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Title: Genetic screening to investigate treatment resistance in MLL-rearranged leukemia
Author: Jamilly, Maximilian
ISNI:       0000 0004 8507 1405
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2019
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Despite the promise of targeted cancer therapies, some subsets of acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) continue to present a dismal prognosis. Translocations in the mixed lineage leukemia (MLL) gene generate MLL fusion proteins and cause MLL-rearranged (MLLr) leukemia, an aggressive subtype of AML and ALL which is common in infants and children. MLLr leukemia is resistant to conventional chemotherapy. I used CRISPR/Cas9-mediated mutagenesis to investigate miRNA function and BH3 mimetic drug sensitivity in MLLr leukemia. I began by testing a published model for the role of a non-coding RNA in MLLr leukemia. The MLL-AF9 fusion protein activates the transcription factors HOXA9 and MEIS1, which are both predicted targets of the oncogenic miRNA miR-196b. I used dual-sgRNA CRISPR/Cas9 knockout to generate and characterise a miR-196b null mutant MLL-AF9 cell line, demonstrating that miR-196b ablation reduces colony-forming capacity but does not affect expression of HOXA9 or MEIS1. Next, I investigated the genetic basis for venetoclax resistance in MLLr leukemia. Venetoclax is a potent inhibitor of the anti-apoptotic protein BCL-2 and is an effective monotherapy in CLL but not in AML or ALL. I optimised a system of pooled CRISPR/Cas9 editing to compare the contributions of BCL-2 family proteins to venetoclax resistance in MLL-AF9 cells. Finally, I used this system to conduct a pooled genome-wide CRISPR/Cas9 screen in MLL-AF9 cells. My results suggest that the 26S proteasome mediates venetoclax resistance in MLLr leukemia. I provide evidence that the proteasome inhibitor bortezomib synergises with venetoclax to kill MLL-AF9 cells in vitro. The work presented in this thesis demonstrates that CRISPR-Cas9 screening is a powerful tool for investigating the causes of resistance to targeted cancer therapy.
Supervisor: Milne, Thomas A. ; Fulga, Tudor A. Sponsor: Engineering and Physical Sciences Research Council ; Biotechnology and Biological Sciences Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Molecular biology