Genetic and epigenetic studies of acute myeloid leukemia and therapeutic possibilities

Sammanfattning: Acute myeloid leukaemia (AML) is malignant tumour that forms in the bone marrow and arises from immature myeloid progenitors. Consequently, this leads to excessive accumulation of dysfunctional blast cells and lack of normal blood cells. The molecular and genetic heterogeneity of the disease is substantial which makes the disease challenging to classify and treat. Although the AML classification is updated continuously and more data and research on AML pathophysiology emerges, first line treatment for the vast majority of AML patients remains a combination of cytarabine and an anthracycline. While most patients attain a complete remission, the majority of AML patients relapse and develop drug resistance. Recently, new drugs have been approved for the treatment of specific AML subtypes. However, there is need for better understanding of disease pathogenesis including better genetic and epigenetic risk factors in order to develop more effective treatment regimens to improve the outcome of the disease. In Study I, we studied off-target effects of APR-246, a drug that originally was developed to restore the activity of mutated TP53 protein. Oxidative stress related genes heme oxygenase-1 (HMOX1, also termed HO-1), SLC7A11 and RIT1 were significantly upregulated. Also, Nrf2 that induces the expression of HO-1 was upregulated and depletion of Nrf2 mRNA resulted in increased cytotoxicity of APR-246. Moreover, blocking Nrf2 from translocating into the nucleus by using PI3K and mTOR inhibitors led to enhanced cell killing. This suggests that a combination of APR-246 with PI3K and mTOR inhibitors improves sensitivity to APR-246 by interfering with the cellular response to ROS activation to achieve better anti-leukemic effects of APR-246. In Study II, we aimed to define the potential of using stroma cells in diagnostic AML samples as a source of germline DNA. To obtain germline DNA, together with DNA from leukemic cells, it is essential to reliably define somatic mutations in AML cells. Consequently, we cultivated and expanded bone marrow stroma cells from vitally frozen mononuclear cells from AML patients with monosomy 7 as well as defined somatic mutations. In vitro expanded bone marrow stroma cells were stable after 6 weeks of culture and were able to differentiate into adipocytes or osteocytes. We could also show that cultivated stroma cells do not harbour the somatic mutations found in the malignant cells. Thus, we could conclude that bone marrow stroma cells from diagnostic bone marrow samples could be used as a source of germline DNA in AML patients. In Study III, we studied the binding occupancy of the chromatin organizer CTCF in AML patient cells and compared it to binding in normal CD34+ cells. We found that AML cells display an aberrant increase of CTCF binding. Motif analysis revealed that gained CTCF sites are enriched for transcription factors such as PU.1, RUNX1 and CEBPA, which is found to be important for normal myeloid development. TET2 mutated AML patients exhibit a greater gain of CTCF occupancy that is mainly annotated to promoters. Generally, gained CTCF sites were found to be hypomethylated and associated with genes that were upregulated in AML. Knockdown of CTCF in K562 cells resulted in loss of CTCF and decreased gene expression of targeted genes as well as loss of RUNX1 binding at common CTCF and RUNX1 binding sites. Knockdown of CTCF also resulted in increased differentiation of K562 cells. In vitro exposure of AML patient cells with azacytidine resulted in major changes in CTCF occupancy where most gained sites restored the binding pattern found in normal CD34+ cells. In conclusion, our results suggest that an aberrant CTCF occupancy in AML can have a role in driving leukemogenic gene expression patterns in AML.