The Experts below are selected from a list of 1767 Experts worldwide ranked by ideXlab platform
Enric Redondo Monte - One of the best experts on this subject based on the ideXlab platform.
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zbtb7a prevents runx1 RUNX1T1 dependent clonal expansion of human hematopoietic stem and progenitor cells
Oncogene, 2020Co-Authors: Georg Leubolt, Enric Redondo Monte, Anja Wilding, Paul Kerbs, Johannes W Bagnoli, Luise HartmannAbstract:: ZBTB7A is frequently mutated in acute myeloid leukemia (AML) with t(8;21) translocation. However, the oncogenic collaboration between mutated ZBTB7A and the RUNX1-RUNX1T1 fusion gene in AML t(8;21) remains unclear. Here, we investigate the role of ZBTB7A and its mutations in the context of normal and malignant hematopoiesis. We demonstrate that clinically relevant ZBTB7A mutations in AML t(8;21) lead to loss of function and result in perturbed myeloid differentiation with block of the granulocytic lineage in favor of monocytic commitment. In addition, loss of ZBTB7A increases glycolysis and hence sensitizes leukemic blasts to metabolic inhibition with 2-deoxy-D-glucose. We observed that ectopic expression of wild-type ZBTB7A prevents RUNX1-RUNX1T1-mediated clonal expansion of human CD34+ cells, whereas the outgrowth of progenitors is enabled by ZBTB7A mutation. Finally, ZBTB7A expression in t(8;21) cells lead to a cell cycle arrest that could be mimicked by inhibition of glycolysis. Our findings suggest that loss of ZBTB7A may facilitate the onset of AML t(8;21), and that RUNX1-RUNX1T1-rearranged leukemia might be treated with glycolytic inhibitors.
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Loss of ZBTB7A Facilitates RUNX1/RUNX1T1-Dependent Clonal Expansion and Sensitizes for Metabolic Inhibition
Blood, 2018Co-Authors: Enric Redondo Monte, Luise Hartmann, Sayantanee Dutta, Georg Leubolt, Anja Wilding, Wolfgang Hiddemann, Lingping Chen-wichmann, Christian Wichmann, Philipp A. GreifAbstract:Abstract ZBTB7A is a transcription factor involved in the regulation of metabolism and hematopoietic linage fate decisions. Recently, we found ZBTB7A mutated in 23% of Acute Myeloid Leukemia (AML) patients with t(8;21) translocation (Hartmann et al., 2016, Nat Commun). However, the oncogenic collaboration between ZBTB7A alterations and the RUNX1/RUNX1T1 fusion in AML t(8;21) remains poorly understood. To study ZBTB7A mutations in the context of RUNX1/RUNX1T1-dependent transformation, we used human CD34+ cells co-transduced with a truncated form of RUNX1/RUNX1T1 and ZBTB7A wild-type (WT) or its mutants (R402C and A175fs). We then followed the evolution of fluorescence marker positive cells over a period of 60 days. While expression of RUNX1/RUNX1T1 alone caused clonal expansion, co-expression of ZBTB7A WT impaired the outgrowth of CD34+ cells (Figure 1a). In contrast, the anti-proliferative effect of ZBTB7A was lost for both of its mutants tested resulting in a rescue of the clonal expansion (Figure 1b). To investigate the effect of ZBTB7A mutations on tumor metabolism, we used CRISPR/Cas9 to knockout (KO) ZBTB7A in the myeloid K562 cell line. As ZBTB7A is a known negative regulator of glycolysis, we treated KO and control cells with the glycolysis inhibitor 2-deoxy-d-glucouse (2-DG). KO cells were more sensitive to 2-DG compared to control cells (mean IC50: 3.06 vs 6.82 mM; p-value=0.087) (Figure 1c). These results are in line with the observed upregulation of glycolytic genes in ZBTB7A-mutant AML t(8;21) and suggest that these patients may benefit from the treatment with metabolic inhibitors. To learn more about deregulation of ZBTB7A target genes we are currently performing RNA-Seq analysis of WT vs KO K562. Moreover, we used the K562 KO model to investigate the impact of loss of ZBTB7A on myeloid differentiation. The baseline expression of the erythroid marker CD235a was reduced in KO cells. Ectopic expression of ZBTB7A WT in the KO cells restored the CD235a levels to a control level, while expression of mutants or vector showed no effect. These findings are in agreement with previous reports of ZBTB7A involvement in erythroid differentiation. To study the effect of ZBTB7A mutations on granulopoiesis, we established HL60 cells stably expressing WT or mutant ZBTB7A. We then differentiated the cells into granulocytes through all-trans retinoic acid (ATRA) treatment. Expression of WT, but not the mutants, resulted in a 4-fold increase of the granulocytic marker CD11b. Additionally, we induced monocytic differentiation through Phorbol 12-myristate 13-acetate (PMA) treatment. The mutant expressing cells showed similar levels of the monocytic marker CD14 as control cells. WT overexpressing cells had a 50% decrease in the number of monocytes. We then used CRISPR/Cas9 to establish ZBTB7A KO HL60, which exhibited a 5.5-fold increase in CD14 compared to control cells (Figure 1d). This data supports a previously unknown negative regulatory role of ZBTB7A in monocytic differentiation. With regards to potential therapeutic applications, we tested the PMA sensitivity of HL60 and found a significantly lower IC50 in absence of ZBTB7A (mean: 124.5 vs 269.8 pM; p-value=0.001). Hence, loss of ZBTB7A may facilitate the pharmacological differentiation of leukemia cells. In conclusion, ZBTB7A mutations in AML t(8;21) display a loss-of-function phenotype. Inactivating mutations of ZBTB7A allow for hCD34+ RUNX1/RUNX1T1-mediated expansion and deregulated tumor metabolism. Finally, loss of ZBTB7A expression perturbs myeloid development and thus may complement the block of differentiation induced by RUNX1/RUNX1T1. These findings open up avenues to novel therapies for ZBTB7A mutated patients including metabolic inhibition and pharmacological differentiation. Disclosures Hiddemann: F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
Vasily V Grinev - One of the best experts on this subject based on the ideXlab platform.
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The contribution of various mechanisms to mRNA diversity of human fusion oncogene RUNX1-RUNX1T1
Journal of the Belarusian State University. Biology, 2019Co-Authors: Ilya M Ilyushonak, Alexandr A Migas, Andrei Yu. Sukhareuski, Aksana D. Schneider, Vasily V GrinevAbstract:In this work, we used a comprehensive set of the fusion oncogene RUNX1-RUNX1T1 alternative exons to analyze the patterns of its mRNA generation. We found that the waste majority of alternative exons are modified variants of canonical exons, and the transcripts, including such exons, have a very low expression level. The «hot regions», including exons 4a, 6, 8b, 9, 11 and 12, produces about 80 % of such variants. Also we described a new transcription start region of RUNX1-RUNX1T1 and provide the evidences of co-expression of the fusion RNAs with normal and shortened 3′-UTRs in leukemic cells.
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runx1 RUNX1T1 controls alternative splicing in the t 8 21 positive acute myeloid leukemia cells
bioRxiv, 2019Co-Authors: Vasily V Grinev, Tatiana V Ramanouskaya, Ilya M Ilyushonak, Richard Clough, Sirintra Nakjang, Job Smink, N Martinezsoria, Constanze Bonifer, Olaf HeidenreichAbstract:SUMMARY The fusion oncogene RUNX1/RUNX1T1 encodes an aberrant transcription factor, which plays a key role in the initiation and maintenance of the t(8;21)-positive acute myeloid leukemia. Here we show that this oncogene is a regulator of the alternative RNA splicing for a sub-set of genes in the leukemia cells. We found two primary mechanisms underlying changes in the production of RNA isoforms: (i) RUNX1/RUNX1T1-mediated regulation of alternative transcription start sites selection in target genes, and (ii) direct or indirect control of the expression of the genes encoding splicing factors. The first mechanism leads to the expression of RNA isoforms with alternative structure of the 5’-UTR regions. The second mechanism generates alternative transcripts with new junctions between internal cassettes and constitutive exons. We also show that RUNX1/RUNX1T1-mediated differential splicing affects several functional groups of genes and produces proteins with unique conserved domain structures. In summary, this study reveals a novel layer of RUNX1/RUNX1T1-dependent transcriptome organization in t(8;21)-positive acute myeloid leukemia.
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RUNX1/RUNX1T1 controls alternative splicing in the t(8;21)-positive acute myeloid leukemia cells
bioRxiv, 2019Co-Authors: Vasily V Grinev, Tatiana V Ramanouskaya, Richard Clough, Sirintra Nakjang, Job Smink, Constanze Bonifer, Ilia M. Ilyushonak, N Martinez-soria, Olaf HeidenreichAbstract:SUMMARY The fusion oncogene RUNX1/RUNX1T1 encodes an aberrant transcription factor, which plays a key role in the initiation and maintenance of the t(8;21)-positive acute myeloid leukemia. Here we show that this oncogene is a regulator of the alternative RNA splicing for a sub-set of genes in the leukemia cells. We found two primary mechanisms underlying changes in the production of RNA isoforms: (i) RUNX1/RUNX1T1-mediated regulation of alternative transcription start sites selection in target genes, and (ii) direct or indirect control of the expression of the genes encoding splicing factors. The first mechanism leads to the expression of RNA isoforms with alternative structure of the 5’-UTR regions. The second mechanism generates alternative transcripts with new junctions between internal cassettes and constitutive exons. We also show that RUNX1/RUNX1T1-mediated differential splicing affects several functional groups of genes and produces proteins with unique conserved domain structures. In summary, this study reveals a novel layer of RUNX1/RUNX1T1-dependent transcriptome organization in t(8;21)-positive acute myeloid leukemia.
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decoding of exon splicing patterns in the human runx1 RUNX1T1 fusion gene
The International Journal of Biochemistry & Cell Biology, 2015Co-Authors: Vasily V Grinev, Alexandr A Migas, Olga A Mishkova, Ilia M. Ilyushonak, Aksana D Kirsanava, Natalia Siomava, Tatiana V Ramanouskaya, Alina V Vaitsiankova, Petr V Nazarov, Laurent VallarAbstract:The t(8;21) translocation is the most widespread genetic defect found in human acute myeloid leukemia. This translocation results in the RUNX1–RUNX1T1 fusion gene that produces a wide variety of alternative transcripts and influences the course of the disease. The rules of combinatorics and splicing of exons in the RUNX1–RUNX1T1 transcripts are not known. To address this issue, we developed an exon graph model of the fusion gene organization and evaluated its local exon combinatorics by the exon combinatorial index (ECI). Here we show that the local exon combinatorics of the RUNX1–RUNX1T1 gene follows a power-law behavior and (i) the vast majority of exons has a low ECI, (ii) only a small part is represented by “exons-hubs” of splicing with very high ECI values, and (iii) it is scale-free and very sensitive to targeted skipping of “exons-hubs”. Stochasticity of the splicing machinery and preferred usage of exons in alternative splicing can explain such behavior of the system. Stochasticity may explain up to 12% of the ECI variance and results in a number of non-coding and unproductive transcripts that can be considered as a noise. Half-life of these transcripts is increased due to the deregulation of some key genes of the nonsense-mediated decay system in leukemia cells. On the other hand, preferred usage of exons may explain up to 75% of the ECI variability. Our analysis revealed a set of splicing-related cis-regulatory motifs that can explain “attractiveness” of exons in alternative splicing but only when they are considered together. Cis-regulatory motifs are guides for splicing trans-factors and we observed a leukemia-specific profile of expression of the splicing genes in t(8;21)-positive blasts. Altogether, our results show that alternative splicing of the RUNX1–RUNX1T1 transcripts follows strict rules and that the power-law component of the fusion gene organization confers a high flexibility to this process.
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RUNX1T1 mtg8 eto gene expression status in human t 8 21 q22 q22 positive acute myeloid leukemia cells
Leukemia Research, 2014Co-Authors: Alexandr A Migas, Olga A Mishkova, Tatiana V Ramanouskaya, Ilya M Ilyushonak, Olga V Aleinikova, Vasily V GrinevAbstract:Abstract The RUNX1 - RUNX1T1 fusion gene, a product of the nonhomologous balanced translocation t(8;21)(q22;q22), is a complex genetic locus. We performed extensive bioinformatic analysis of transcription initiation as well as transcription termination sites in this locus and predicted a number of different RUNX1T1 transcripts. To confirm and quantify the RUNX1T1 gene expression, we analyzed samples from seven acute myeloid leukemia (AML) patients and from the Kasumi-1 cell line. We found variable activity of the four predicted RUNX1T1 promoters located downstream of the chromosome breakpoint. Nineteen alternative RUNX1T1 transcripts were identified by sequencing at least seventeen of which predictably can be translated into functional proteins. While the RUNX1T1 gene is not expressed in normal hematopoietic cells, it may participate in t(8;21)(q22;q22)-dependent leukemic transformation due to its multiple interactions in cell regulatory network particularly through synergistic or antagonistic effects in relation to activity of RUNX1 - RUNX1T1 fusion gene.
Woongyang Park - One of the best experts on this subject based on the ideXlab platform.
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abstract 1723 elucidation of novel therapeutic targets for acute myeloid leukemias with runx1 RUNX1T1 fusion
Cancer Research, 2019Co-Authors: Sejong Chun, Woongyang ParkAbstract:The RUNX1-RUNX1T1 fusion is a frequent chromosomal alteration in acute myeloid leukemias (AMLs). Although RUNX1-RUNX1T1 fusion protein has pivotal roles in the development of AMLs with the fusion, RUNX1-RUNX1T1 fusion protein is difficult to target since it lacks kinase activities. Here, we used a sophisticated bioinformatic tool to elucidate targetable signaling pathways in AMLs with RUNX1-RUNX1T1 fusion. After analysis of 93 AMLs from the TCGA database, the expressions of 293 genes were significantly correlated with the expression of the RUNX1-RUNX1T1 fusion gene. Based on the 293 genes, the COX, VEGFR, PDGFR, and FGFR pathways are predicted to be specifically activated in AMLs with RUNX1-RUNX1T1 fusion. Compared with the AML cells without the fusion, the specific activations of the AML cell lines with RUNX1-RUNX1T1 fusion were confirmed by the increased phosphorylation of VEGFRs, PDGFRs, and FGFRs. Moreover, the in vitro proliferation of AML cells with RUNX1-RUNX1T1 fusion decreased significantly more than that of AML cells without the fusion when the pathways were inhibited pharmacologically. The results indicate that novel targetable signaling pathways could be identified by the analysis of the gene expression features of AMLs with non-targetable genetic alterations. The elucidation of specific molecular targets for AMLs that have a specific genetic alteration would promote personalized treatment of AMLs and improve treatment outcomes for AML patients in clinic. Note: This abstract was not presented at the meeting. Citation Format: Jae Won Yun, Yoon Kyung Bae, So Yeong Cho, Hee-Jin Kim, Do-Hyun Nam, Sun-Hee Kim, Sejong Chun, Kyeung Min Joo, Hye Won Lee, Woong-Yang Park. Elucidation of novel therapeutic targets for acute myeloid leukemias with RUNX1 - RUNX1T1 fusion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1723.
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elucidation of novel therapeutic targets for acute myeloid leukemias with runx1 RUNX1T1 fusion
International Journal of Molecular Sciences, 2019Co-Authors: Sejong Chun, Woongyang ParkAbstract:The RUNX1-RUNX1T1 fusion is a frequent chromosomal alteration in acute myeloid leukemias (AMLs). Although RUNX1-RUNX1T1 fusion protein has pivotal roles in the development of AMLs with the fusion, RUNX1-RUNX1T1, fusion protein is difficult to target, as it lacks kinase activities. Here, we used bioinformatic tools to elucidate targetable signaling pathways in AMLs with RUNX1-RUNX1T1 fusion. After analysis of 93 AML cases from The Cancer Genome Atlas (TCGA) database, we found expression of 293 genes that correlated to the expression of the RUNX1-RUNX1T1 fusion gene. Based on these 293 genes, the cyclooxygenase (COX), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR) pathways were predicted to be specifically activated in AMLs with RUNX1-RUNX1T1 fusion. Moreover, the in vitro proliferation of AML cells with RUNX1-RUNX1T1 fusion decreased significantly more than that of AML cells without the fusion, when the pathways were inhibited pharmacologically. The results indicate that novel targetable signaling pathways could be identified by the analysis of the gene expression features of AMLs with non-targetable genetic alterations. The elucidation of specific molecular targets for AMLs that have a specific genetic alteration would promote personalized treatment of AMLs and improve clinical outcomes.
Luise Hartmann - One of the best experts on this subject based on the ideXlab platform.
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zbtb7a prevents runx1 RUNX1T1 dependent clonal expansion of human hematopoietic stem and progenitor cells
Oncogene, 2020Co-Authors: Georg Leubolt, Enric Redondo Monte, Anja Wilding, Paul Kerbs, Johannes W Bagnoli, Luise HartmannAbstract:: ZBTB7A is frequently mutated in acute myeloid leukemia (AML) with t(8;21) translocation. However, the oncogenic collaboration between mutated ZBTB7A and the RUNX1-RUNX1T1 fusion gene in AML t(8;21) remains unclear. Here, we investigate the role of ZBTB7A and its mutations in the context of normal and malignant hematopoiesis. We demonstrate that clinically relevant ZBTB7A mutations in AML t(8;21) lead to loss of function and result in perturbed myeloid differentiation with block of the granulocytic lineage in favor of monocytic commitment. In addition, loss of ZBTB7A increases glycolysis and hence sensitizes leukemic blasts to metabolic inhibition with 2-deoxy-D-glucose. We observed that ectopic expression of wild-type ZBTB7A prevents RUNX1-RUNX1T1-mediated clonal expansion of human CD34+ cells, whereas the outgrowth of progenitors is enabled by ZBTB7A mutation. Finally, ZBTB7A expression in t(8;21) cells lead to a cell cycle arrest that could be mimicked by inhibition of glycolysis. Our findings suggest that loss of ZBTB7A may facilitate the onset of AML t(8;21), and that RUNX1-RUNX1T1-rearranged leukemia might be treated with glycolytic inhibitors.
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Loss of ZBTB7A Facilitates RUNX1/RUNX1T1-Dependent Clonal Expansion and Sensitizes for Metabolic Inhibition
Blood, 2018Co-Authors: Enric Redondo Monte, Luise Hartmann, Sayantanee Dutta, Georg Leubolt, Anja Wilding, Wolfgang Hiddemann, Lingping Chen-wichmann, Christian Wichmann, Philipp A. GreifAbstract:Abstract ZBTB7A is a transcription factor involved in the regulation of metabolism and hematopoietic linage fate decisions. Recently, we found ZBTB7A mutated in 23% of Acute Myeloid Leukemia (AML) patients with t(8;21) translocation (Hartmann et al., 2016, Nat Commun). However, the oncogenic collaboration between ZBTB7A alterations and the RUNX1/RUNX1T1 fusion in AML t(8;21) remains poorly understood. To study ZBTB7A mutations in the context of RUNX1/RUNX1T1-dependent transformation, we used human CD34+ cells co-transduced with a truncated form of RUNX1/RUNX1T1 and ZBTB7A wild-type (WT) or its mutants (R402C and A175fs). We then followed the evolution of fluorescence marker positive cells over a period of 60 days. While expression of RUNX1/RUNX1T1 alone caused clonal expansion, co-expression of ZBTB7A WT impaired the outgrowth of CD34+ cells (Figure 1a). In contrast, the anti-proliferative effect of ZBTB7A was lost for both of its mutants tested resulting in a rescue of the clonal expansion (Figure 1b). To investigate the effect of ZBTB7A mutations on tumor metabolism, we used CRISPR/Cas9 to knockout (KO) ZBTB7A in the myeloid K562 cell line. As ZBTB7A is a known negative regulator of glycolysis, we treated KO and control cells with the glycolysis inhibitor 2-deoxy-d-glucouse (2-DG). KO cells were more sensitive to 2-DG compared to control cells (mean IC50: 3.06 vs 6.82 mM; p-value=0.087) (Figure 1c). These results are in line with the observed upregulation of glycolytic genes in ZBTB7A-mutant AML t(8;21) and suggest that these patients may benefit from the treatment with metabolic inhibitors. To learn more about deregulation of ZBTB7A target genes we are currently performing RNA-Seq analysis of WT vs KO K562. Moreover, we used the K562 KO model to investigate the impact of loss of ZBTB7A on myeloid differentiation. The baseline expression of the erythroid marker CD235a was reduced in KO cells. Ectopic expression of ZBTB7A WT in the KO cells restored the CD235a levels to a control level, while expression of mutants or vector showed no effect. These findings are in agreement with previous reports of ZBTB7A involvement in erythroid differentiation. To study the effect of ZBTB7A mutations on granulopoiesis, we established HL60 cells stably expressing WT or mutant ZBTB7A. We then differentiated the cells into granulocytes through all-trans retinoic acid (ATRA) treatment. Expression of WT, but not the mutants, resulted in a 4-fold increase of the granulocytic marker CD11b. Additionally, we induced monocytic differentiation through Phorbol 12-myristate 13-acetate (PMA) treatment. The mutant expressing cells showed similar levels of the monocytic marker CD14 as control cells. WT overexpressing cells had a 50% decrease in the number of monocytes. We then used CRISPR/Cas9 to establish ZBTB7A KO HL60, which exhibited a 5.5-fold increase in CD14 compared to control cells (Figure 1d). This data supports a previously unknown negative regulatory role of ZBTB7A in monocytic differentiation. With regards to potential therapeutic applications, we tested the PMA sensitivity of HL60 and found a significantly lower IC50 in absence of ZBTB7A (mean: 124.5 vs 269.8 pM; p-value=0.001). Hence, loss of ZBTB7A may facilitate the pharmacological differentiation of leukemia cells. In conclusion, ZBTB7A mutations in AML t(8;21) display a loss-of-function phenotype. Inactivating mutations of ZBTB7A allow for hCD34+ RUNX1/RUNX1T1-mediated expansion and deregulated tumor metabolism. Finally, loss of ZBTB7A expression perturbs myeloid development and thus may complement the block of differentiation induced by RUNX1/RUNX1T1. These findings open up avenues to novel therapies for ZBTB7A mutated patients including metabolic inhibition and pharmacological differentiation. Disclosures Hiddemann: F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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ZBTB7A mutations in acute myeloid leukaemia with t(8;21) translocation
Nature Communications, 2016Co-Authors: Luise Hartmann, Sayantanee Dutta, Sabrina Opatz, Sebastian Vosberg, Katrin Reiter, Georg Leubolt, Klaus H. Metzeler, Tobias Herold, Stefanos A. Bamopoulos, Kathrin BräundlAbstract:The t(8;21) translocation is one of the most frequent cytogenetic abnormalities in acute myeloid leukaemia (AML) and results in the RUNX1/RUNX1T1 rearrangement. Despite the causative role of the RUNX1/RUNX1T1 fusion gene in leukaemia initiation, additional genetic lesions are required for disease development. Here we identify recurring ZBTB7A mutations in 23% (13/56) of AML t(8;21) patients, including missense and truncating mutations resulting in alteration or loss of the C-terminal zinc-finger domain of ZBTB7A. The transcription factor ZBTB7A is important for haematopoietic lineage fate decisions and for regulation of glycolysis. On a functional level, we show that ZBTB7A mutations disrupt the transcriptional repressor potential and the anti-proliferative effect of ZBTB7A. The specific association of ZBTB7A mutations with t(8;21) rearranged AML points towards leukaemogenic cooperativity between mutant ZBTB7A and the RUNX1/RUNX1T1 fusion.
Georg Leubolt - One of the best experts on this subject based on the ideXlab platform.
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zbtb7a prevents runx1 RUNX1T1 dependent clonal expansion of human hematopoietic stem and progenitor cells
Oncogene, 2020Co-Authors: Georg Leubolt, Enric Redondo Monte, Anja Wilding, Paul Kerbs, Johannes W Bagnoli, Luise HartmannAbstract:: ZBTB7A is frequently mutated in acute myeloid leukemia (AML) with t(8;21) translocation. However, the oncogenic collaboration between mutated ZBTB7A and the RUNX1-RUNX1T1 fusion gene in AML t(8;21) remains unclear. Here, we investigate the role of ZBTB7A and its mutations in the context of normal and malignant hematopoiesis. We demonstrate that clinically relevant ZBTB7A mutations in AML t(8;21) lead to loss of function and result in perturbed myeloid differentiation with block of the granulocytic lineage in favor of monocytic commitment. In addition, loss of ZBTB7A increases glycolysis and hence sensitizes leukemic blasts to metabolic inhibition with 2-deoxy-D-glucose. We observed that ectopic expression of wild-type ZBTB7A prevents RUNX1-RUNX1T1-mediated clonal expansion of human CD34+ cells, whereas the outgrowth of progenitors is enabled by ZBTB7A mutation. Finally, ZBTB7A expression in t(8;21) cells lead to a cell cycle arrest that could be mimicked by inhibition of glycolysis. Our findings suggest that loss of ZBTB7A may facilitate the onset of AML t(8;21), and that RUNX1-RUNX1T1-rearranged leukemia might be treated with glycolytic inhibitors.
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Loss of ZBTB7A Facilitates RUNX1/RUNX1T1-Dependent Clonal Expansion and Sensitizes for Metabolic Inhibition
Blood, 2018Co-Authors: Enric Redondo Monte, Luise Hartmann, Sayantanee Dutta, Georg Leubolt, Anja Wilding, Wolfgang Hiddemann, Lingping Chen-wichmann, Christian Wichmann, Philipp A. GreifAbstract:Abstract ZBTB7A is a transcription factor involved in the regulation of metabolism and hematopoietic linage fate decisions. Recently, we found ZBTB7A mutated in 23% of Acute Myeloid Leukemia (AML) patients with t(8;21) translocation (Hartmann et al., 2016, Nat Commun). However, the oncogenic collaboration between ZBTB7A alterations and the RUNX1/RUNX1T1 fusion in AML t(8;21) remains poorly understood. To study ZBTB7A mutations in the context of RUNX1/RUNX1T1-dependent transformation, we used human CD34+ cells co-transduced with a truncated form of RUNX1/RUNX1T1 and ZBTB7A wild-type (WT) or its mutants (R402C and A175fs). We then followed the evolution of fluorescence marker positive cells over a period of 60 days. While expression of RUNX1/RUNX1T1 alone caused clonal expansion, co-expression of ZBTB7A WT impaired the outgrowth of CD34+ cells (Figure 1a). In contrast, the anti-proliferative effect of ZBTB7A was lost for both of its mutants tested resulting in a rescue of the clonal expansion (Figure 1b). To investigate the effect of ZBTB7A mutations on tumor metabolism, we used CRISPR/Cas9 to knockout (KO) ZBTB7A in the myeloid K562 cell line. As ZBTB7A is a known negative regulator of glycolysis, we treated KO and control cells with the glycolysis inhibitor 2-deoxy-d-glucouse (2-DG). KO cells were more sensitive to 2-DG compared to control cells (mean IC50: 3.06 vs 6.82 mM; p-value=0.087) (Figure 1c). These results are in line with the observed upregulation of glycolytic genes in ZBTB7A-mutant AML t(8;21) and suggest that these patients may benefit from the treatment with metabolic inhibitors. To learn more about deregulation of ZBTB7A target genes we are currently performing RNA-Seq analysis of WT vs KO K562. Moreover, we used the K562 KO model to investigate the impact of loss of ZBTB7A on myeloid differentiation. The baseline expression of the erythroid marker CD235a was reduced in KO cells. Ectopic expression of ZBTB7A WT in the KO cells restored the CD235a levels to a control level, while expression of mutants or vector showed no effect. These findings are in agreement with previous reports of ZBTB7A involvement in erythroid differentiation. To study the effect of ZBTB7A mutations on granulopoiesis, we established HL60 cells stably expressing WT or mutant ZBTB7A. We then differentiated the cells into granulocytes through all-trans retinoic acid (ATRA) treatment. Expression of WT, but not the mutants, resulted in a 4-fold increase of the granulocytic marker CD11b. Additionally, we induced monocytic differentiation through Phorbol 12-myristate 13-acetate (PMA) treatment. The mutant expressing cells showed similar levels of the monocytic marker CD14 as control cells. WT overexpressing cells had a 50% decrease in the number of monocytes. We then used CRISPR/Cas9 to establish ZBTB7A KO HL60, which exhibited a 5.5-fold increase in CD14 compared to control cells (Figure 1d). This data supports a previously unknown negative regulatory role of ZBTB7A in monocytic differentiation. With regards to potential therapeutic applications, we tested the PMA sensitivity of HL60 and found a significantly lower IC50 in absence of ZBTB7A (mean: 124.5 vs 269.8 pM; p-value=0.001). Hence, loss of ZBTB7A may facilitate the pharmacological differentiation of leukemia cells. In conclusion, ZBTB7A mutations in AML t(8;21) display a loss-of-function phenotype. Inactivating mutations of ZBTB7A allow for hCD34+ RUNX1/RUNX1T1-mediated expansion and deregulated tumor metabolism. Finally, loss of ZBTB7A expression perturbs myeloid development and thus may complement the block of differentiation induced by RUNX1/RUNX1T1. These findings open up avenues to novel therapies for ZBTB7A mutated patients including metabolic inhibition and pharmacological differentiation. Disclosures Hiddemann: F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.
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ZBTB7A mutations in acute myeloid leukaemia with t(8;21) translocation
Nature Communications, 2016Co-Authors: Luise Hartmann, Sayantanee Dutta, Sabrina Opatz, Sebastian Vosberg, Katrin Reiter, Georg Leubolt, Klaus H. Metzeler, Tobias Herold, Stefanos A. Bamopoulos, Kathrin BräundlAbstract:The t(8;21) translocation is one of the most frequent cytogenetic abnormalities in acute myeloid leukaemia (AML) and results in the RUNX1/RUNX1T1 rearrangement. Despite the causative role of the RUNX1/RUNX1T1 fusion gene in leukaemia initiation, additional genetic lesions are required for disease development. Here we identify recurring ZBTB7A mutations in 23% (13/56) of AML t(8;21) patients, including missense and truncating mutations resulting in alteration or loss of the C-terminal zinc-finger domain of ZBTB7A. The transcription factor ZBTB7A is important for haematopoietic lineage fate decisions and for regulation of glycolysis. On a functional level, we show that ZBTB7A mutations disrupt the transcriptional repressor potential and the anti-proliferative effect of ZBTB7A. The specific association of ZBTB7A mutations with t(8;21) rearranged AML points towards leukaemogenic cooperativity between mutant ZBTB7A and the RUNX1/RUNX1T1 fusion.
