Medical Biology - Theses
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ItemIdentifying and characterising novel regulators of TRAIL-induced cell death and cholangitis-like liver injury( 2022)Primary sclerosing cholangitis (PSC) is a progressive, idiopathic cholangiopathy characterised by chronic inflammation of the biliary epithelium and cholestasis. PSC promotes fibrotic scarring of the intrahepatic and extrahepatic bile ducts often leading to premature death due to irreversible liver damage. Chronic persistent inflammation in the biliary tree further predisposes to the development of malignant cholangiocarcinoma (CCA). Tumour necrosis factor (TNF)-Related Apoptosis Inducing Ligand (TRAIL)/TRAIL-receptor-mediated signalling was shown to play a substantial role in the pathogenesis of human sclerosing cholangitis-like disease in mice with TRAIL- mediated apoptosis contributing to the disease. However, the etiology and exact pathogenic mechanisms of TRAIL-dependent PSC, or inflammation-associated cholangiocarcinogenesis are largely unclear. Various genetic and environmental factors have been reported to play role in the pathogenesis of PSC. Recently, mutations in ZFYVE19 gene (protein name: ANCHR) were described as a novel cause of neonatal sclerosing cholangitis and hepatic fibrosis termed ZFYVE19 disease. However, the mechanism by which ZFYVE19/ANCHR is involved in the pathogenesis of sclerosing cholangiopathy in these patients has not been yet explored. In Chapters 3 & 4 of my thesis, I identify and characterise two novel regulators of TRAIL-induced cell death, the Abscission/NoCut Checkpoint Regulator (ANCHR/ZFYVE19) and its interacting protein E3 ligase Mind Bomb 2 (MIB2) and show that the loss of ANCHR or MIB2 sensitises TRAIL-resistant cancer cells to caspase-8- dependent death. Moreover, loss of ANCHR alone sensitises CCA cells to death in vitro. Given that the loss of ZFYVE19/ANCHR in a cohort of patients was associated with PSC and cholestasis, and TRAIL/TRAIL-R-mediated apoptosis has been suggested to play an essential role in a PSC-like disease in mice, I further interrogate the physiological consequences of ANCHR loss in mice, particularly focusing on TRAIL-mediated cell death in the liver. In Chapters 3 & 4 I demonstrate a role for ANCHR in limiting TRAIL-induced cell death in vivo and show that loss of ANCHR in mice sensitises to TRAIL-mediated liver cell death. A significant increase in liver cell death in Zfyve19 knock-out mutant mice is observed compared to the wild-type mice after anti-TRAIL-R2 monoclonal antibody (MD5- 1) injection, with concurrent increase in cholangiocyte cell death, suggesting a role for ANCHR in limiting the TRAIL-mediated cholangitis in mice by limiting TRAIL-induced cell death in the liver. Lastly, in the Chapter 5 of this thesis, I demonstrate, as a proof-of-concept, that ANCHR and MIB2 can be efficiently targeted and degraded using the emerging degradation tag (dTAG) PROteolysis-TArgeting Chimera molecules (PROTACs). Our preliminary results serve as a basis for future research and suggest that anti-apoptotic ANCHR and MIB2 are feasible targets for target-specific protein degradation for development of future TRAIL therapeutics. Overall, this thesis expands our understanding on how TRAIL-signalling is regulated and provides a mechanism for the interplay between ZFYVE19/ANCHR loss and TRAIL- mediated PSC-like liver disease. Furthermore, our studies provide correlative evidence for the relationship between the PSC pathology seen in patients carrying bi-allelic nonsense mutations in the ZFYVE19 gene and an overactive TRAIL signalling or overactive liver sensitivity to endogenous TRAIL.