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Title: Gene editing of BTK using CRISPR/Cas9 to study drug resistance in acute myeloid leukaemia
Author: Märken, Melanie
ISNI:       0000 0004 7964 2371
Awarding Body: University of Liverpool
Current Institution: University of Liverpool
Date of Award: 2019
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Acute myeloid leukaemia (AML) has a current poor 5-year survival rate of only 27%, making the finding of new therapeutic avenues for treatment crucial. Bruton's tyrosine kinase (BTK) is best known for its role in B-cell receptor signalling but is found to be expressed and constitutively phosphorylated in AML cell lines and patient-derived cells. Targeting BTK in B-cell malignancies with small molecule BTK inhibitor ibrutinib is clinically effective, however a small proportion of CLL and MCL relapse during the therapy and become refractory to ibrutinib. This resistance is mainly due to a cysteine to serine mutation in BTK at position 481 (C481S) that disrupts the irreversible, covalent binding between ibrutinib and the protein. As little is known about the role of BTK and its potential as a therapeutic target in AML, we studied the effects and functional consequences of pharmacological inhibition of BTK using ibrutinib in AML cell lines. Using the CRISPR/Cas9 system we generated the BTKC481S mutation in AML cell lines to explore the functional impact of this mutation on ibrutinib treatment as well as other related protein networks. Whilst no effect of ibrutinib on cell proliferation and viability was observed in a cell culture model using THP-1 cells, we demonstrate that the BTKC481S mutation led to reduced ibrutinib binding affinity as well as reversible binding compared to irreversible binding observed in BTKWT cells. BTK inhibition correlated with a decrease in p- PLC?2(Y1217) but had no observable effect on p-AKT(S473) or pp42/ 44(T202/Y204). Kinase substrate enrichment analysis showed a decrease in NEK2 activity and increased activity in kinases belonging to the RSK family in THP- 1 BTKWT and BTKC481S cells. An increase in abundance of PKC family members was also seen in THP-1 BTKC481S cells. Taken together this data demonstrates the CRISPR/Cas9 system can be utilised to model and study drug resistance mechanisms.
Supervisor: Macewan, David ; Slupsky, Joseph Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral