The role of PRMT5 and its interaction with LMO2 Complex in T-ALL

Saleman, Fatma (2022). The role of PRMT5 and its interaction with LMO2 Complex in T-ALL. University of Birmingham. Ph.D.

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Abstract

T-cell acute lymphoblastic leukaemia (T-ALL) is an aggressive haematological malignancy that is commonly characterised by overexpression of oncogenic factors, such as LIM-only 2 (LMO2). LMO2 is normally downregulated during T-cell development, and aberrant expression of LMO2 causes T-ALL leukaemia. A key feature of LMO2 is its function as part of a multi-protein complex, where it directly interacts with transcription factors that are essential during normal haematopoietic development, such as TAL1, GATA, LDB1 and E proteins. Therapeutic regimens for T-ALL patients include repeated treatments with intensive cytotoxic multi-chemotherapy agents. Although chemotherapy has significantly improved the overall 5-year survival rate, adult patients often show poor outcomes. Most patients with T-ALL suffer from toxic side effects and secondary complications. It is important to understand the molecular basis of the role the LMO2 complex plays in the development of T-ALL treatment, as this may aide to develop new targeted therapies.
Investigating proteins that interact with LMO2 in human T-ALL cell lines, using pulldown assays followed by mass spectrometry, revealed protein arginine methyltransferase 5 (PRMT5) as a potential interacting partner of LMO2. The aim of this project was to characterise and investigate the function of this interaction in T-ALL, which can be a therapeutic target for T-ALL. We hypothesised that the interaction between LMO2 and PRMT5 brings PRMT5 to the DNA, where it can be involved in the regulation of gene expression. We confirmed the expression of PRMT5 in human T-ALL cell lines and mouse myeloid progenitors and its interaction with LMO2 using pulldown assays. Arginine methylation of non-histone proteins by PRMT5 is important for controlling many cellular processes. Using pulldown and mass spectrometry, we identified possible PRMT5 substrates. Among these, RUNX1 was identified and found to be dimethylated at arginine R244. The RUNX1 transcription factor is essential for adult haematopoiesis. ChIP-sequencing analysis, using antibodies recognising PRMT5, LMO2 and RUNX1, showed overlapping peaks in T-ALL, confirming a functional relationship between these proteins. We found specific binding patterns involving different complex members, which regulate gene expression that is involved in T-cell function and development.
Inhibition of PRMT5 activity or overexpression of catalytically inactive PRMT5 showed the requirement of PRMT5 enzymatic activity for cell proliferation and preservation of genomic stability. In addition, the differentiation of mouse myeloid progenitors showed the requirement of PRMT5 for cell proliferation and that loss of PRMT5 enzymatic activity leads to a defect in myeloid differentiation, as well as chromosomal instability. Our project shows the interaction of the LMO2 multiprotein complex with both PRMT5 and RUNX1. The identification of dimethylated arginine RUNX1 indicates a possible regulatory function for PRMT5, which may be related to their interaction with the LMO2 complex. Their collaborative functions in regulating gene expression in T-ALL may help to identify specific therapeutic targets for T-ALL treatment.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

Supervisor(s):