Genetic defects in myeloid malignancies and preleukemic conditions

Myeloid neoplasms (MNs) are malignant hematopoietic disorders that can be subdivided into acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) based on blast percentage. In recent years, progress has been made in uncovering the landscape of genetic alterations frequently occurring in MNs. This thesis has focused on the impact and consequences of genetic mutations at preleukemic stages. Furthermore, the value of individual aberrancies for the MN phenotype, disease maintenance and response to therapy was investigated. Therapy-related myeloid neoplasms (t-MNs) are malignant hematopoietic disorders that develop following treatment with chemotherapy and irradiation therapy. Reconstruction based on genetic mutations showed that preleukemic clones can be present years before t-MN diagnosis. Ringsideroblasts (RS) represent an aberrant form of erythroid differentiation which is particularly characteristic for MDS. We demonstrated that the genetic basis for the RS-phenotype in AML is different from MDS, however underlying mechanisms share similarities. MNs containing TP53 mutations are notorious for poor outcomes following chemotherapy. Our data indicate that treatment using hypomethylating agents may be beneficial in AML patients with TP53 mutations. Furthermore, we have demonstrated that genetic predisposition may underlie MN diagnosis in adult cases, which may present as donor cell leukemia. The process of leukemic transformation induced by genetic mutations involves disruption of transcriptional networks. Our observations show that transcriptional co-activator CITED2 is essential for leukemic cell survival in a subset of AML patients

CITED2 affects leukemic cell survival by interfering with p53 activation

CITED2 (CBP/p300-interacting-transactivator-with-an-ED-rich-tail 2) is a regulator of the acetyltransferase CBP/p300 and elevated CITED2 levels are shown in a number of acute myeloid leukemia (AML). To study the in vivo role of CITED2 in AML maintenance, AML cells were transduced with a lentiviral construct for RNAi-mediated knockdown of CITED2. Mice transplanted with CITED2-knockdown AML cells (n = 4) had a significantly longer survival compared to mice transplanted with control AML cells (P <0.02). In vitro, the reduction of CITED2 resulted in increased p53-mediated apoptosis and CDKN1A expression, whereas BCL2 levels were reduced. The activation of p53 upon CITED2 knockdown is not a direct consequence of increased CBP/p300-activity towards p53, since no increased formation of CBP/p300/p53 complexes was demonstrated and inhibition of CBP/p300-activity could not rescue the phenotype of CITED2-deficient cells. Instead, loss of CITED2 had an inhibitory effect on the AKT-signaling pathway, which was indicated by decreased levels of phosphorylated AKT and altered expression of the AKT-pathway regulators PHLDA3 and SOX4. Notably, simultaneous upregulation of BCL2 or downregulation of the p53-target gene PHLDA3 rescued the apoptotic phenotype in CITED2-knockdown cells. Furthermore, knockdown of CITED2 led to a decreased interaction of p53 with its inhibitor MDM2, which results in increased amounts of total p53 protein. In summary, our data indicate that CITED2 functions in pathways regulating p53 activity and therefore represents an interesting target for AML therapy, since de novo AML cases are characterized by an inactivation of the p53 pathway or deregulation of apoptosis-related genes

Molecular regulation of normal and leukemic hematopoietic cells by CITED2

One of the major problems in acute myeloid leukemia (AML) is the inability to target leukemic stem cells (LSCs), resulting in recurrent and often resistant disease. Therefore, understanding the regulation of healthy hematopoietic as well as leukemic cells is vital to solve this problem. The transcriptional co-activator CITED2 is shown to be essential for normal hematopoiesis, and is also associated with leukemia. However, knowledge regarding the mechanisms by which CITED2 functions is limited. This study aims to clarify the regulatory mechanism of CITED2, emphasizing on the tumour suppressor protein p53 as a possible downstream target for inhibition. In normal hematopoietic stem and progenitor cells, knockdown of CITED2 resulted in decreased proliferation and a differentiation-shift towards myeloid, as revealed by long term cultures and colony-forming cell assays. The response to the chemotherapeutic agent cytarabine was investigated in the leukemic NB4 cell line by means of Annexin-V staining (apoptosis) and MTS assays (cell viability). Chemotherapy-induced apoptosis was reduced after overexpression of CITED2, and this effect was also observed by means of improvement in cell viability. On the other hand, cell viability upon cytarabine treatment was impaired in CITED2 knockdown cells and induction of chemotherapy-induced apoptosis was most effective in cells with CITED2 downregulation. Both observations could be rescued with additional downregulation of p53, functionally demonstrating that CITED2 interferes with chemotherapy-induced apoptosis in a p53-dependent manner. In addition, mass spectrometry analysis of protein-interaction partners for CITED2 demonstrates various ways in which CITED2 can be linked to p53, showing exciting new proteins for further research possible involved in CITED2 function. In conclusion, this project further explores the role of CITED2 in regulation of hematopoietic cells under both normal and leukemic conditions. CITED2 was shown to be a potential inhibitor of p53 function, interesting since malfunction of p53 is a common feature in cancers. These findings could help aid our understanding of mechanisms responsible for therapeutic resistance in leukemia.

Ring sideroblasts in AML are associated with adverse risk characteristics and have a distinct gene expression pattern

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Early detection and evolution of preleukemic clones in therapy-related myeloid neoplasms following autologous SCT

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