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Articles

Gfi1b: a key player in the genesis and maintenance of acute myeloid leukemia and myelodysplastic syndrome

Department of Haematology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Department of Haematology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Department of Haematology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany;Department of Endocrinology, Diabetes and Metabolism, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Department of Haematology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Department of Haematology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Department of Haematology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Department of Haematology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Department of Haematology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Department of Haematology, Haemostaseology, Oncology, and Stem Cell Transplantation, Medical University of Hannover, Germany
Department of Haematology, Haemostaseology, Oncology, and Stem Cell Transplantation, Medical University of Hannover, Germany
Department of Haematology, Haemostaseology, Oncology, and Stem Cell Transplantation, Medical University of Hannover, Germany
Department of Haematology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Department of Haematology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Department of Haematology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Department of Haematology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Institute for Cell Biology, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany;Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden
Institute for Transfusion Medicine, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Department of Haematology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Department of Haematology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany
Department of Haematology, West German Cancer Center, University Hospital Essen, University of Duisburg-Essen, Essen, Germany;Department of Medicine A, Hematology, Oncology and Pneumology, University Hospital Münster, Germany
Vol. 103 No. 4 (2018): April, 2018 https://doi.org/10.3324/haematol.2017.167288

Abstract

Differentiation of hematopoietic stem cells is regulated by a concert of different transcription factors. Disturbed transcription factor function can be the basis of (pre)malignancies such as myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). Growth factor independence 1b (Gfi1b) is a repressing transcription factor regulating quiescence of hematopoietic stem cells and differentiation of erythrocytes and platelets. Here, we show that low expression of Gfi1b in blast cells is associated with an inferior prognosis of MDS and AML patients. Using different models of human MDS or AML, we demonstrate that AML development was accelerated with heterozygous loss of Gfi1b, and latency was further decreased when Gfi1b was conditionally deleted. Loss of Gfi1b significantly increased the number of leukemic stem cells with upregulation of genes involved in leukemia development. On a molecular level, we found that loss of Gfi1b led to epigenetic changes, increased levels of reactive oxygen species, as well as alteration in the p38/Akt/FoXO pathways. These results demonstrate that Gfi1b functions as an oncosuppressor in MDS and AML development.

Introduction

Myelodysplastic syndrome (MDS) is characterized by disturbed function of the myeloid compartment of the bone marrow (BM),1 leading in some cases to acute myeloid leukemia (AML).2 AML is characterized by an accumulation of immature myeloid blasts in the BM.2 Hematopoietic development, among other functions, is regulated by transcription factors (TFs).3 Functional dysregulation of several TFs54 can induce malignant transformation. The hematopoietic Growth factor independence 1b (Gfi1b) regulates dormancy and proliferation6 of hematopoietic stem cells (HSCs), the development of erythroid and megakaryocytic cells,107 as well as B and T cells.1311 Constitutive deletion of Gfi1b in mice is embryonically lethal at day E15 due to bleeding and anemia.9 Conditional loss of Gfi1b leads to a significant expansion of functional HSCs in the BM and peripheral blood.6 In human primary hematopoietic progenitors, forced expression of GFI1B results in expansion of immature erythroblasts and repression of myeloid differentiation.14 Gfi1b exerts its function by recruiting histone modifying enzymes, such as CoREST, G9a, LSD1 or HDACs, to induce deacetylation of H3K9, demethylation of H3K4 and/or methylation of H3K9.1815 We report that a reduced level or absence of GFI1B negatively influences the prognosis of MDS/AML patients. Moreover, we present evidence that loss/reduced expression of Gfi1b promotes AML development in different murine models of human AML. Furthermore, reduced expression of Gfi1b in murine models of human leukemia leads to a higher number of leukemic stem cells (LSCs). On a molecular level, aberrant regulation of the ROS/p38/Akt/FoXO pathway as a consequence of reduced Gfi1b level might contribute to these phenotypic changes.

Methods

Study samples

Characteristics of different patient cohorts have been described previously.2519

Boundaries of GFI1B expression

To set boundaries for GFI1B expression levels in AML and MDS patients, we correlated expression levels with the survival outcome of patients.

Mice

Gfi1bfl/fl and Gfi1bEGFP/, MxCre, NUP98/HOXD13 and Kras mice have been described previously.28266 Mice were housed in specific pathogen-free conditions in the animal facility of University Hospital Essen. All mouse experiments were performed with the approval of the local ethics committee for animal use (authorization n. G1196/11).

Poly(I:C) treatment

MxCretg mice harboring the poly(I:C) inducible Cre recombinase gene under the control of the Mx1 promoter were crossed to Gfi1bfl/fl mice. To conditionally delete the Gfi1b alleles in the NUP98/HOXD13 MDS mouse model, Gfi1bfl/flMxCretg NUP98/HOXD13tg mice were injected intraperitoneally (i.p.), as shown previously.6 For Gfi1bfl/flMxCretgKras+/fl mice, two poly(I:C) injections were sufficient to activate the Kras oncogene and delete the Gfi1b alleles. As a control, Gfi1bfl/fl or Gfi1bwt/wt mice not carrying the MxCre were injected with poly(I:C). Three weeks after transplantation of MLL-AF9-transduced lineage negative (Lin-) BM cells from Gfi1bfl/flMxCretg or Gfi1bfl/flMxCrewt mice, primary recipient mice were injected with poly(I:C) 4 times every second day.

Isolation, retroviral transduction, and transplantation of murine hematopoietic progenitor cells

Mouse leukemia was induced by transplanting Lin- BM cells that were retrovirally transduced with the MLL-AF9 oncofusion gene as well as the GFP-encoding gene, as previously described.274 For the limiting dilution assay, different numbers of leukemic cells were retransplanted into sublethally irradiated (3 Gy) secondary recipient mice (3–4 mice/group). The frequency of functional LSCs was determined using ELDA software.29

ChIP and ChIP–Seq analyses

Chromatin Immunoprecipitation (ChIP) and ChIP-Seq analyses were performed as previously described.274 Data are available from: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE88934

Online Supplementary Appendix

Details on the experimental procedures and figures are available in the Online Supplementary Appendix.

Results

Low level of GFI1B are indicative of an inferior prognosis of MDS and AML patients

To obtain a first insight into the role of GFI1B in AML prognosis, we analyzed two well-annotated published data sets.252119 In these sets, CD34 leukemic cells and CD34 control HSCs were used. CD34 leukemic cells represent a fraction in which LSCs are enriched, whereas CD34 cells from healthy donors represent a fraction of cells in which HSCs are enriched.3021 GFI1B showed lower expression in CD34 AML blasts compared to CD34 control HSCs (Figure 1A). MDS can progress to AML, and therefore, we wanted to elucidate how GFI1B expression changes during the progression of MDS to AML. Again, GFI1B showed a lower expression in AML blasts compared to GFI1B expression in CD34 cells from the BM of patients with MDS (Figure 1B).

Figure 1.Correlation between different GFI1B expression levels and myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) prognosis. (A) Expression of GFI1B in CD34+ AML cells (n=269) compared to CD34+ control cells (n=8) based on the patient cohort published by Valk et al.;21 P≤0.0001. (B) Expression of GFI1B in CD34+ MDS cells (n=23) compared to CD34+ AML cells (n=501) based on the patient cohort published by Wouters et al.;19 P≤0.0001. (C) Expression of GFI1B in leukemic stem cells (LSCs)20 of different AML subtypes compared to normal hematopoietic stem cells (HSCs) or common myeloid progenitor cells (CMPs) in published gene expression arrays.31

We also analyzed an independent data set, which provided whole genome expression data for LSCs in different types of AML as well as different human hematopoietic progenitor cells.2520 GFI1B showed a lower expression in human LSCs of different AML subtypes compared to its expression in normal human myeloid progenitors (GMPs) or HSCs (Figure 1C).20 GMPs and HSCs are two fractions from which LSCs arise in mice and humans.31

We analyzed whether GFI1B level might also be informative regarding the prognosis of MDS and AML patients. Based on available expression data of GFI1B and the associated survival data, we could distinguish two distinct populations with regard to GFI1B expression (Figure 2A). A low level of GFI1B (see Methods section and Online Supplementary Appendix for details) in leukemic blast cells was associated with inferior outcome with regard to overall survival (OS) of all AML patients (Figure 2B) as well as OS and event-free survival (EFS) in the group of patients with no overt cytogenetic aberrations (Figure 2C and D). We also performed a multivariate analysis, including additional factors such as age, sex and cytogenetic status, as well as mutational status of certain genes. There was a tendency for a very low GFI1B level to be an independent prognostic marker (P=0.12), but this did not reach a level of significance (data not shown). Low GFI1B expression might be associated with an inferior prognosis, but other confounding factors contribute to this association. Finally, we examined whether low GFI1B expression (the lowest 5% compared with the highest 20% of expression levels) was associated with a certain gene expression signature to obtain a first insight into how GFI1B might influence prognosis. We performed Signaling Pathway Enrichment using Experimental Datasets (SPEED) analysis (see Online Supplementary Appendix) on two separate studies,2221 for which expression data of the full length GFI1B and associated clinical data were available. Low level of GFI1B expression was associated with a reactive oxygen species (ROS)-mediated signature pathway as well as activation of mitogen-activated protein kinase (MAPK), JAK, TGFB and TLR signaling pathways (Figure 2E).

Figure 2.Different Gfi1B levels are indicative of prognosis of myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) patients. (A) High expression of GFI1B in human AML cells (n=116) compared to lower expression of GFI1B (0-15%) in AML cells (n=394) based on the patient cohort published by Verhaak et al.22 (B) Overall survival (OS) from patients described in Verhaak et al.22 with regard to GFI1B expression (P=0.0443). (C) Overall survival (OS) from patients described in Verhaak et al.22 (restricted to cytogenetically normal patients) with regard to GFI1B expression; P=0.0407. (D) Same as in (C) but with regard to event-free survival (EFS); P=0.0350. (E) Analysis of signaling pathways with low GFI1B expression (the lowest 5% compared with the highest 20% of expression levels) in a bigger dataset from Verhaak et al.22 Analysis was performed by Signaling Pathway Enrichment using Experimental Datasets (SPEED) analysis. Pathways such as reactive oxygen species (ROS; H2O2), MAPK, JAK, TGFB and TLR are highly significant. (F) High expression of GFI1B (31–100%) in human MDS patients (n=32) compared to lower expression of GFI1B (0–30%) in MDS patients (n=85) based on the patient cohort published by Papaemmanuil et al.23 (G) EFS of patients described in Papaemmanuil et al.23 with regard to GFI1B expression; P=0.032.

We also examined whether GFI1B expression level influences survival and disease progression from MDS to AML using a separate set of data.2723 Again, we could distinguish two different populations with regard to GFI1B expression (low and high) (Figure 2F): low expression of GFI1B correlated with poor EFS (Figure 2G).

Anguita et al.32 observed a positive correlation between the expression of a mutated form of GFI1B, which acts in a dominant-negative manner, and the expression of MLLT3 and a negative correlation with regard to SPI1. In addition, Chowdhury et al. described a negative correlation between GFI1B expression and MEIS1.33 In our patient cohorts, we also found an inverse correlation between GFI1B expression level and SPI1 expression as well as MEIS1 and a positive correlation with MLLT3 (Online Supplementary Figure S1).

Reduced expression level or loss of Gfi1b promotes progression of MDS to AML in a murine MDS model

To investigate a connection between Gfi1b level and AML, we used different mouse strains and models of human leukemia. We used one strain in which both Gfi1b alleles can be conditionally deleted in the hematopoietic system upon poly(I:C) administration, resembling absence of Gfi1b expression (Gfi1bfl/flMxCretg).6 In a second mouse model, one coding allele of Gfi1b is replaced by enhanced green fluorescence protein (EGFP) cDNA (Gfi1bEGFP/wt),13 which leads to a lower expression level of Gfi1b (see below). Finally, wild-type mice were used to model normal/high Gfi1b expression. To study whether reduced Gfi1b expression accelerates MDS to AML progression, we crossed the above-mentioned mouse strains with NUP98/HOXD13tg mice, which represent a model for human MDS/AML.34

We first used the Gfi1b:EGFP knock-in reporter mouse strain and crossed these mice with NUP98/HOXD13tg mice (Figure 3A). Loss of one allele of Gfi1b shortened the latency period of AML development (Figure 3B). In BM cells derived from heterozygous leukemic mice, the expression of Gfi1b mRNA and Gfi1b protein levels were reduced to approximately 50% compared to BM cells from Gfi1b leukemic mice (Online Supplementary Figure S2A and B). Furthermore, we found that the EGFP expression level and hence Gfi1b expression level was significantly lower in the myeloid blasts when the disease onset was within the first 250 days compared to Gfi1b expression in blasts from mice that developed overt leukemia more than 250 days after birth (Figure 3C). The leukemic cells from Gfi1bwt/wt or Gfi1bEGFP/wt animals showed no significant differences with regard to surface marker expression, spleen size, white blood cell and platelet counts, or cytological appearance, but showed significant differences with regard to hemoglobin and red blood cells (Figure 3D, Online Supplementary Figure S2C-F and data not shown), which might be due to a potential dose-dependent role of Gfi1b in erythropoiesis.96

Figure 3.Low level or absence of Gfi1b accelerates the progression of myelodysplastic syndrome (MDS) to acute myeloid leukemia (AML) in the NUP98/HOXD13 MDS mouse model. (A) Crossing of the Gfi1bwt/wt and Gfi1bEGFP/wt mouse strains with the NUP98/HOXD13 mouse model. (B) Survival of Gfi1bwt/wt and Gfi1bEGFP/wtNUP98/HOXD13 transgenic mice; P=0.0039. Number of mice succumbing to AML is indicated. (C) Mean fluorescence intensity (MFI) of the GFP expression level (and hence Gfi1b promoter activity) in Gfi1bEGFP/wt mice that died of AML before 250 days (n=7) or after 250 days (n=5); P=0.0272. (D) Wright-Giemsa staining of bone marrow (BM) cytospins from representative Gfi1bEGFP/wt and Gfi1bwt/wt NUP98/HOXD13 leukemic mice (bar=20 μm). (E) Crossing of the Gfi1bfl/flMxCrewt and Gfi1bfl/flMxCretg mouse strains with the NUP98/HOXD13 mouse model. After Cre-mediated deletion of the Gfi1b gene upon poly(I:C) administration, the mice were monitored for signs of leukemia. (F) Survival of Gfi1bfl/flMxCrewt(Gfi1bwt/wt) and Gfi1bfl/flMxCretg (Gfi1bKO/KO) mice transgenically expressing NUP98/HOXD13 after poly(I:C) administration; P<0.0001. Number of mice succumbing to AML is indicated. (G) Polymerase chain reaction genotyping of DNA from BM cells of poly(I:C)-injected Gfi1bfl/flMxCrewt and Gfi1bfl/flMxCretgNUP98/HOXD13 leukemic mice. (H) Wright-Giemsa staining of BM cytospins from representative poly(I:C)-injected Gfi1bfl/flMxCrewt and Gfi1bfl/flMxCretgNUP98/HOXD13 leukemic mice (bar=20 μm). (I) The frequency of monocytes (Mac-1hiGr-1int) (left panel, ****P<0.0001), granulocytes (Mac-1hiGr-1hi) (middle panel, *P=0.0206) and CD117+ (c-Kit) cells (right panel, ****P<0.0001) in the BM of mice described in (F) (n=15 for Gfi1bfl/flMxCrewt mice; n=13 for Gfi1bfl/flMxCretgNUP98/HOXD13 mice).

We next examined how complete absence of Gfi1b influences MDS to AML progression. We used the Gfi1b conditional knockout mouse model (Gfi1bfl/flMxCretg), whereby the expression of Gfi1b can be conditionally abrogated in the hematopoietic system upon poly(I:C) administration6 (Figure 3E). The absence of Gfi1b resulted in a substantially earlier onset of AML with a median survival time of approximately 50 days (P<0.0001) (Figure 3F). Cre-mediated excision was verified to be efficient in leukemic Gfi1bfl/flMxCretgNUP98/HOXD13tg mice after poly(I:C) administration with non-excised Gfi1b alleles below detection levels (Figure 3G), and this was associated with practically no expression of Gfi1b mRNA and protein (Online Supplementary Figure S2A and B).6 Leukemic cells from Gfi1bfl/flMxCretg and Gfi1bfl/flMxCretg animals showed no significant differences in spleen size, white blood cells or cytological appearance but significant differences in hemoglobin, red blood cells and platelet counts (Figure 3H and Online Supplementary Figure S2G-J and data not shown), which might be due to a dose-dependent role of Gfi1b in erythropoiesis.96 The absence of Gfi1b led to a reduced frequency of myeloid cells (Figure 3I, left, middle, and Online Supplementary Figure S3A–C). CD117 (c-Kit) was uniformly higher expressed on all Gfi1b-deficient blast cells (derived from Gfi1bfl/flMxCretg) mice compared to Gfi1b expressing blasts (Gfi1bfl/flMxCrewt) (Figure 3I, right). Finally, there was no difference with regard to apoptosis in NUP98/HOXD13tg mice (Online Supplementary Figure S3D). In our murine model of MDS/AML development, we did not observe a positive correlation between Gfi1b and Mllt3 expression nor a negative correlation between Gfi1b and Spi1 expression, which might be disease context-dependent and thus not reproducible in all types of AML (Online Supplementary Figure S3A and B). We also analyzed the expression level of Meis1, since Chowdhury et al. observed a negative correlation between GFI1B and MEIS1.33 We were able to confirm this finding for this model of AML (Online Supplementary Figure S4C).

Loss of Gfi1b promotes the progression of myeloproliferative disorder in a conditional Kras mouse model

To validate the results above in a second model, we used mice conditionally expressing a mutated form of Kras. RAS mutations are found in 5–10% of AML patients.2 These mice harbor a transcriptional stop codon flanked by loxP sites upstream of a mutated Kras allele and, after removal of the stop codon, develop myeloproliferative disorder.35 We crossed these mice with Gfi1bfl/flMxCretg or Gfi1bwt/wtMxCretg mice, and after poly(I:C) administration, we observed mice for the emergence of disease (Figure 4A).

Figure 4.Absence of Gfi1b accelerates the progression of myeloproliferative disorder and acute myeloid leukemia (AML). (A) Crossing of the Gfi1bwt/wtMxCretg and Gfi1bfl/flMxCretg mouse strains with the Kras+/fl mouse model. (B) Survival of Gfi1bwt/wtMxCretg and Gfi1bfl/flMxCretg mice transgenically expressing Kras after poly (I:C) administration; ****P<0.0015. Numbers indicate the number of mice succumbing to AML. (C) Wright-Giemsa staining of bone marrow (BM) cytospins from representative Gfi1bwt/wtMxCretg and Gfi1bfl/flMxCrewt leukemic mice transgenically expressing Kras after poly(I:C) administration (bar=20 μm). (D) Flow cytometric analysis of the BM from the leukemic mice shown in (B) with regard to Gr-1 and Mac-1 expression. (E) Isolation, transduction and transplantation of lineage-negative (Lin-) cells from Gfi1bfl/flMxCrewt and Gfi1bfl/flMxCretg mice with MLL-AF9-expressing retrovirus. After Cre-mediated deletion of the Gfi1b gene upon poly(I:C) administration, the mice were monitored for signs of leukemia. (F) Survival of the mice transplanted with Gfi1bfl/flMxCrewt and Gfi1bfl/flMxCretg MLL-AF9 transduced cells; ***P=0.0002. Number of mice succumbing to AML is indicated. (G) Wright-Giemsa staining of BM cytospins from the leukemic mice described in (F) (bar=20 μm). (H) Flow cytometric analysis of the BM from the leukemic mice described in (F) with regard to Gr-1 and Mac-1 expression.

While Gfi1bwt/wtMxCretgKras+/fl mice developed a lethal myeloproliferative disorder with a median survival of approximately 25 days, loss of Gfi1b significantly shortened the latency period of the disease to a median survival of approximately seven days (Figure 4B). There was no difference with regard to cytological appearance, number of myeloid cells or level of apoptosis (Figure 4C and D and Online Supplementary Figure S5A–D). We also did not observe any significant difference with regard to white blood counts, platelet counts or spleen size but a significant difference in hemoglobin and red blood cells between Gfi1bfl/flMxCretg and Gfi1bwt/wtMxCretg animals (Online Supplementary Figure S5E–H), which might be due to the role of Gfi1b in erythropoiesis.96

Loss of Gfi1b promotes the progression of AML initiated by retroviral MLL-AF9 expression

The Mixed Lineage Leukemia (MLL) gene is a common target for chromosomal translocations.2 MLL-AF9 is a fusion protein frequently occurring in a subset of AML patients,2 and its expression in hematopoietic progenitors has been linked to the induction of AML in mice.36 As a third AML mouse model, we thus used mice that developed AML through the induction of MLL-AF9 expression, the product of the t(9;11)(q22;p23) translocation. Lin- BM cells derived from Gfi1bwt/wtMxCretg or Gfi1bfl/flMxCretg mice were transduced with a retrovirus expressing MLL-AF9 and transplanted into lethally irradiated C57BL/6J mice. For Cre-mediated excision of Gfi1b in the transplanted cells, mice were injected with poly(I:C) three weeks after transplantation (Figure 4E). Poly(I:C)-injected mice with MLL-AF9-transduced Gfi1bfl/flMxCretg (Gfi1b-deficient) cells succumbed faster to leukemia than mice injected with poly(I:C) and transplanted with MLL-AF9-transduced Gfi1bwt/wtMxCretg (Gfi1b-expressing) cells (Figure 4F). However, there were no major qualitative differences concerning cytological findings and or blood parameters (Figure 4G and data not shown). We did not observe a significant change in the number of overall myeloid cells or apoptosis level in the different settings (Figure 4H and Online Supplementary Figure S6A–D).

Loss of Gfi1b increases the number of LSCs

Loss of Gfi1b leads to an expansion in the number of functional HSCs;6 therefore we investigated whether the same applies to LSCs. We performed a limiting dilution assay by transplanting MLL-AF9 leukemic BM cells derived from poly(I:C)-treated Gfi1bfl/flMxCrewt or Gfi1bfl/flMxCretg leukemic mice into sublethally irradiated congenic mice (Figure 5A). Gfi1b-deficient MLL-AF9 BM cells had a LSC frequency of 1:3500 compared with an LSC frequency of 1:63000 cells in Gfi1b-expressing leukemic cells (Figure 5B). The increased number of functional LSCs in Gfi1b-deficient leukemic cells could explain why loss of Gfi1b accelerated disease progression, as it has already been shown that a higher number of LSCs is associated with a poor prognosis of leukemia patients.37 However, this hypothesis needs to be confirmed in independent experiments.

Figure 5.Loss of Gfi1b increases the stemness of LSCs. (A) In vivo limiting dilution assay for determination of functional LSCs. The indicated numbers of poly (I:C)-treated Gfi1bfl/flMxCrewt and Gfi1bfl/flMxCretg MLL-AF9 transduced cells were retransplanted into sublethally irradiated mice. (B) Survival of the mice transplanted with different numbers of poly(I:C)-treated Gfi1bfl/flMxCrewt MLL-AF9 and Gfi1bfl/flMxCretg MLL-AF9 transduced cells. (C) Determination of functional LSCs by limiting dilution assay.

Loss of Gfi1b induces gene expression changes supporting AML development

To further study the molecular function of Gfi1b in AML, we performed whole genome gene expression analysis using Gfi1b-expressing and Gfi1b-deficient NUP98/HOXD13tg leukemic mice (Figure 6A). This model was used since the difference between Gfi1b-deficient and Gfi1b-expressing leukemic cells was most striking in the NUP98/HOXD13tg mouse model. Using gene set enrichment analysis (GSEA), loss of Gfi1b was associated with a signature showing enrichment of genes involved in AML development as well as regulation of stemness (Figure 6B). This is of interest since we observed an increase in the number of LSCs upon deletion of Gfi1b.

Figure 6.Loss of Gfi1b induces gene expression changes supporting acute myeloid leukemia (AML) development. (A) Microarray analysis was performed with Gfi1bfl/flMxCrewtNUP98/HOXD13 and Gfi1bfl/flMxCretgNUP98/HOXD13 leukemic cells. (B) Based on the results of the micro-array analysis, a Gene set enrichment analysis (GSEA) of leukemic cells from Gfi1bfl/flMxCrewtNUP98/HOXD13tgan d Gfi1bfl/flMxCretgNUP98/HOXD13tg mice w as performed. As a result, Gfi1b deficient leukemic cells showed an enrichment of the gene set of VALK AML cluster 8 with a normalized enrichment score (NES) of 2.1 and false discovery rate (FDR) of q-val=0.00191213. Gfi1b deficient cells showed also an enrichment for RAMALHO STEMNESS with an NES=2.41 and an FDR q-val=0.(C) ChIP and ChIP-Seq analysis was performed with Gfi1bfl/flMxCrewtNUP98/HOXD13 and Gfi1bfl/flMxCretgNUP98/HOXD13 leukemic cells. ChIP-Seq analysis for differences in the frequency of H3K9 acetylation of Gfi1b-deficient (Gfi1bfl/flMxCretg) leukemic blasts from NUP98/HOXD13tg mice compared to leukemic cells with normal Gfi1b expression (Gfi1bfl/flMxCrewt). (D) GSEA of genes with an elevated acetylation level in Gfi1b deficient mice are associated with regulation of cell growth NES=1.78 and FDR q-val=0.055857178 and GNF2_MAP2K3 NES=2.02 and FDR q-val=1.7715618E-4. (E) Upon analyzing the differentially acetylated genes and using the MSigDB Pathway approach, we found significant enrichment of different pathways, among them the p38 pathway. P-value was used to rank the enrichment.

Gfi1b recruits different histone-modifying enzymes, among them HDACs,16 to its target genes. This in turn leads to deacetylation of H3K9, which leads to epigenetic silencing of the particular Gfi1b target genes.16 We, therefore, analyzed the genome-wide H3K9 acetylation level of leukemic blasts from Gfi1b-expressing and Gfi1b-deficient NUP98/HOXD13tg leukemic mice. Loss of Gfi1b leads to a genome-wide increase in H3K9 acetylation level (Figure 6C). In a subsequent step, we analyzed those genes, which showed an elevated level of H3K9 acetylation in Gfi1b-deficient leukemic cells compared to the H3K9 acetylation level of the same genes found in Gfi1b-expressing leukemic cells. Using GSEA, we found a significant enrichment of gene sets associated with the regulation of cell growth and MAPK signaling (Figure 6D). We also performed a Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis of those genes, which exhibited differentially H3K9 acetylated promoter areas in Gfi1b-expressing and Gfi1b-deficient leukemic cells. We found a number of processes involved in erythroid regulation (Online Supplementary Figure S7A and B), which is a main function of Gfi1b and hence underscores the validity of our results.1696 Finally, we analyzed the differentially acetylated genes in Gfi1b-deficient and Gfi1b-expressing leukemic cells and compared these gene sets based on the gene sets provided by the Molecular Signatures Database (MSigDB). Using this approach, we repeatedly found signatures associated with p38 (Figure 6E).

We observed increased H3K9 acetylation of the promoter area of genes involved in stem cell function in Gfi1b-deficient leukemic cells, and these epigenetic changes correlated with the gene-expression changes described above (Figure 6B). As described, gene expression arrays revealed an enrichment of a stem cell/leukemic stem cell gene signature in Gfi1b-deficient leukemic cells. To validate these results, we selected 14 different genes (Abcg2, Gata3, Itga2, Thy1, Cd24a, Pecam1, Prom1, Plaur, Klf4, Mycn, Ptch1, Pecam1, Sav1, and Notch1), which were differentially expressed by more than 2-fold between Gfi1b-expressing and Gfi1b-deficient leukemic mice in the gene expression arrays. We selected these genes based on their diverse role in regulating stem cell function. We then examined these genes and confirmed that these genes were also differentially expressed using RT-PCR (Online Supplementary Figure S8A).

Loss of Gfi1b leads to increased ROS levels and decreased levels of activated p38

To obtain further insight into the molecular mechanism behind our observation we compared the whole genome gene expression pattern in murine Gfi1b-expressing and Gfi1b-deficient leukemic cells based on an AltAnalyze approach (see Online Supplementary Appendix). Using the same algorithm, we compared the gene expression pattern found in AML blasts with low GFI1B expression and high GFI1B expression (data obtained from published studies from Valk et al.21 and Verhaak et al.22). For the analysis of the human dataset, we analyzed the 10% of patients with the lowest and the 20% of patients with the highest GFI1B expression level in order to have enough observations from which to draw any conclusions. Then we compared which pathways were similarly deregulated in the human and murine leukemia sets. ROS and MAPK signaling were among the pathways differentially expressed between both murine and human Gfi1b/GFI1B-deficient/low and Gfi1b/GFI1B high-expressing leukemic cells (Figure 7A). As ROS plays an important role in the pathogenesis of AML,38 we examined the level of ROS. For non-malignant HSCs, it was shown that HSCs with low ROS had a high self-renewal capacity.39 In contrast, HSCs with elevated ROS were mostly located in the vascular niche, had a reduced self-renewal capacity, and were more restricted with regard to their differentiation potential.39 Based on this and our previous report that loss of Gfi1b leads to higher level of ROS in HSCs,6 we examined whether ROS level might differ between Gfi1b-deficient and Gfi1b expressing LSCs. Due to the difficulty of defining a distinct LSC population in each set of AML samples, we used CD117 (c-Kit) expression as a surrogate marker to define a population which is enriched for LSCs. CD117 expression has been used to identify a fraction that is enriched for LSCs.40 We identified two distinct populations in the CD117 blast cells that differ with regard to their ROS expression (a population with low ROS expression and a population with high ROS expression). Loss of Gfi1b led to an increased level of ROS (defined as the mean fluorescent intensity, MFI) in both ROS-low and ROS-high populations (Figure 7B–D and Online Supplementary Figure S8B).

Figure 7.Loss of Gfi1b deregulates acute myeloid leukemia (AML) signaling pathway. (A) Analysis to see which pathways were enriched in GFI1B low-expressing blast cells in two independent sets of myelodysplastic syndrome (MDS) or AML patients compared to the expression pattern found in GFI1B high-expressing MDS/AML blast cells. The same approach was repeated for Gfi1b-expressing and Gfi1b-non-expressing leukemic cells from our mice experiments. As an overlap, enrichment was observed in pathways of JAK-STAT-, MAPK- and ROS-related signaling. (B) Representative flow cytometric analysis of bone marrow (BM) from Gfi1bfl/flMxCretgNUP98/HOXD13tg mice compared to Gfi1bfl/flMxCrewtNUP98/HOXD13tg mice showing the gating strategy for determining ROS low and ROS high levels. (C) Mean fluorescence intensity (MFI) for ROS in the ROS-low population of c-Kit+ blast cells derived from Gfi1bfl/flMxCrewtNUP98/HOXD13tg (n=6) and Gfi1bfl/flMxCretgNUP98-HOXD13tg (n=5); *P=0.0488. (D) Mean fluorescence intensity (MFI) for ROS in the ROS-high population of c-Kit+ blast cells derived from Gfi1bfl/flMxCrewtNUP98-HOXD13tg (n=6) and Gfi1bfl/flMxCretgNUP98/HOXD13tg(n=5); *P=0.0191. (E) Flow cytometric analysis of p38 MAPK (pT180/pY182) in CD117+ blast cells derived from Gfi1bfl/flMxCrewtNUP98/HOXD13tg (n=5) and Gfi1bfl/flMxCretg NUP98/HOXD13tg (n=6); *P=0.0144. (F) Flow cytometric analysis of Akt (pS473) in c-Kit+ blast cells derived from Gfi1bfl/flMxCrewtNUP98/HOXD13tg (n=4) and Gfi1bfl/flMxCretgNUP98/HOXD13tg (n=5); **P=0.0040. (G) FoXO3 protein level was detected in nuclear extraction (NER)- and cytoplasmic extraction (CER)-derived BM cells from Gfi1bfl/flMxCretgNUP98/HOXD13tg and Gfi1bfl/flMxCrewtNUP98/HOXD13tg. (H) Working model hypothesis: normal levels of Gfi1b lead to reduced ROS levels, resulting in normal maturation and differentiation of progenitor cells. Loss of Gfi1b in leukemic cells is associated with higher ROS levels, which have been shown to promote AML development. However, through a still undefined mechanism, this results in lower levels of p38 MAPK and pAkt and higher levels of unphosphorylated FoXO3, which might explain the increased number of functional leukemic stem cells in the Gfi1b-deficient AML population. LSC: leukemic stem cells.

Altered activity of the AKT pathway in Gfi1b-deficient AML

Elevated levels of ROS promote AML development, but ROS also activates various redox-sensitive signaling transduction cascades,41 including the MAPK pathway, which limits the stemness of the affected cells, at least in a non-malignant setting.42 In the presence of ROS, the MAPK pathway component p38 is activated, which subsequently results in an exhaustion of the HSC population.43 It has also been shown that activation of p38 limits oncogenic transformation.44 Despite a higher level of ROS, in our models, Gfi1b-deficient leukemic cells have escaped p38 activation, indicated by a decreased level of phosphorylated p38 compared to Gfi1bfl/flMxCrewtNUP98/HOXD13tg (Figure 7E). The fact that Gfi1b might directly or indirectly regulate p38 is also supported by the analysis of differentially acetylated genes in Gfi1b-deficient and Gfi1b-expressing leukemic cells. Because decreased p38 levels are associated with higher oncogenic potential,44 this could partially explain the higher number of functional LSCs we observed in the Gfi1b-deficient leukemic cells. Activation of p38 leads to an increased level of Akt45 Akt activity inversely correlates with the number of LSCs in AML.46 We thus examined the connection between loss of Gfi1b and Akt and found that the level of phosphorylated Akt is reduced in Gfi1b-deficient leukemia (Figure 7F). Akt represses the function of FoXO3, and since FoXO3 acts as an oncogene in AML,46 we determined the FoXO3 protein level. Gfi1b-deficient leukemic cells showed an increased expression of FoXO3 protein in the nucleotide (NER) and cytoplasmatic (CER) cell fraction compared to the expression level of FoXO3 in Gfi1b-expressing leukemic cells (Figure 7G). To obtain an insight into whether this increased level of FoXO3 also has any functional consequences, we re-examined the whole genome expression datasets in Gfi1b-expressing and Gfi1b-deficient leukemic cells and found an enrichment of FoXO3 binding sites among the promoter areas of those genes, which were differentially expressed between cells with absence of Gfi1b expression and cells with intact expression of Gfi1b (Online Supplementary Figure S9), showing that altered level of FoXO3 might be one additional explanation for our observations (Figure 7H).

Discussion

In the datasets analyzed by us, GFI1B was expressed at a lower level in LSCs compared to the control. Low GFI1B was also indicative of an inferior prognosis for MDS and AML patients, with the caveat that these statements are based on retrospective studies. Larger prospective studies would be required to make such a claim on a solid basis. We previously reported that low GFI1 expression level in AML blasts was associated with poor outcome and here we report that low GFI1B expression levels were associated with poor survival. This might appear surprising since GFI1 and GFI1B repress each other, but in our cohorts we observed that low GFI1B expression level can also be associated with low GFI1 expression (data not shown), therefore, the reciprocal regulation between GFI1 and GFI1B might be different in leukemic cells. We postulate that GFI1B plays a dose-dependent role in human/murine AML pathogenesis. Anguita et al. showed that a mutated dominant-negative form of GFI1B contributes to AML development. These reports show how altering the function of GFI1B can influence normal and malignant development. Recent studies have highlighted the role of different isoforms of GFI1B in the course of erythroid and megakaryocytic development.49476 It remains to be elucidated whether altering the expression of these isoforms might also contribute to AML development.

Loss of Gfi1b in our murine models increased the number of LSCs on a functional level. These data are in line with our previous reports that loss of Gfi1b leads to an expansion of functional HSCs.6 On a molecular level, loss of Gfi1b resulted in an increased level of H3K9ac among its target genes, which is in line with other reports regarding the epigenetic function of Gfi1b.1611 Among these target genes are a number of genes involved in the regulation of leukemogenesis and stem cell regulation, indicating that the absence of Gfi1b leads to a gene expression signature that directly or indirectly contributes to an increased number of LSCs.

Both murine and human data also indicated a possible connection between Gfi1b and ROS/p38/Akt signaling. P38 and Akt activation limit oncogenic and stemness potential.464443 Conceivably, lower expression of these proteins would increase the oncogenic potential. P38 and Akt were down-regulated in Gfi1b-deficient leukemic cells in vivo. In line with this, Saleque et al. demonstrated that Gfi1b is involved in the regulation of p38 and that reduced Gfi1b levels are associated with lower p38 signaling.50 In addition to the ROS/p38/Akt/FoXO3 signaling cascade, other pathways were altered. It remains to be elucidated which role these pathways might play in the pathogenesis of human and murine AML. In addition, how Gfi1b/GFI1B regulates ROS, p38, Akt and FoXO3 levels remains to be analyzed.

In summary, epigenetic changes and alteration of the ROS/p38/Akt/FoXO signaling cascade might facilitate the progression of normal hematopoietic cells to LSCs. In the future, testing will be needed to determine whether alteration of the ROS pathway could be a targeted therapeutic approach to treat AML patients with low GFI1B expression.

Acknowledgments

We thank the animal facility of University Hospital Essen.

Footnotes

  • Check the online version for the most updated information on this article, online supplements, and information on authorship & disclosures: www.haematologica.org/content/103/4/614
  • FundingThis work was supported by the Deutsche Forschungsgemeinschaft and the IFORES program of University Hospital Essen.
  • Received June 29, 2017.
  • Accepted January 5, 2018.

References

  1. Tefferi A, Vardiman JW. Myelodysplastic syndromes. N Engl J Med. 2009; 361(19):1872-1885. PubMedhttps://doi.org/10.1056/NEJMra0902908Google Scholar
  2. Dohner H, Estey E, Grimwade D. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017; 129(4):424-447. PubMedhttps://doi.org/10.1182/blood-2016-08-733196Google Scholar
  3. Calero-Nieto FJ, Ng FS, Wilson NK. Key regulators control distinct transcriptional programmes in blood progenitor and mast cells. EMBO J. 2014; 33(11):1212-1226. PubMedhttps://doi.org/10.1002/embj.201386825Google Scholar
  4. Hones JM, Botezatu L, Helness A. GFI1 as a novel prognostic and therapeutic factor for AML/MDS. Leukemia. 2016; 30(6):1237-1245. https://doi.org/10.1038/leu.2016.11Google Scholar
  5. Gentner B, Pochert N, Rouhi A. MicroRNA-223 dose levels fine tune proliferation and differentiation in human cord blood progenitors and acute myeloid leukemia. Exp Hematol. 2015; 43(10):858-868. Google Scholar
  6. Khandanpour C, Sharif-Askari E, Vassen L. Evidence that growth factor independence 1b regulates dormancy and peripheral blood mobilization of hematopoietic stem cells. Blood. 2010; 116(24):5149-5161. PubMedhttps://doi.org/10.1182/blood-2010-04-280305Google Scholar
  7. Laurent B, Randrianarison-Huetz V, Marechal V, Mayeux P, Dusanter-Fourt I, Dumenil D. High-mobility group protein HMGB2 regulates human erythroid differentiation through trans-activation of GFI1B transcription. Blood. 2010; 115(3):687-695. PubMedhttps://doi.org/10.1182/blood-2009-06-230094Google Scholar
  8. Foudi A, Kramer DJ, Qin J. Distinct, strict requirements for Gfi-1b in adult bone marrow red cell and platelet generation. J Exp Med. 2014; 211(5):909-927. PubMedhttps://doi.org/10.1084/jem.20131065Google Scholar
  9. Saleque S, Cameron S, Orkin SH. The zinc-finger proto-oncogene Gfi-1b is essential for development of the erythroid and megakaryocytic lineages. Genes Dev. 2002; 16(3):301-306. PubMedhttps://doi.org/10.1101/gad.959102Google Scholar
  10. Vassen L, Beauchemin H, Lemsaddek W, Krongold J, Trudel M, Moroy T. Growth factor independence 1b (gfi1b) is important for the maturation of erythroid cells and the regulation of embryonic globin expression. PLoS One. 2014; 9(5):e96636. PubMedhttps://doi.org/10.1371/journal.pone.0096636Google Scholar
  11. van der Meer LT, Jansen JH, van der Reijden BA. Gfi1 and Gfi1b: key regulators of hematopoiesis. Leukemia. 2010; 24(11):1834-1843. PubMedhttps://doi.org/10.1038/leu.2010.195Google Scholar
  12. Schulz D, Vassen L, Chow KT. Gfi1b negatively regulates Rag expression directly and via the repression of FoxO1. J Exp Med. 2012; 209(1):187-199. PubMedhttps://doi.org/10.1084/jem.20110645Google Scholar
  13. Vassen L, Okayama T, Moroy T. Gfi1b:green fluorescent protein knock-in mice reveal a dynamic expression pattern of Gfi1b during hematopoiesis that is largely complementary to Gfi1. Blood. 2007; 109(6):2356-2364. PubMedhttps://doi.org/10.1182/blood-2006-06-030031Google Scholar
  14. Osawa M, Yamaguchi T, Nakamura Y. Erythroid expansion mediated by the Gfi-1B zinc finger protein: role in normal hematopoiesis. Blood. 2002; 100(8):2769-2777. PubMedhttps://doi.org/10.1182/blood-2002-01-0182Google Scholar
  15. Vassen L, Fiolka K, Moroy T. Gfi1b alters histone methylation at target gene promoters and sites of gamma-satellite containing heterochromatin. EMBO J. 2006; 25(11):2409-2419. PubMedhttps://doi.org/10.1038/sj.emboj.7601124Google Scholar
  16. Saleque S, Kim J, Rooke HM, Orkin SH. Epigenetic regulation of hematopoietic differentiation by Gfi-1 and Gfi-1b is mediated by the cofactors CoREST and LSD1. Mol Cell. 2007; 27(4):562-572. PubMedhttps://doi.org/10.1016/j.molcel.2007.06.039Google Scholar
  17. McGhee L, Bryan J, Elliott L. Gfi-1 attaches to the nuclear matrix, associates with ETO (MTG8) and histone deacetylase proteins, and represses transcription using a TSA-sensitive mechanism. J Cell Biochem. 2003; 89(5):1005-1018. PubMedhttps://doi.org/10.1002/jcb.10548Google Scholar
  18. Duan Z, Zarebski A, Montoya-Durango D, Grimes HL, Horwitz M. Gfi1 coordinates epigenetic repression of p21Cip/WAF1 by recruitment of histone lysine methyltransferase G9a and histone deacetylase 1. Mol Cell Biol. 2005; 25(23):10338-10351. PubMedhttps://doi.org/10.1128/MCB.25.23.10338-10351.2005Google Scholar
  19. Wouters BJ, Lowenberg B, Erpelinck-Verschueren CA, van Putten WL, Valk PJ, Delwel R. Double CEBPA mutations, but not single CEBPA mutations, define a subgroup of acute myeloid leukemia with a distinctive gene expression profile that is uniquely associated with a favorable outcome. Blood. 2009; 113(13):3088-3091. PubMedhttps://doi.org/10.1182/blood-2008-09-179895Google Scholar
  20. Rapin N, Bagger FO, Jendholm J. Comparing cancer vs normal gene expression profiles identifies new disease entities and common transcriptional programs in AML patients. Blood. 2014; 123(6):894-904. PubMedhttps://doi.org/10.1182/blood-2013-02-485771Google Scholar
  21. Valk PJ, Verhaak RG, Beijen MA. Prognostically useful gene-expression profiles in acute myeloid leukemia. N Engl J Med. 2004; 350(16):1617-1628. PubMedhttps://doi.org/10.1056/NEJMoa040465Google Scholar
  22. Verhaak RG, Wouters BJ, Erpelinck CA. Prediction of molecular subtypes in acute myeloid leukemia based on gene expression profiling. Haematologica. 2009; 94(1):131-134. PubMedhttps://doi.org/10.3324/haematol.13299Google Scholar
  23. Papaemmanuil E, Gerstung M, Malcovati L. Clinical and biological implications of driver mutations in myelodysplastic syndromes. Blood. 2013; 122(22):3616-3627. PubMedhttps://doi.org/10.1182/blood-2013-08-518886Google Scholar
  24. Gerstung M, Pellagatti A, Malcovati L. Combining gene mutation with gene expression data improves outcome prediction in myelodysplastic syndromes. Nat Commun. 2015; 6:5901. PubMedhttps://doi.org/10.1038/ncomms6901Google Scholar
  25. Bagger FO, Sasivarevic D, Sohi SH. BloodSpot: a database of gene expression profiles and transcriptional programs for healthy and malignant haematopoiesis. Nucleic Acids Res. 2016; 44(D1):D917-924. PubMedhttps://doi.org/10.1093/nar/gkv1101Google Scholar
  26. Al-Matary YS, Botezatu L, Opalka B. Acute myeloid leukemia cells polarize macrophages towards a leukemia supporting state in a Growth factor independence 1 dependent manner. Haematologica. 2016; 101(10):1216-1227. PubMedhttps://doi.org/10.3324/haematol.2016.143180Google Scholar
  27. Botezatu L, Michel LC, Helness A. Epigenetic therapy as a novel approach for GFI136N-associated murine/human AML. Exp Hematol. 2016; 44(8):713-726 e714. Google Scholar
  28. Khandanpour C, Krongold J, Schuette J. The human GFI136N variant induces epigenetic changes at the Hoxa9 locus and accelerates K-RAS driven myeloproliferative disorder in mice. Blood. 2012; 120(19):4006-4017. PubMedhttps://doi.org/10.1182/blood-2011-02-334722Google Scholar
  29. Hu Y, Smyth GK. ELDA: Extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods. 2009; 347(1–2):70-78. PubMedhttps://doi.org/10.1016/j.jim.2009.06.008Google Scholar
  30. Bonnet D, Dick JE. Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell. Nat Med. 1997; 3(7):730-737. PubMedhttps://doi.org/10.1038/nm0797-730Google Scholar
  31. Goardon N, Marchi E, Atzberger A. Coexistence of LMPP-like and GMP-like Leukemia Stem Cells in Acute Myeloid Leukemia. Cancer Cell. 2011; 19(1):138-152. PubMedhttps://doi.org/10.1016/j.ccr.2010.12.012Google Scholar
  32. Anguita E, Gupta R, Olariu V. A somatic mutation of GFI1B identified in leukemia alters cell fate via a SPI1 (PU.1) centered genetic regulatory network. Dev Biol. 2016; 411(2):277-286. Google Scholar
  33. Chowdhury AH, Ramroop JR, Upadhyay G, Sengupta A, Andrzejczyk A, Saleque S. Differential transcriptional regulation of meis1 by Gfi1b and its co-factors LSD1 and CoREST. PLoS One. 2013; 8(1):e53666. PubMedhttps://doi.org/10.1371/journal.pone.0053666Google Scholar
  34. Lin YW, Slape C, Zhang Z, Aplan PD. NUP98–HOXD13 transgenic mice develop a highly penetrant, severe myelodysplastic syndrome that progresses to acute leukemia. Blood. 2005; 106(1):287-295. PubMedhttps://doi.org/10.1182/blood-2004-12-4794Google Scholar
  35. Chan IT, Kutok JL, Williams IR. Conditional expression of oncogenic K-ras from its endogenous promoter induces a myeloproliferative disease. J Clin Invest. 2004; 113(4):528-538. PubMedhttps://doi.org/10.1172/JCI200420476Google Scholar
  36. Krivtsov AV, Twomey D, Feng Z. Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature. 2006; 442(7104):818-822. PubMedhttps://doi.org/10.1038/nature04980Google Scholar
  37. Meyer LH, Eckhoff SM, Queudeville M. Early relapse in all is identified by time to leukemia in NOD/SCID mice and is characterized by a gene signature involving survival pathways. Cancer Cell. 2011; 19(2):206-217. PubMedhttps://doi.org/10.1016/j.ccr.2010.11.014Google Scholar
  38. Hole PS, Darley RL, Tonks A. Do reactive oxygen species play a role in myeloid leukemias?. Blood. 2011; 117(22):5816-5826. PubMedhttps://doi.org/10.1182/blood-2011-01-326025Google Scholar
  39. Jang YY, Sharkis SJ. A low level of reactive oxygen species selects for primitive hematopoietic stem cells that may reside in the low-oxygenic niche. Blood. 2007; 110(8):3056-3063. PubMedhttps://doi.org/10.1182/blood-2007-05-087759Google Scholar
  40. Wang Y, Krivtsov AV, Sinha AU. The Wnt/beta-catenin pathway is required for the development of leukemia stem cells in AML. Science. 2010; 327(5973):1650-1653. PubMedhttps://doi.org/10.1126/science.1186624Google Scholar
  41. Hole PS, Zabkiewicz J, Munje C. Overproduction of NOX-derived ROS in AML promotes proliferation and is associated with defective oxidative stress signaling. Blood. 2013; 122(19):3322-3330. PubMedhttps://doi.org/10.1182/blood-2013-04-491944Google Scholar
  42. Essers MA, Offner S, Blanco-Bose WE. IFNalpha activates dormant haematopoietic stem cells in vivo. Nature. 2009; 458(7240):904-908. PubMedhttps://doi.org/10.1038/nature07815Google Scholar
  43. Ito K, Hirao A, Arai F. Reactive oxygen species act through p38 MAPK to limit the lifespan of hematopoietic stem cells. Nat Med. 2006; 12(4):446-451. PubMedhttps://doi.org/10.1038/nm1388Google Scholar
  44. Bulavin DV, Fornace AJ. p38 MAP kinase’s emerging role as a tumor suppressor. Adv Cancer Res. 2004; 92:95-118. PubMedhttps://doi.org/10.1016/S0065-230X(04)92005-2Google Scholar
  45. Park S, Chapuis N, Tamburini J. Role of the PI3K/AKT and mTOR signaling pathways in acute myeloid leukemia. Haematologica. 2010; 95(5):819-828. PubMedhttps://doi.org/10.3324/haematol.2009.013797Google Scholar
  46. Sykes SM, Lane SW, Bullinger L. AKT/FOXO signaling enforces reversible differentiation blockade in myeloid leukemias. Cell. 2011; 146(5):697-708. PubMedhttps://doi.org/10.1016/j.cell.2011.07.032Google Scholar
  47. Polfus LM, Khajuria RK, Schick UM. Whole-Exome Sequencing Identifies Loci Associated with Blood Cell Traits and Reveals a Role for Alternative GFI1B Splice Variants in Human Hematopoiesis. Am J Hum Genet. 2016; 99(3):785. Google Scholar
  48. Schulze H, Schlagenhauf A, Manukjan G. Recessive grey platelet-like syndrome with unaffected erythropoiesis in the absence of the splice isoform GFI1B-p37. Haematologica. 2017; 102(9):e375-e378. PubMedhttps://doi.org/10.3324/haematol.2017.167957Google Scholar
  49. Vassen L, Khandanpour C, Ebeling P. Growth factor independent 1b (Gfi1b) and a new splice variant of Gfi1b are highly expressed in patients with acute and chronic leukemia. Int J Hematol. 2009; 89(4):422-430. PubMedhttps://doi.org/10.1007/s12185-009-0286-5Google Scholar
  50. Sengupta A, Upadhyay G, Sen S, Saleque S. Reciprocal regulation of alternative lineages by Rgs18 and its transcriptional repressor Gfi1b. J Cell Sci. 2016; 129(1):145-154. PubMedhttps://doi.org/10.1242/jcs.177519Google Scholar

Article Information

Vol. 103 No. 4 (2018): April, 2018 : Articles
 
DOI
https://doi.org/10.3324/haematol.2017.167288
Pubmed
29326122
Pubmed Central
PMC5865438
 
Published
2018-04-01
Published By
Ferrata Storti Foundation, Pavia, Italy
Print ISSN
0390-6078
Online ISSN
1592-8721
 

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