Investigating the Role of zDHHC Palmitoyl Transferases in the Transendothelial Migration of T-Cell Acute Lymphoblastic Leukaemia

Abstract

T-cell acute lymphoblastic leukaemia (T-ALL) arises from aberrant proliferation of immature T-cell precursors, with prognosis worsened upon metastasis to organs such as the central nervous system (CNS).
During inflammation, T-cells adhere to and pass through the endothelium to enter tissues, a process known as transendothelial migration (TEM). This well-defined event is also involved in T-ALL metastasis. TEM requires interactions with transmembrane receptors on the endothelial cell surface. One mechanism of regulating membrane localisation is palmitoylation, a reversible post-translational modification catalysed by zinc-finger Asp-His-His-Cys domain (zDHHC) palmitoyl acyltransferases (PATs).
An siRNA screen conducted by the laboratory in human umbilical vein endothelial cells (HUVECs) revealed that depletion of several zDHHCs altered VE-cadherin localisation – an endothelial cell-specific adhesion molecule and marker of junction stability. zDHHC4 and zDHHC18 specifically were found to alter T-ALL cell TEM. This project aimed to explore the involvement of additional zDHHCs in T-ALL metastasis by analysing their roles in T-ALL cell adhesion to and TEM across endothelial cells.
A static adhesion assay using siRNA pools targeting all 23 zDHHCs identified the top and bottom two zDHHCs that alter T-ALL Jurkat cell adhesion to HUVECs: zDHHC9 and zDHHC17 knockdown reduced adhesion, whereas zDHHC13 and zDHHC14 knockdown increased adhesion. Interestingly, these zDHHCs did not alter Jurkat cell adhesion under pulsatile flow conditions. Preliminary data suggest zDHHC17 alters TEM, whereas confocal imaging of VE-cadherin localisation indicated that zDHHC9 and zDHHC14 depletion decrease and increase endothelial junctional stability respectively. Acyl biotin exchange and liquid chromatography-mass spectroscopy was also conducted to identify potential palmitoylated endothelial cell substrates, resulting in the selection of 142 ‘proteins of interest’ for future investigation.
While methods used in this project require further optimisation, findings indicate that different zDHHCs play distinct roles in the adhesion and transmigration of T-ALL cells. Specific zDHHCs or their substrates could therefore be targeted to help reduce T-ALL metastasis.

Date of Award	10 Dec 2024
Original language	English
Awarding Institution	University of Bristol
Supervisor	Anne J Ridley (Supervisor) & Ann Williams (Supervisor)