Epigenetic-Mediated Apoptosis in Aggressive B-Cell Lymphomas
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Epigenetic-mediated apoptosis in aggressive B-cell lymphomas Inaugural-Dissertation to obtain the academic degree Doctor rerum naturalium (Dr. rer. nat.) submitted to the Department of Biology, Chemistry and Pharmacy of Freie Universität Berlin by Chidimma Agatha Akpa 2019 This dissertation was prepared at the Medical University, Berlin Institute of Pathology Department of Experimental Hematopathology under the supervision of Prof. Dr. Michael Hummel from January 2016 till November 2019 1st Reviewer: Prof. Dr. Michael Hummel 2nd Reviewer: Prof. Dr. Rupert Mutzel Date of defense: 16.03.2020 2 Directory Summary .................................................................................................................................... 7 Zusammenfassung ..................................................................................................................... 9 CHAPTER ONE ...................................................................................................................... 11 BACKGROUND .................................................................................................................. 11 1.1 Lymphomas .................................................................................................................... 11 1.1.1 Burkitt lymphoma (BL) ........................................................................................... 11 1.1.2 Diffuse large B-cell lymphoma (DLBCL) ............................................................... 12 1.2 Epigenetics and lymphoma ............................................................................................. 13 1.2.1 Polycomb group proteins (PGPs) ............................................................................. 15 1.2.2 Enhancer of zeste homolog 1/2 (EZH1 / EZH2) ..................................................... 16 1.2.3 EZH2 inhibitors ....................................................................................................... 17 1.3 3-Deazaneplanocin A (DZNep) and S-adenosylhomocysteine hydrolase (AHCY) ...... 18 1.4 Aims of the study ............................................................................................................ 19 CHAPTER TWO ..................................................................................................................... 20 MATERIALS AND METHODS ......................................................................................... 20 2.1 Cell lines and cell culture ............................................................................................... 20 2.2 Drug preparation ............................................................................................................. 20 2.3 Flow cytometry ............................................................................................................... 20 2.4 Generation of a DZNep-resistant BLUE-1 variant ......................................................... 21 2.5 Inhibitory concentration 50 (IC50) analysis .................................................................... 22 2.6 Cell fractionation and Western blotting .......................................................................... 22 2.7 RNA extraction and real-time reverse transcriptase polymerase chain reaction (RT- PCR) ..................................................................................................................................... 22 2.7.1 Proliferation assay .................................................................................................... 22 2.7.2 AHCY gene expression assay ................................................................................... 23 2.8 DNA extraction, polymerase chain reaction (PCR) and Sanger sequencing ................. 24 2.9 Fluorescence in situ hybridization (FISH) studies ......................................................... 25 3 2.10 Whole exome sequencing (WES) ................................................................................. 26 2.11 Copy number variation (CNV) assay ........................................................................... 26 2.12 Global DNA methylation analysis ................................................................................ 26 2.13 Cytogenetics, OncoScan CNV assay and metabolomics assay .................................... 27 2.14 Immunohistochemistry (IHC) ....................................................................................... 27 2.15 Stable AHCY transfection of cell lines ........................................................................ 28 2.15.1 AHCY Knockdown ................................................................................................ 28 2.15.2 AHCY overexpression ........................................................................................... 28 2.16 Statistical analysis ......................................................................................................... 28 CHAPTER THREE ................................................................................................................. 29 RESULTS ............................................................................................................................. 29 3.1. DZNep causes apoptosis in lymphoma cell lines .......................................................... 29 3.2. Sensitivity to DZNep is independent of the EZH2 mutation status, lymphoma type, and MYC, BCL2 and BCL6 translocations .................................................................................. 33 3.3 Consequence of mutated EZH2 on H3K27me3 in B-cell lymphoma cell lines ............. 37 3.4 DZNep induces apoptosis by the inhibition of EZH2 and downregulation of H3K27me3 in lymphoma ......................................................................................................................... 37 3.5 DZNep suppresses the proliferation of lymphoma cells................................................. 39 3.6 Comparison of the apoptotic effect of DZNep and EPZ-6438 ....................................... 40 3.7 Characterization of the generated DZNep-resistant clone .............................................. 43 3.8 Identification of biomarkers underlying the resistance of BLUE-1R10 ......................... 48 3.9 Validation of the detected AHCY copy number gain ...................................................... 51 3.9.1 Validation of AHCY copy number gain at the DNA level ....................................... 51 3.9.2 Validation of AHCY copy number gain at the chromosomal level .......................... 56 3.9.3 Validation of AHCY copy number gain at the transcriptional level ......................... 57 3.9.4 Validation of AHCY copy number gain at the protein level .................................... 58 3.9.5 Validation of AHCY copy number in primary patient samples ................................ 60 3.10 Effect of DZNep on AHCY expression ........................................................................ 61 3.11 Metabolomics ............................................................................................................... 62 4 3.12. Global DNA methylation profile of BLUE-1, BLUE-1K10 and BLUE-1R10 ........... 63 3.13. Functional validation of AHCY copy number gain (AHCY knockdown assays) ........ 68 3.14. Functional validation of AHCY copy number gain (AHCY overexpression assays) .. 74 3.15. AHCY alterations in other DZNep-resistant cell lines ................................................. 76 3.16. Analysis of publicly available genomic data sets for alterations of AHCY ................. 77 CHAPTER FOUR ................................................................................................................... 78 DISCUSSION ....................................................................................................................... 78 4.1 DZNep induces apoptosis and inhibits proliferation in B-cell lymphoma irrespective of the lymphoma type, the EZH2 mutation state and the presence of MYC, BCL2 and BCL6 translocations ........................................................................................................................ 78 4.1.1 Many cell lines are sensitive to DZNep while some are resistant ........................... 78 4.1.2 Sensitivity of B-cell lymphoma cell lines to DZNep does not depend on the EZH2 mutation status and MYC, BCL2 and BCL6 translocations ............................................... 79 4.1.3 Impact of mutated and wild-type EZH2 on H3K27me3 in B-cell lymphoma cell lines ................................................................................................................................... 80 4.1.4 DZNep inhibits proliferation of B-cell lymphoma cell lines ................................... 80 4.1.5 DZNep shows a stronger apoptotic effect in B-cell lymphoma cell lines compared to EPZ-6438 ...................................................................................................................... 80 4.2 Alterations of the AHCY gene could be linked with the resistance of B-cell lymphoma to DZNep-mediated apoptosis .................................................................................................. 81 4.2.1 Characterization of the DZNep-resistant clone possessing acquired resistance to DZNep ............................................................................................................................... 81 4.2.2 Detection of biomarkers determining DZNep resistance ........................................