Development and application of methods for mass spectrometric analysis of acute myeloid leukemia derived cell lines to identify receptor tyrosine kinase inhibitor resistance mechanisms and serum biomarkers
Başlık çevirisi mevcut değil.
- Tez No: 597098
- Danışmanlar: DR. PEER-HENDRIK KUHN
- Tez Türü: Doktora
- Konular: Anatomi, Patoloji, Anatomy, Pathology
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2017
- Dil: İngilizce
- Üniversite: Technische Universität München
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 135
Özet
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Özet (Çeviri)
The secretome comprises soluble proteins and small molecules that either are secreted or proteolytically released from cells. These soluble factors are crucial for intercellular communication and tissue homeostasis. Perturbations in the secretome can lead to diseases such as cancer, neurodegenerative or autoimmune disorders. Therefore, the analysis of secretomes could help to learn more about disease-associated perturbations in intercellular communication and thereof derived treatment strategies and diagnostic biomarkers. Secretome protein enrichment with click sugars (SPECS) is a novel technology that enables the mass spectrometric (MS) analysis of secretomes in various cell types in vitro even in the presence of fetal calf serum. One aspect of my thesis dealt with the development of an improved SPECS method that finally outperformed the old protocol in terms of number, intensity and sequence coverage of identified glycoproteins. This most likely will allow a reduction of required input material for MS analysis in the future. Besides optimizing the SPECS method, I applied SPECS to study receptor tyrosine kinase (RTK) signalling in acute myeloid leukemia (AML). AML is a neoplasia of the hematopoietic system with aggressive and heterogeneous characteristics difficult to cure with chemotherapy due to the occurrence of chemo resistance. Therefore, after chemotherapy, targeted inhibition of RTKs such as FLT3 has been developed considering frequent mutations of FLT3 and c-kit in AML. However, both monotherapy and combinations of RTK inhibitors with chemotherapy suffer from fast resistance formation and disease recurrence. Hence, AML requires a better understanding of its molecular biology and better biomarkers for disease monitoring. To this aim, I first analysed the secretome and surface proteome of 6 FAB subtype-related AML cell lines including RTK expression. These data provide a rich resource to be tested as serum biomarkers to monitor AML disease load and AML subtype identification. Second, to learn more about resistance development towards a targeted therapy with the FLT3 inhibiting TKI Sorafenib, I analysed the secretome and surface proteome of the FLT3 mutation carrying MV4-11 cell line in response to Sorafenib using SPECS and mass spectrometry. The MS data demonstrate that in Sorafenib treated cells survival and proliferation related receptors such as FLT3 itself, ICAM3, CD84 and CSFR1 were significantly increased while negative modulators of RTK signalling like PTPRJ, PTPRC and apoptosis inducing factors such as FAS were significantly decreased demonstrating that the AML surface proteome can react very dynamic to compensate FLT3 signalling. As secretome analysis provides a rich resource of biomarkers, I established a first MS method for serum anlaysis of AML patients. Comparing the serum of two patients before and after chemotherapy with complete remission showed that the proteins CD44 and MPO correlate well with disease load, being both established markers in leukemic blast FACS analysis. Finally, I established CRISPR-Cas9 gene editing in AML cell lines to study an exclusive loss of FLT3 or c-kit signalling and established the Gal4-VP16 driven expression and purification of RTK ligands in our lab such as FLT3L and c-Kit ligand. Both tools will be essential for future studies on FLT3 and c-kit.
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