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Modelling and targeting epigenetic regulators in acute leukemia

Leonards, Katharina. Modelling and targeting epigenetic regulators in acute leukemia. 2016, Doctoral Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_12144

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Abstract

A complex network of transcription factors controls self-renewal and differentiation of hematopoietic stem cells (HSC). Mutations or translocations of these epigenetic regulators may result in malignant transformation leading to acute leukemia. A significant fraction of childhood acute myeloid leukemia (AML) patients carry a translocation involving the nuclear receptor-binding SET domain protein 1 (NSD1) histone methyltransferase. To understand its function we ablated the gene in the hematopoietic system of the mouse. Surprisingly, all “Nsd1-null” (Vav1-iCre;Nsd1fl/fl) mice developed a lethal, malignant accumulation of CD71dim/+ TER119- erythroid progenitor cells with aberrant clonogenic activity and impaired terminal maturation of self-renewing erythroblasts in vitro, a phenotype that resembles human acute erythroleukemia. The lack of Nsd1 also reduced the number of HSC starting during fetal liver hematopoiesis. Although gene expression signatures revealed reduced mRNA expression of the erythroid master transcription factor Gata1, erythroblasts of Vav1-iCre;Nsd1fl/fl mice expressed constitutively high levels of GATA1 protein. Interestingly, the cells were significantly impaired in activation but still able to repress several known GATA1 targets. Strikingly, retroviral overexpression of Gata1 induced terminal maturation of Vav1-iCre;Nsd1fl/fl proerythroblasts which was associated with activation of GATA1 target genes.
Knockdown of NSD1 in human adult or cord-blood derived CD34+ HSC cells also impaired erythroid differentiation associated with increased protein levels of GATA1. In addition, we found high GATA1 protein levels in several human erythroleukemia cell lines suggesting a key role in aberrant erythroid differentiation. Currently ongoing experiments aim to mechanistically understand Nsd1-mediated GATA1 regulation and erythroid differentiation. Preliminary observations with peptide array-based in vitro methylation assays suggest the possibility for direct methylation of GATA1 by NSD1. We also found aberrant expression of erythroid- associated transcription factor complex members with increased levels of GATA1 and ETO2 but reduced levels of TAL1, E2A and LBD1. Moreover, significant changes in global histone H3K36 methylation were seen in proerythroblasts lacking Nsd1. Taken together, our data so far revealed Nsd1 as a novel regulator of normal and malignant erythropoiesis. Ongoing studies may not only provide mechanistic insights of aberrant transcriptional regulation leading to erythroleukemia but could also set the base to develop novel therapies against this rare but very aggressive disease that is currently incurable in most patients.
Next to histone methyltransferases like NSD1, histone acetyltransferases like CBP/p300 are recurrently involved in AML-associated chromosomal translocations and also serve as co-activators of other fusion oncogenes, suggesting therapeutic potential of specific targeting of CBP/p300. We characterized the anti-leukemic potential of I-CBP112, a novel small molecule chemical probe that selectively binds the CBP/p300 bromodomain (BRD). BRDs belong to a diverse family of evolutionary conserved protein-interaction modules recognizing acetylated lysine residues and thereby mediating recruitment of proteins to macromolecular complexes. We found that I-CBP112 significantly impaired the clonogenic activity of a series of murine cell lines immortalized by the MLL-CBP fusion and other leukemic fusion oncogenes (MLL-AF9, MLL-ENL, NUP98-HOXA9) in a dose-dependent manner. Similar to the murine cells, we found that I-CBP112 did not cause immediate cytotoxic effects but impaired colony formation and induced cellular differentiation of a series of 18 human leukemic cell lines. Likewise, I-CBP112 also reduced colony formation of human primary AML blasts but not of normal CD34+ HSC. Importantly, combination of I-CBP112 with another BRD inhibitor targeting BET proteins (JQ1) or with a chemotherapeutic agent (Doxorubicin) revealed clear synergistic effects on cell survival of the AML cell lines. Extreme limited dilution assays in methylcellulose, as well as bone marrow transplantation experiments revealed that I-CBP112 significantly impaired self-renewal of leukemic stem cells in vitro and reduced the leukemia-initiating potential in vivo. Taken together, we found that selective interference with the CBP/p300 BRD by I-CBP112 has the potential to selectively target leukemic stem cells and opens the way for novel combinatory “BRD inhibitor” therapies for AML. In addition to I-CBP112, we tested a pan- bromodomain inhibitor (“bromosporine”, BSP) broadly targeting BRDs including BET. Evaluation of BSP in BET- inhibitor sensitive and non-sensitive leukemic cell-lines revealed strong anti-proliferative activity in semi-solid medium. Similar to treatment with JQ1 (a selective BET inhibitor) BSP arrested in S- cell cycle phase suggesting BET-mediated effects. Finally, non-selective targeting of BRDs by BSP identified BETs as master regulators of primary transcription response in leukemia.
Collectively the experiments of this thesis investigated the role of epigenetic regulators in normal and malignant hematopoiesis and explored strategies for selective interference as novel anti-leukemic therapies.
Advisors:Schwaller, Jürg and Affolter, Markus and Mercher, Thomas
Faculties and Departments:03 Faculty of Medicine > Departement Biomedizin > Department of Biomedicine, University Children's Hospital > Childhood Leukemia (Schwaller)
UniBasel Contributors:Schwaller, Jürg and Affolter, Markus
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:12144
Thesis status:Complete
Bibsysno:Link to catalogue
Number of Pages:1 Online-Ressource (222 Seiten)
Language:English
Identification Number:
Last Modified:22 Apr 2018 04:32
Deposited On:18 May 2017 08:49

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