Understanding the molecular mechanisms of AML development and treatment using phosphoproteomics
AuthorHeckmann, Denise Annette
Document TypePhD thesis
Access StatusOpen Access
© 2020 Denise Annette Heckmann
AML (Acute Myeloid Leukemia) is a rapidly progressing cancer of the blood and bone marrow where the accumulation of abnormal myeloid cells crowds out healthy blood cells. Despite the improvements in understanding the biology of AML, these advancements are not reflected in the trajectory of the survival rate. One reason for the poor translation of research results into novel therapies, is the genetic and epigenetic heterogeneity of the disease, which hampers the development of reliable AML models. Mouse models of AML are designed to mimic human disease by expressing known oncogenes in the hematopoietic system of mice. Although the oncogene-specific disease pathology appears to reflect what is observed in human patients, the translation of novel treatments into the clinic has often been unsuccessful. Expression of the hematopoietic transcription factor EVI1 has been identified as a marker for poor prognosis in human AML patients. In this thesis, I aimed to develop a mouse model, that recapitulates a particularly aggressive and treatment-resistant form of AML by overexpressing EVI1 together with the fusion-oncogene MLL-AF9. Although I successfully generated EVI-expressing AMLs, the presence of the transcription factor did not affect disease features such as latency, disease pathology, morphology and immunophenotype of leukemic cells. However, when cultured in vitro, leukemic cells expressing EVI1 were more resistant to Smac mimetic combination treatments and the chemotherapeutic agent Ara-C, therefore reflecting the treatment resistance observed in human patients. Although some targeted treatments are now available, chemotherapy remains the standard of care therapy for AML. Therefore, to improve disease outcome, novel treatments are desperately needed. The IAPs (Inhibitor of Apoptosis Proteins) have been identified as an attractive therapeutic target in a number of cancers including AML. Smac mimetics, a class of drugs that specifically inhibit IAPs, have been found to selectively induce cell death in leukemic cells when combined with an inhibitor of the MAP kinase p38 both in vivo and in vitro. To understand the molecular mechanisms behind this synergistic killing, I studied the phosphoproteome of treated cells using mass spectrometry and revealed that the PI3K/Akt/mTOR survival pathway was activated following Smac mimetic treatment. Upon p38 inhibition, this survival signaling did not occur. Furthermore, CSF1R was identified as a potential regulator of the PI3K/Akt/mTOR pathway. In support of this finding, the combination of a Smac mimetic and CSF1R inhibitor, resulted in synergistic cell killing in vitro. To study proteins, gene tagging provides a valuable tool for a range of applications including real-time monitoring of target protein activities, modifications of proteins and protein-protein interactions. The CRISPR/Cas9 technology is a powerful tool for modifying any DNA of interest and enables the tagging of endogenous genes. However, the targeted insertion of foreign DNA into cell lines is challenging. I aimed to generate endogenously FLAG-tagged proteins in cell lines by targeting components of the TNF pathway using the CRISPR-Cas9 knock-in technology. Although ssDNA containing the FLAG sequence was inserted into the genome of mouse dermal fibroblasts (MDFs) at the correct site, next generation sequencing revealed that this process was both inefficient and error-prone and resulted in the introduction of mutations. Nonetheless, I was able to enrich for tagged RIPK1 following FLAG pull-down, which was detectable via both immunohistochemistry and mass spectrometry.
KeywordsPhosphoproteomics; AML; p38; kinase inhibitor; EVI1; CRISPR; TNF; SMAC mimetics
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