Abstract
Anaplastic Wilms’ tumour has poor prognosis and is characterised by a high frequency of missense mutations in TP53. Despite these potential gain-of-function (GOF) mutations having been reported in the early 1990’s, the biological effects of GOF mutant p53 (mutp53) protein in anaplastic Wilms’ tumour has never been examined.In this study, we demonstrate that GOF mutp53 is critical to anaplastic Wilms’ tumour survival, with depletion causing growth arrest, autophagy induction, and apoptotic cell death. RNA sequencing following mutp53 depletion identifies putative downstream mutp53 targets. These include genes encoding several epigenetic writers and erasers, such as histone methyltransferase EHMT2/G9a; EHMT2 is overexpressed in Wilms’ tumour patient samples, with a positive correlation between staging and expression. Further analyses demonstrate that mutp53 co-opts G9a in a newly identified oncogenic axis to methylate and silence wtp53 target genes, including CDKN1A. Mutp53 is suggested to be a key driver mutation as it acts upstream of several reported Wilms’ tumour oncogenes and tumour suppressor genes; activities of mutp53 include transactivation of oncogene MYCN and repression of tumour suppressor gene REST.
Genetic and pharmacological inhibition studies validated G9a as a potential therapeutic target in anaplastic Wilms’ tumour cell lines. G9a small molecule inhibitor UNC0638 promptly induced autophagy with regulation of induction genes, including GABARAPL1. However, autophagic flux is impaired as autophagosome-lysosome fusion is inhibited; this accumulation of autophagic vesicles is suggested to cause apoptosis. UNC0638 treatment also rapidly and effectively switches the activity of R248Q and G245S mutants to wtp53-like as wtp53 targets, such as CDKN1A, were derepressed. G9a inhibition causes transcriptional downregulation of TP53 demonstrating that G9a activity enhances mutp53 activity. RNA sequencing following UNC0638 treatment identified multiple overlapping pathways with the mutp53 datasets, including the regulation of cholesterol biosynthesis pathway genes, which is important for lysosomal function during autophagy.
These results indicate a new vital oncogenic axis in anaplastic Wilms’ tumour through which the activities of GOF mutp53 can be therapeutically targeted.
| Date of Award | 18 Jun 2024 |
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| Original language | English |
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| Supervisor | Karim T A Malik (Supervisor) & Stefan Roberts (Supervisor) |