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Building an in-vitro model of clonal evolution to study mechanisms of therapy resistance in chronic lymphocytic leukaemia.


Project Description

Background:
Progressive disease in chronic lymphocytic leukaemia (CLL) follows a natural history of treatment followed by relapse where the malignant clone takes on increasing aggressive behaviour1. Previous work by others has linked malignant cell resistance in such cases to genetic clonal evolution2, identifying a profile of genes that becomes mutated within subpopulations of the malignant cells. This phenomenon is best characterized in populations of patients receiving front line therapy for CLL [fludarabine, cyclophosphamide, rituximab (FCR)] where clones bearing 17p deletions and TP53 mutations become dominant at disease relapse3. Malignant cell clones bearing these deletions/mutations already exist in patients at diagnosis of CLL and correlate with very poor prognostic outcome, but whether these clones can be identified and tracked at single cell level during treatment is unknown. Moreover, the reasons why these cells behave in a more aggressive fashion at disease relapse are also unclear. This project aims to provide new knowledge into these areas by building an in-vitro model of therapy resistance for testing on CLL cell samples derived from clinical trials. Our approach will be to use the CLL cell line HG3, a line derived from EBV-transformation of CLL cells that have a wild-type configuration at 17p and TP534. CRISPR will be used to delete the TP53 gene to create hemi- and homozygous clones bearing this deletion and mimic the MDR of 17p- CLL cells, and then use these cells, along with wild type cells, to develop further clones that are resistant to fludarabine and cyclophosphamide within different microenviromental settings. Analysis of the resistant and sensitive cell lines by mRNAseq, miRseq and gene methylation analysis will then provide insight into potential therapy resistance mechanisms, as well as provide a gene expression signature that can be exploited by mass cytometry to identify and track clonal evolution using PLAYR, a method we have recently developed for use here in Liverpool5. Ultimately, the gene expression signature we identify from this in-vitro model can then be applied to primary samples of CLL cells from patients to identify and track clonal expansion of resistant cells in vivo.

Project Aims:
1. To create a CLL cell line (HG3) bearing deletion of the TP53 gene using CRISPR that will model 17p deletion in primary CLL cells. (The principle of this aim is currently being investigated and will potentially be ready at the beginning of this project)
2. Generate fludarabine/cyclophosphamide resistant cells (wt and TP53del) for comparative analysis and generation of insight into resistance mechanisms to these therapeutic agents.
3. Track expansion of the TP53del-HG3 cells in populations of wt cells using mass cytometry.
Scientific Objectives:
To create an in-vitro model of clonal evolution in CLL to give insight into the processes of therapy resistance and aggressive cell behaviour at disease relapse in primary CLL.

Training and Support: The student will have direct access to training in cell culture, CRISPR and mass cytometry by the immediate Slupsky/Kalakonda group, which is pioneering mass cytometry at the UoL and has significant experience with CRISPR and the manufacture of drug-resistant cell lines. Training in computational approaches to data analysis is a feature that will also be provided. The PhD student will have direct access to the primary supervisor for at least 2h/wk (1;1 and in lab meetings) where aspects of experimental design, write up and thesis preparation are discussed. As part of a lab team, the student will be encouraged to openly discuss their data and have it challenged as it is generated. All students are supervised in adherence to the UoL policy for supervision.

The student on this project will work in close collaboration with an existing project “Kinome profiling and mass cytometry as tools to detect rewiring of B cell receptor signalling in the malignant cells of chronic lymphocytic leukaemia patients taking ibrutinib” that is funded by Northwest Cancer Research.

Qualifications and Experience:
Applicants will have a first class or upper second class honours degree (or equivalent) in the biological sciences and some laboratory research experience. You may have additional masters level research experience in cell biology, biochemistry or cancer research. All applicants must satisfy the appropriate University English language requirements. For EU and international students this is an IELTS score of 6.5 with no band score lower than 5.5.

To apply please send your CV, cover letter and the names and addresses of at least two references to . For application enquires please contact Joseph Slupsky () or Nagesh Kalakonda ().

Funding Notes

This 3-year PhD studentship covers tuition fees, research support costs and a stipend at UKRI standard rates (£14,999 p.a. for 2019/20). The studentship is open to UK, EU and international candidates.

References

1. Kipps TJ, Stevenson FK, Wu CJ, Croce CM, Packham G, et al., Chronic lymphocytic leukaemia. Nat Rev Dis Primers. 2017, 3:17008.
2. Landau DA, Tausch E, Taylor-Weiner AN, Stewart C, Reiter JG, et al., Mutations driving CLL and their evolution in progression and relapse. Nature. 2015, 526:525-30.
3. Malcikova J, Stano-Kozubik K, Tichy B, Kantorova B, Pavlova S, et al., Detailed analysis of therapy-driven clonal evolution of TP53 mutations in chronic lymphocytic leukemia. Leukemia. 2015, 29:877-85.
4. Rosén A, Bergh AC, Gogok P, Evaldsson C, Myhrinder AL, et al., Lymphoblastoid cell line with B1 cell characteristics established from a chronic lymphocytic leukemia clone by in vitro EBV infection. Oncoimmunology. 2012, 1:18-27.
5. Duckworth AD, Gherardini PF, Sykorova M, Yasin F, Nolan GP, et al. Multiplexed profiling of RNA and protein expression signatures in individual cells using flow and mass cytometry. Nature Protocols (2019, in press).

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