Genetic screening to investigate treatment resistance in MLL-rearranged leukemia
<p>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) leuke...
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| Format: | Thesis |
| Language: | English |
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2019
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| _version_ | 1826315596700057600 |
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| author | Jamilly, M |
| author2 | Milne, TA |
| author_facet | Milne, TA Jamilly, M |
| author_sort | Jamilly, M |
| collection | OXFORD |
| description | <p>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.</p> |
| first_indexed | 2024-03-06T18:39:36Z |
| format | Thesis |
| id | oxford-uuid:0c708cd6-8f39-457b-be40-aa4001e84f3a |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-12-09T03:29:07Z |
| publishDate | 2019 |
| record_format | dspace |
| spelling | oxford-uuid:0c708cd6-8f39-457b-be40-aa4001e84f3a2024-12-01T11:51:51ZGenetic screening to investigate treatment resistance in MLL-rearranged leukemiaThesishttp://purl.org/coar/resource_type/c_db06uuid:0c708cd6-8f39-457b-be40-aa4001e84f3aMolecular biologyEnglishORA Deposit2019Jamilly, MMilne, TAFulga, TA<p>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.</p> |
| spellingShingle | Molecular biology Jamilly, M Genetic screening to investigate treatment resistance in MLL-rearranged leukemia |
| title | Genetic screening to investigate treatment resistance in MLL-rearranged leukemia |
| title_full | Genetic screening to investigate treatment resistance in MLL-rearranged leukemia |
| title_fullStr | Genetic screening to investigate treatment resistance in MLL-rearranged leukemia |
| title_full_unstemmed | Genetic screening to investigate treatment resistance in MLL-rearranged leukemia |
| title_short | Genetic screening to investigate treatment resistance in MLL-rearranged leukemia |
| title_sort | genetic screening to investigate treatment resistance in mll rearranged leukemia |
| topic | Molecular biology |
| work_keys_str_mv | AT jamillym geneticscreeningtoinvestigatetreatmentresistanceinmllrearrangedleukemia |