early lethality in scl-null embryos, specification of hematopoietic cells occurred in homozygous mutant embryos, indicating that direct DNA binding is dispensable for this process. Lethality was forestalled to later in development, although some mice survived to adulthood. Anemia was documented throughout development and in adulthood. Cellular and molecular studies showed requirements for SCL direct DNA binding in red cell maturation and indicated that scl expression is positively autoregulated in terminally differentiating erythroid cells. Thus, different mechanisms of SCL's action predominate depending on the developmental/cellular context: indirect DNA binding activities and/or sequestration of other nuclear regulators are sufficient in specification processes, whereas direct DNA binding functions with transcriptional autoregulation are critically required in terminal maturation processes.

scholarly journals Block of C/EBPα function by phosphorylation in acute myeloid leukemia with FLT3 activating mutations

2006 ◽  
Vol 203 (2) ◽  
pp. 371-381 ◽  
Author(s):  
Hanna S. Radomska ◽  
Daniela S. Bassères ◽  
Rui Zheng ◽  
Pu Zhang ◽  
Tajhal Dayaram ◽  
...  
Keyword(s):  
Myeloid Leukemia ◽  
Myelogenous Leukemia ◽  
Granulocytic Differentiation ◽  
Extracellular Signal Regulated Kinase ◽  
Activating Mutations ◽  
Flt3 Inhibitor ◽  
Extracellular Signal ◽  
Enhancer Binding Protein ◽  
Acute Myelogenous ◽  
Differentiation Block

Mutations constitutively activating FLT3 kinase are detected in ∼30% of acute myelogenous leukemia (AML) patients and affect downstream pathways such as extracellular signal–regulated kinase (ERK)1/2. We found that activation of FLT3 in human AML inhibits CCAAT/enhancer binding protein α (C/EBPα) function by ERK1/2-mediated phosphorylation, which may explain the differentiation block of leukemic blasts. In MV4;11 cells, pharmacological inhibition of either FLT3 or MEK1 leads to granulocytic differentiation. Differentiation of MV4;11 cells was also observed when C/EBPα mutated at serine 21 to alanine (S21A) was stably expressed. In contrast, there was no effect when serine 21 was mutated to aspartate (S21D), which mimics phosphorylation of C/EBPα. Thus, our results suggest that therapies targeting the MEK/ERK cascade or development of protein therapies based on transduction of constitutively active C/EBPα may prove effective in treatment of FLT3 mutant leukemias resistant to the FLT3 inhibitor therapies.

Abstract 080: Eya4 Induces Hypertrophy Via Regulation Of p27kip1

2013 ◽  
Vol 113 (suppl_1) ◽  
Author(s):  
Tatjana Williams ◽  
Moritz Hundertmark ◽  
Peter Nordbeck ◽  
Sabine Voll ◽  
Melanie Muehlfelder ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Cardiac Hypertrophy ◽  
Molecular Mechanisms ◽  
Pressure Overload ◽  
Dominant Negative ◽  
Heart Weight ◽  
Dominant Negative Effect ◽  
Mutant Form ◽  
Cardiac Phenotype ◽  
Truncating Mutation

Introduction: E193, a truncating mutation in the transcription cofactor Eyes absent 4 (Eya4) causes hearing impairment followed by heart failure. Here we identified the Eya4 dependent molecular mechanisms leading to the cardiac phenotype in the E193 mutation. Methods and Results: First we showed in vitro that the cyclin-dependent kinase inhibitor protein p27kip1 is a direct target of Eya4/Six1 and is suppressed upon Eya4 overexpression, whereas E193 has a dominant negative effect, releasing Eya4 mediated suppression of p27. We next generated transgenic mice with cardiac specific constitutive overexpression of full-length Eya4 or the mutant form E193. While E193 transgenic mice developed age-dependent DCM, Eya4 mice displayed cardiac hypertrophy already under basal conditions as judged by increases in heart weight and cardiomyocyte cross-sectional areas along with increases in myocardial dimension and mass. These two distinct cardiac phenotypes were even more aggravated upon pressure overload suggesting Eya4 is a regulator of cardiac hypertrophy. We also observed that the activity of Casein Kinase 2-α and the phosphorylation status of HDAC2 were significantly upregulated in the Eya4 transgenic mice, while they were significantly reduced in E193 mice, under baseline conditions and pressure overload. We were also able to identify a new human mutation (E215) with a phenotype comparable to the one seen in E193 patients. Conclusion: Our results implicate that Eya4/Six1 regulates cardiac hypertrophic reactions via p27/CK2-α/HDAC2 and indicate that truncating mutations in Eya4 interfere with this newly established signalling pathway.

scholarly journals Leukemia-Related Transcription Factor TEL Is Negatively Regulated through Extracellular Signal-Regulated Kinase-Induced Phosphorylation

2004 ◽  
Vol 24 (8) ◽  
pp. 3227-3237 ◽  
Author(s):  
Kazuhiro Maki ◽  
Honoka Arai ◽  
Kazuo Waga ◽  
Ko Sasaki ◽  
Fumihiko Nakamura ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Dominant Negative Effect ◽  
Phosphorylation Sites ◽  
3T3 Cells ◽  
Nih 3T3 ◽  
Nih 3T3 Cells

ABSTRACT TEL is an ETS family transcription factor that possesses multiple putative mitogen-activated protein kinase phosphorylation sites. We here describe the functional regulation of TEL via ERK pathways. Overexpressed TEL becomes phosphorylated in vivo by activated ERK. TEL is also directly phosphorylated in vitro by ERK. The inducible phosphorylation sites are Ser213 and Ser257. TEL binds to a common docking domain in ERK. In vivo ERK-dependent phosphorylation reduces trans-repressional and DNA-binding abilities of TEL for ETS-binding sites. A mutant carrying substituted glutamates on both Ser213 and Ser257 functionally mimics hyperphosphorylated TEL and also shows a dominant-negative effect on TEL-induced transcriptional suppression. Losing DNA-binding affinity through phosphorylation but heterodimerizing with unmodified TEL could be an underlying mechanism. Moreover, the glutamate mutant dominantly interferes with TEL-induced erythroid differentiation in MEL cells and growth suppression in NIH 3T3 cells. Finally, endogenous TEL is dephosphorylated in parallel with ERK inactivation in differentiating MEL cells and is phosphorylated through ERK activation in Ras-transformed NIH 3T3 cells. These data indicate that TEL is a constituent downstream of ERK in signal transduction systems and is physiologically regulated by ERK in molecular and biological features.

The AML1-ETO fusion protein promotes the expansion of human hematopoietic stem cells

Blood ◽  
2002 ◽  
Vol 99 (1) ◽  
pp. 15-23 ◽  
Author(s):  
James C. Mulloy ◽  
Jörg Cammenga ◽  
Karen L. MacKenzie ◽  
Francisco J. Berguido ◽  
Malcolm A. S. Moore ◽  
...  
Keyword(s):  
Stem Cells ◽  
Fusion Protein ◽  
Progenitor Cells ◽  
Protein Interactions ◽  
Dominant Negative ◽  
Myelogenous Leukemia ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

The acute myelogenous leukemia–1 (AML1)–ETO fusion protein is generated by the t(8;21), which is found in 40% of AMLs of the French-American-British M2 subtype. AML1-ETO interferes with the function of the AML1 (RUNX1, CBFA2) transcription factor in a dominant-negative fashion and represses transcription by binding its consensus DNA–binding site and via protein-protein interactions with other transcription factors. AML1 activity is critical for the development of definitive hematopoiesis, and haploinsufficiency of AML1 has been linked to a propensity to develop AML. Murine experiments suggest that AML1-ETO expression may not be sufficient for leukemogenesis; however, like the BCR-ABL isoforms, the cellular background in which these fusion proteins are expressed may be critical to the phenotype observed. Retroviral gene transfer was used to examine the effect of AML1-ETO on the in vitro behavior of human hematopoietic stem and progenitor cells. Following transduction of CD34+ cells, stem and progenitor cells were quantified in clonogenic assays, cytokine-driven expansion cultures, and long-term stromal cocultures. Expression of AML1-ETO inhibited colony formation by committed progenitors, but enhanced the growth of stem cells (cobblestone area-forming cells), resulting in a profound survival advantage of transduced over nontransduced cells. AML1-ETO–expressing cells retained progenitor activity and continued to express CD34 throughout the 5-week long-term culture. Thus, AML1-ETO enhances the self-renewal of pluripotent stem cells, the physiological target of many acute myeloid leukemias.

Disordered Expression of HIPK Family Members in MDS and AML.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4311-4311
Author(s):  
Chunhong Gu ◽  
Helen J. Zheng ◽  
Lap Shu Chan ◽  
Joseph Brandwein ◽  
Suzanne Kamel-Reid ◽  
...  
Keyword(s):  
Protein Kinases ◽  
Drug Exposure ◽  
Dominant Negative ◽  
Myelogenous Leukemia ◽  
Inhibition Rate ◽  
Monocytic Differentiation ◽  
Granulocytic Differentiation ◽  
Empty Vector ◽  
Rate Of Growth ◽  
Significant Difference

Abstract The homeodomain interacting protein kinases (HIPKs) are a group of three nuclear serine/threonine protein kinases originally identified as corepressors for various homeodomain-containing transcription factors. The physiological roles of these kinases are largely unknown, though HIPK1 was reported to be a p53-binding protein and might play a role in tumorgenesis. HIPK2 promotes apoptosis via pathways including p53, transcriptional corepressor CtBP, and Wnt-1 signalling. HIPK2 maps to chromosome 7q32-34 and resides within one of the minimal deleted regions of 7q in acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS). Finally, HIPK3 was first identified as a putative multidrug resistant protein and was reported to be elevated by JNK in prostate cancer cells, thus contributing to increased resistance to Fas-mediated apoptosis. We have utilized Real-Time PCR to investigate the expression of HIPKs in bone marrow from patients with AML (19 patients), MDS (15 patients), chronic myelomonocytic leukemia (CMML, 5 patients), and 11 normal CD34+ mobilized peripheral blood progenitors. The mRNA expression of HIPK1 was higher in AML (P=0.00002), CMML (P=0.02), and MDS (P=0.029) compared to normal. There was also a significant difference in HIPK1 expression between AML and MDS, higher in AML (P=0.009). HIPK2 expression was variable in AML, MDS and CMML patients, while in normal CD34+ cells its expression was consistently low. HIPK3 expression was high in AML compared to normal (P=0.019), but in CMML and MDS its expression was similar to normal. We utilized the AML cell lines HL-60, NB4, and U937 to analyze changes in HIPKs expression during myeloid differentiation. HIPK2 expression significantly increased during ATRA-induced granulocytic differentiation of HL-60 and NB4 cells, but no significant change was seen in Vitamin D3-induced monocytic differentiation of U937 cells. Subgroup analysis of public domain microarray data( Valk et al, NEJM, 2004) indicates that HIPK2 expression is lower in AML patients with -7/-7q compared to other AML patients. We hypothesize that loss of HIPK2 expression contributes to the chemoresistance of -7/-7q AML by impairing normal apoptosis. As an initial test of this hypothesis we have studied the effect of forced expression of HIPK2 on sensitivity of COS-7 cells to daunorubicin, cytarabine and etoposide. COS-7 cells transfected with HIPK2 are more sensitive to daunorubicin with the inhibition rate of growth of 28.31% at 1.6 μg/ml daunorubicin after 48h incubation, while the inhibition rate of COS-7 cell transfected with dominant negative (DN)-HIPK2 (18.80%) was similar to cells transfected with an empty vector (14.96%) and untransfected cells (15.12%) under the same condition of drug exposure. Similar results were found for cytarabine, where the inhibition rate of growth was 17.40% (HIPK2), 9.00% (DN-HIPK2), 6.41% (empty vector), 5.77% (untransfected control). No change of sensitivity was found for etoposide. Conclusions: The anti-apoptotic kinases HIPK1 and HIPK3 are highly expressed in AML, and might have roles in leukemogenesis. HIPK2 shows heterogeneous expression in AML but is underexpressed in AML patients with -7q, and mediates sensitivity to cytarabine- and daunorubicin-induced apoptosis. The possible roles of HIPKs in normal and leukemic hematopoiesis require further investiagtion.

BCR/ABL-Mediated Myeloid Expansion Is Promoted by C/EBPβ, a Regulator of Emergency Granulopoiesis,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3747-3747
Author(s):  
Yoshihiro Hayashi ◽  
Hideyo Hirai ◽  
Hisayuki Yao ◽  
Satoshi Yoshioka ◽  
Sakiko Satake ◽  
...  
Keyword(s):  
Median Survival ◽  
Molecular Mechanisms ◽  
Conflicts Of Interest ◽  
Flow Cytometric Analysis ◽  
Myeloid Cells ◽  
Expression Level ◽  
Hematopoietic Stem ◽  
Emergency Situations ◽  
Proliferation And Differentiation ◽  
Myeloid Progenitors

Abstract Abstract 3747 Chronic phase chronic myeloid leukemia (CP-CML) is characterized by the increase of myeloid cells in the peripheral blood (PB) and bone marrow (BM). We have previously shown that the C/EBPβ transcription factor is required for emergency granulopoiesis, increased proliferation and differentiation of granulocytic precursors in emergency situations such as infection (Hirai H et al., Nature Immunol. 2006). Enhanced myelopoiesis is a common feature between emergency situations and CP-CML. However, little is known about the roles of C/EBPβ in the pathogenesis of CP-CML. The aim of this study is to elucidate the regulation and function of C/EBPβ in BCR/ABL-mediated myeloid expansion. We first assessed the expression level of C/EBPβ in hematopoietic stem cells and myeloid progenitors in BM obtained from healthy donors or CP-CML patients. The transcript of C/EBPβ is expressed at significantly higher level in common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) in CP-CML BM than those in normal BM. When BCR/ABL was retrovirally transduced into a mouse hematopoietic stem cell line, EML, C/EBPβ expression was significantly upregulated. Treatment of the EML-BCR/ABL cells with imatinib mesylate normalized the expression level of C/EBPβ. These data suggested that C/EBPβ was upregulated in response to the downstream signaling of BCR/ABL. In order to investigate the function of C/EBPβ in BCR/ABL-mediated myeloid expansion, BCR/ABL was retrovirally introduced into BM cells obtained from 5-FU treated C/EBPβ knockout (KO) mice and their properties were compared with those of BCR/ABL-transduced BM cells from wild type (WT) mice. When the transduced cells were cultured in cytokine-free semisolid methylcellulose medium, the number and the size of the colonies of C/EBPβ KO cells were significantly smaller. Flow cytometric analysis of the colony-forming cells revealed that the BCR/ABL-transduced C/EBPβ KO BM cells gave rise to higher frequency of c-kit+ cells and lower CD11b+ cells than BCR/ABL-transduced WT BM cells (%c-kit+ cells=8.2±3.0% vs. 11.3±3.5%, p=0.002, %CD11b+ cells=75.1±2.1% vs. 90.0±4.2%, p=0.003). In addition, BCR/ABL-transduced C/EBPβ KO BM cells revealed higher replating efficiency than BCR/ABL-transduced WT BM cells. To investigate the role of C/EBPβ in leukemogenesis, BCR/ABL-transduced BM cells from C/EBPβ KO mice or WT mice were transplanted into lethally irradiated recipient mice. In mice transplanted with BCR/ABL-transduced C/EBPβ KO cells, the increase of white blood cell count was delayed (Figure) and higher frequency of c-kit+ cells were observed in the BM at day 19 post transplantation (16.0±2.6% vs. 5.5±4.6%, p=0.01). Spleen size of mice transplanted with BCR/ABL-transduced WT cells is much larger than that of BCR/ABL-transduced C/EBPβ KO cells (Figure). The median survival of mice transplanted with BCR/ABL-transduced WT cells was 19 days. In contrast, the median survival of mice transplanted with BCR/ABL-transduced C/EBPβ KO cells was 31 days (p=0.0005). In summary, C/EBPβ is upregulated by BCR/ABL and the absence of C/EBPβ resulted in delayed proliferation and differentiation of myeloid cells both in vitro and in vivo. Our results suggest that C/EBPβ is involved in the BCR/ABL-mediated myeloid expansion in CP-CML and that C/EBPβ can be the novel molecular target for the therapy of CML. We are currently investigating the molecular mechanisms which mediate the upregulation of C/EBPβ and the direct targets of C/EBPβ in CP-CML. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Loss-of-Function and Gain-of-Function Consequences of GATA2 Disease Mutations

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2519-2519
Author(s):  
Koichi Ricardo Katsumura ◽  
Peng Liu ◽  
Charu Mehta ◽  
Kyle J Hewitt ◽  
Alexandra Soukup ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Colony Formation ◽  
Molecular Mechanisms ◽  
Differentially Expressed ◽  
Homozygous Mutation ◽  
Loss Of Function ◽  
Granulocytic Differentiation ◽  
Gain Of Function ◽  
Hematopoietic Stem ◽  
Disease Mutations

The master regulator of hematopoiesis GATA2 controls generation and function of hematopoietic stem and progenitor cells, and heterozygous GATA2 mutations create a predisposition to develop immunodeficiency, myelodysplasia, and acute myeloid leukemia (Spinner et al. Blood, 2014; Dickinson et al. Blood, 2014; Churpek and Bresnick J. Clin. Invest. 2019). Although mechanisms that trigger the transition of a non-pathogenic GATA2 mutation into overt pathology are enigmatic, a paradigm has arisen in which GATA2 mutations are considered to be loss-of-function. We developed a genetic rescue assay to quantify the function of wild type GATA2 and GATA2 disease mutants when expressed at near-physiological levels in primary progenitor cells and demonstrated that GATA2 disease mutations abrogate certain biological and molecular activities, while enabling others (Katsumura et al., 2018, PNAS). We isolated lineage-negative (Lin-) or Lin-Kit+ cells from fetal liver of mice with a homozygous mutation of the Gata2 -77 enhancer, which downregulates Gata2 expression by ~80%. The mutant progenitor cells are largely defective in erythroid, megakaryocytic and granulocytic differentiation and exhibit a predominant monocytic differentiation fate (Johnson et al., 2015, Science Adv.). We compared GATA2 and GATA2 disease mutant activities in the rescue system using a colony formation assay. GATA2, R307W mutant (in N-finger) and T354M mutant (in DNA-binding C-finger) rescued myeloid colony formation and promoted granulocyte proliferation. Surprisingly, R307W and T354M induced more CFU-GM than GATA2. GATA2 and R307W, but not T354M, rescued BFU-E. These data indicated that GATA2 disease mutations were not strictly inhibitory, and in certain contexts, mutant activities exceeded that of GATA2. To extend these results, we subjected -77+/+ or -77-/- Lin- cells to a short-term ex vivo liquid culture, expressed GATA2, R307W, or T354M and used RNA-seq to elucidate progenitor cell transcriptomes. While -77+/+ Lin- cells generate erythroid and myeloid cells, -77-/- Lin- cells are competent for myeloid, but not erythroid, differentiation. Comparison of -77+/+ and -77-/- cell transcriptomes revealed 3064 differentially expressed genes (>2-fold). 1824 genes were >2-fold higher in -77+/+ cells, and 1240 genes were >2-fold higher in -77-/- cells. GATA2 expression in -77-/- cells activated 834 genes >2-fold and repressed 503 genes >2-fold. 60-65% of these genes overlapped with genes differentially expressed between -77+/+ cells and -77-/- cells. R307W expression activated 661 genes >2-fold and repressed 523 genes >2-fold. T354M expression activated 468 genes >2-fold and repressed 575 genes >2-fold. The genes regulated by mutants included GATA2-regulated genes and certain genes that were not GATA2-regulated. Multiple genes were hypersensitive to the mutants, relative to GATA2, and the mutants ectopically regulated certain genes. However, R307W and T354M did not universally regulate an identical gene cohort. For example, both R307W and T354M activated Ncam1, Nrg4, and Mpo more strongly than GATA2. R307W, but not T354M, activated Ear2 and Ces1d more strongly than GATA2. By contrast, T354M, but not R307W, activated Ctsg, Epx, and Rab38 more strongly than GATA2. Both R307W and T354M repressed macrophage genes similarly to GATA2, but they lacked the capacity to activate mast cell genes, differing from GATA2. To elucidate molecular mechanisms underlying GATA2 mutant activities, we leveraged our prior discovery that p38 or ERK kinases induce multi-site GATA2 phosphorylation (Katsumura et al. Blood. 2017). We tested whether these kinases mediate the ectopic transcriptional regulatory activity of GATA2 disease mutants. p38 inhibition attenuated aberrant regulation of Ear2 and Ces1d by R307W (p < 0.05), and mutation of S192 to S192A decreased R307W-induced CFU-GM formation by 49% (p < 0.05). In aggregate, these results indicate that GATA2 disease mutants exert context-dependent activities to regulate transcription and differentiation, activities can be signal-dependent and certain activities are distinct from GATA2. It is attractive to consider the pathogenic consequences of GATA2 disease mutant gain-of-function activities, and an important implication is GATA2 mutation-associated hematologic diseases might not solely reflect haploinsufficiency. Disclosures No relevant conflicts of interest to declare.

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