Unraveling Novel Genes that Are Essential for Acute Myeloid Leukemia and Normal Hematopoiesis

• Main Author: Turköz, Gözde
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2018
• Subjects: Antigens; Apoptosis; Biochemistry; Bioinformatics; Blood; Bone marrow; Cancer therapies; Cell cycle; Cell growth; Cellular biology; Chemotherapy; Chromosomes; Classification; Cloning; DNA methylation; Down syndrome; Enzymes; Epigenetics; Gene expression; Genetics; Genomes; Kinases; Leukemia; Medical prognosis; Morphology; Mutation; Pathogenesis; Patients; Proteins; Therapy; Tumors
• ISBN: 9798744411930

Acute myeloid leukemia (AML) is the most common hematological malignancy among adults. This disease results from an abnormal expansion of myeloid progenitors that are blocked in differentiation. Malignant transformation arises from various genetic and epigenetic changes in hematopoietic stem cells (HSCs) that accumulate with age. The overall aim of this thesis was to discover and characterize novel factors that are essential for AML and normal hematopoiesis.In study I and study III, our aim was to discover and characterize novel genes that are selectively essential for AML. For this reason, we performed high throughput large-scale shRNA-based screens on human and mouse AML cell lines, and on non-transformed mouse hematopoietic Factor-Dependent Continuous Paterson Laboratories (FDCP mix) cell line as control cells. Based on the screening results, we have identified Chromatin Remodelling Factor 4 (CHD4) and General Transcription Factor II-I Repeat Domain-Containing Protein (GTF2IRD1) as the most promising hits, showing a highly significant selective importance in AML cell growth. Loss of function studies including both shRNA or CRISPR- Cas9 technology in normal mouse and human hematopoietic cells and various leukemia cell lines showed that either CHD4 or GTF2IRD1 were selectively required for AML cell growth, however their loss did not significantly affect normal hematopoietic cells. The importance of GTF2IRD1 or CHD4 was found to be conserved in primary AML cells, including leukemia-initiating cells, a using niche-like co-culture system. The importance of CHD4 in childhood AML was further supported by the fact that the shRNA-targeted childhood primary AML cells displayed a significantly lower level of engraftment when transplanted into a xenograft mouse model for AML. In addition, the downregulation of GTF2IRD1 in primary AML samples from both childhood and adult samples significantly prevented disease progression, compared to controls. The importance of CHD4 or GTF2IRD1 in AML was found to be associated with cell cycle progression via MYC and its target genes. Finally, using a heterologous reporter system we showed that GTF2IRD1 acts as a transcriptional repressor, us. In conclusion, we have shown that GTF2IRD1 is a transcriptional repressor required for AML maintenance both in vitro and in vivo, but not for normal hematopoietic cells. Our data demonstrated that CHD4 is a novel epigenetic factor required for the maintenance of childhood AML.In study II, we aimed to determine the role of Euchromatic Histone Lysine Methyltransferase 1 (EHMT1) in AML and to investigate the link to its homolog, EHMT2. Our in vitro data showed that both EHMT1 and EHMT2 were required for growth of various leukemic cell lines. CRISPR-Cas9 based individual knock-out of EHMT1 and EHMT2 prevented AML cell growth to a comparable degree, while double knock-out of EHMT1/2 did not show any additive or synergistic effect in growth of AML cell lines. Moreover, downregulation of EHMT1 using shRNA caused a remarkable reduction in engraftment of both childhood and adult primary AML cells in recipient mice, suggesting that downregulation of EHMT1 can prevent AML disease progression. Although downregulation of EHMT1 or EHMT2 caused a block in the G0 phase of the cell cycle, the apoptotic rate was not significantly increased.