Genetic screening to investigate treatment resistance in MLL-rearranged leukemia

Thesis

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.

Authors: Jamilly, M

• DOI: 10.5287/ora-1zx9wmvoz
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: oncology; CRISPR; molecular biology; miRNA; mixed-lineage leukemia; apoptosis; leukemia; drug discovery
• Subjects: Molecular biology