Unravelling the Role of ZEB1 in Chronic Lymphocytic Leukaemia Progression: Pathways to Novel Therapeutic Insights

Primary supervisor: Sergey Krysov, Queen Mary University of London

Secondary supervisor: Maxim Freydin, Queen Mary University of London

Project

Thousands of new CLL cases are diagnosed in the UK annually, making CLL the most common blood malignancy (CRUK stats). Despite recent advances in targeted treatment that has significantly decreased the mortality rates a remarkable proportion of patients develop resistance to the existing first and second lines of therapy, and in a growing number of patients, there is no more alternative to the failed therapy (Mato. 2022. Clin Cancer Res).

Our pathobiology research has revealed significant involvement of Epithelial-mesenchymal transition (EMT) factors, with initial findings highlighting the novel role of the ‘classical’ EMT regulator ZEB1 in modulating clinically relevant factors, including ZAP70, CXCR4 and others in CLL. ZEB1 is a transcription factor usually associated with EMT, cell motility, apoptosis, proliferation and cell ‘stemness’. Interestingly, it is mostly forgotten that the first observation of ZEB1 has been made in B-cells, in which it regulates immunoglobulin transcription and B-cell development (Genetta T. et al. (1994); Postigo, et. al. (2000)). We characterised EMT-like signatures in CLL analysing EMT associated gene and protein expression. Notably, we found EMT involvement in CLL proliferative centres demonstrating a remarkable disparity in ZEB1 and other EMT protein factors’ expression (EMT-like signature) between patients with favourable vs. poor prognoses. Distinct profiles were observed between CLL proliferative centres (high ZEB1) and circulating CLL cells (low or no ZEB1). We observed expression changes upon different stimulation modalities (BCR activation, CpG proliferative signal, Ibrutinib treatment). Importantly, our preliminary data from RNA and ChIP-seq analysis suggests ZEB1 directly regulates genes that are known to prognosticate CLL progression. Preliminary analysis supports our hypothesis, showing ZEB1 positively correlates with ZAP70, CD79B, IKZF3, CXCR4 expressions in CLL, but not in healthy controls (GSE13164, CLL=448, healthy control=74, ).

Using knockdown/overexpression techniques alongside bioinformatics, we aim to elucidate the links between EMT-like activation in CLL cells, CLL progression, and clinical outcomes by examining ZEB1’s role in both normal B-cell development and CLL. This study will offer insights into EMT role in normal B-cell biology and understanding CLL evolution, facilitating identification, development and production of novel biological therapies.

This PhD project focuses on exploring ZEB1-dependent mechanisms in CLL through the analysis of transcription datasets from 150 patients, encompassing diagnostic samples and post-treatment samples, including those with developed resistance. Additionally, the project will utilize in vitro overexpression assays in B-cell lines and primary CLL samples and studies in conditionally knockout animal model (A genetically engineered mouse line with a conditional Zeb1 knockout allele features two loxP sites flanking Exon 6, not altering expression until Cre-mediated recombination induces a loss-of-function mutation. This allele, maintained homozygous in a C57BL/6 background, allows tissue-specific inactivation when bred with the Cre-line). Additionally, in series of functional assays (Proliferation, Viability, Motility, Transcription and SHM activation), we aim to elucidate the influence of ZEB1 on critical cellular processes identifying EMT-dependent therapeutic targets in CLL and validate small molecule inhibitors for targeted therapy that we develop in collaboration with Target Discovery Institute in Oxford.

References

1. Mato, A.R., et al. (2022). Recognizing Unmet Need in the Era of Targeted Therapy for CLL/SLL: “What’s Past is Prologue?” (Shakespeare). Clin Cancer Res, 28(4), 603-608.
2. Genetta T. et al. (1994). Displacement of an E-box-binding repressor by basic helix-loop-helix proteins: implications for B-cell specificity of the immunoglobulin heavy-chain enhancer. Mol Cell Biol., 6153-63.
3. Postigo, et al. (2000). Differential expression and function of members of the zfh-1 family of zinc finger/homeodomain repressors. PNAS, 97(12): 6391-6396