Acute myeloid leukaemia (AML) is an aggressive cancer with a poor prognosis, for which mainstream treatments have not changed for decades. To identify novel therapeutic targets in AML, I have optimized a genome-wide clustered regularly interspaced short palindromic repeats (CRISPR) screening platform and use it to identify genetic vulnerabilities in AML cells. This work led to the identification of 492 AML-specific cell-essential genes, including several established therapeutic targets such as that represent new clinically actionable candidates. I have validated selected genes using DOT1L, BCL2, MEN1 and many other genes genetic and pharmacological inhibition, and chose candidates for downstream studies. Both the epigenetic modifier KAT2A and SRPK1 as promising KAT2A and spliceosome kinase SRPK1 inhibition demonstrated anti-AML activity by inducing myeloid differentiation and apoptosis, and suppressed the growth of primary human AMLs while sparing normal hemopoietic stem-progenitor cells. My findings propose that KAT2A and SRPK1 inhibition should be investigated as new therapeutic strategies in AML and also provide a large number of novel genetic vulnerabilities of this leukaemia that can be pursued in downstream studies. As these screens were performed in immortalised AML cell lines, I then went on to develop a method for the performance of dropout screens in genotypically-defined primary murine AMLs developed in our lab and arising in Cas9-expressing mice. Through this work, I successfully carried out the first such screen in AML cells driven by mutant Npm1 (NPM1c) and Flt3-ITD, the commonest two-mutation combination in human AML. Downstream analysis of the results revealed the excellent potential of this type of screen and enabled me to investigate the molecular effects on mutant Npm1, which are currently poorly understood. Overall, my results demonstrate that unbiased CRISPR dropout screens can identify novel therapeutic targets in cancer while, in parallel, revealing novel biological insights.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:745012 |
| Date | January 2017 |
| Creators | Tzelepis, Konstantinos |
| Contributors | Vassiliou, George |
| Publisher | University of Cambridge |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.repository.cam.ac.uk/handle/1810/271130 |
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