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Project title
Investigating MYB regulated transcriptional circuits to uncover novel leukaemia susceptibilities
Supervisors names
Owen Williams
David Michod
Background
Acute myeloid leukaemia (AML) is an aggressive disease that remains among the deadliest childhood cancers, despite advances in modern chemotherapy and disease management, indicating an urgent need for new non-toxic therapies that are effective against AML. The molecular heterogeneity of relapsed paediatric AML suggests that novel therapies targeting pathways required in general for AML progression, and for which a disease-specific therapeutic window exists, stand the best chance of rapid and safe integration into the clinic. The transcription factor MYB represents an ideal therapeutic target in this context (1). A broad range of AML subtypes exhibit elevated sensitivity to modulation of MYB expression and function in comparison to normal blood cells. However, direct pharmacological targeting of MYB is complicated by the lack of druggable pockets in its protein structure, in common with other transcription factors. Previously, we used an alternative approach to target it by deriving a MYB-dependent gene expression signature, from a conditional AML model, for connectivity map analysis to identify MYB-targeting drugs (2,3). Further opportunities to therapeutically target the dependence of AML on MYB function may be revealed by dissecting its essentiality. With this in mind, we performed a CRISPR/Cas9 drop-out screen in AML cells with enforced overexpression of MYB, to identify novel MYB-associated dependencies. This analysis identified several genes belonging to discrete molecular pathways positively or negatively associated with MYB function.
Aims and objectives
The aim of this project is to identify novel AML susceptibilities associated with MYB function. Project objectives are as follows:
1) Validate MYB-associated positive and negative gene enrichment using CRISPR/Cas9 competition assays;
2) Determine the susceptibility to and molecular consequences of targeted disruption of validated genes and pathways across a range of AML cell subtypes;
3) Establish the therapeutic efficacy of targeting these dependencies pharmacologically in AML cells lines and a panel of patient-derived xenograft (PDX) AML samples.
Methods
The project will use a range of molecular techniques to modulate target gene expression in AML cells, such as lentiviral transduction, CRISPR/Cas9 gene knockout and shRNA knockdown, and use examine the consequences through gene expression analysis (RNA-seq and QPCR), immunoprecipitation and chromatin immunoprecipitation, flow cytometry and haematopoietic cell culture. Haematopoietic cell cultures, such as colony assays and mesenchymal stem cell co-cultures will be used to investigate the efficacy of target gene modulation and pharmacological inhibition of associated pathways in AML PDX cells. We have already generated a bank of paediatric AML PDX samples for use in these experiments from patients diagnosed at GOSH and have begun to incorporate AML PDX models from refractory and relapsed samples generated as part of the CWCUK/CRUK-funded nationwide prospective precision oncology study (REVEALL).
Timeline
- Objective 1: month 6-9
- Objective 2: month 6-24
- Objective 3: month 18-32
References
1. Pattabiraman DR, Gonda TJ. Role and potential for therapeutic targeting of MYB in leukemia. Leukemia 2013;27:269-77.
2. Walf-Vorderwülbecke, V., Pearce, K., Brooks, T., Hubank, M., van den Heuvel-Eibrink, M. M., Zwaan, C. M., Adams, S., Edwards, D., Bartram, J., Samarasinghe, S., Ancliff, P., Khwaja, A., Goulden, N., Williams, G., de Boer, J., Williams, O. Targeting acute myeloid leukemia by drug-induced c-MYB degradation. Leukemia 2018;32:882-9.
3. Clesham, K., Walf-Vorderwülbecke, V., Gasparoli, L., Virely, C., Cantilena, S., Tsakaneli, A., Inglott, S., Adams, S., Samarasinghe, S., Bartram, J., Williams, G., de Boer, J., Williams, O. Identification of a c-MYB-directed therapeutic for acute myeloid leukemia. Leukemia 2022;36:1541-9.
Contact
Owen Williams, owen.williams@ucl.ac.uk