Global characterization of oncogenic kinase signaling

Aberrant activation and expression of the proto-oncogene Src have been observed in various human malignancies. Despite major advancements in kinase-targeted cancer therapies, targeting Src has only achieved limited clinical success, thus highlighting the urgent need to better understand the complex signalling network governed by Src and design novel therapeutic strategies. In this thesis, a novel approach integrating mass spectrometry-based quantitative kinome profiling and siRNA-based functional genomics screens was applied to provide insights into this problem. Firstly, a novel broad-spectrum kinase inhibitor CTx-0294885 was developed and characterized as a kinase-capturing tool in order to increase the kinome coverage of the profiling technique. Using CTx-0294885 as a single reagent and in combination with others led to the identification of 235 and 261 kinases from MDA-MB-231 cells, respectively. Coupling the kinase enrichment with phosphopeptide enrichment enabled the identification of 799 kinase-derived phosphosites, with ~10% localized within the activation loop. Secondly, the global impact of constitutively active Src on the expressed kinome in MCF-10A cells was characterized using the optimized MS-based kinome profiling technique. In total, 302 kinases and 1,553 phosphorylation sites were identified. Applying a cut-off of ≥ 1.5-fold change unveiled a complex network comprised of 67 kinase members from all major kinase groups. While 62 kinases exhibited changes at the phosphorylation level, only 9 responded to Src activation at the protein expression level. In addition to the well-characterized Src interaction partners such as AKT, EGFR, and FAK, this Src-regulated kinome also included oncogenes (e.g. IKKE, TNK2, DAPK1 and ROCK1) implicated in triple negative breast cancer, and many poorly characterized kinases and phosphorylation sites. Lastly, high throughput RNAi screens were carried out in order to identify Src-regulated kinases critical for cell proliferation in 2D cell culture and acini formation in 3D cell culture. The essentiality of the same kinase varied dramatically under different culturing conditions. The 3D screens identified of 9 kinases (e.g. SGK1, DAPK1, NEK7, and LIMK2) essential for Src-induced transformation, while MAK4K5 promoted acini growth. This study not only characterized the Src-regulated kinome involved in oncogenesis for the first time, but also provided novel therapeutic targets for clinical development.

Grantor dc:publisher

UNSW, Sydney

Year dc:date

2019

Author dc:creator

• Zhang, Luxi

• Functional genomics
• Src signalling
• Kinome profiling
• Chemical proteomics
• Triple negative breast cancer

Statement dc:rights

• open access
• CC BY-NC-ND 3.0
• free_to_read

Licence

• https://purl.org/coar/access_right/c_abf2
• https://creativecommons.org/licenses/by-nc-nd/3.0/au/

Language dc:language

EN

Identifier

https://doi.org/10.26190/unsworks/21399

OAI identifier oai:identifier

oai:unsworks.library.unsw.edu.au:1959.4/63337