Dissecting chromatin and cytoskeletal regulatory functions of SETD2 in ccRCC

Clear cell renal cell carcinoma (ccRCC) is the most frequent renal tumour subtype and is highly metastatic. ccRCC is characterized by frequent inactivation of the von Hippel-Lindau (VHL) gene and biallelic inactivation of VHL occurs at the earliest stage of tumour formation. However, inactivation of VHL alone is not sufficient to cause ccRCC, suggesting that other genes are likely to be important in its development. Biallelic loss of SETD2 occurs in up to 20% of primary human RCC tumours and is associated with more progressive disease and metastatic phenotype. Previous research has demonstrated that SETD2 not only is the sole enzyme responsible for the trimethylation of lysine 36 on histone H3 (H3k36me3) but also functions as a microtubule- and actin- methyltransferase. The methylation of α-tubulin at lysine 40 (α-TubK40me3) and actin at lysin 68 (ActK68me3) is necessary for cell mobility, actin dynamics and genomic stability. However, the contribution of loss of SETD2 to tumour formation and progression is poorly understood. We generated and analysed a panel of isogenic ccRCC cell lines harbouring individual and combined mutations in VHL and SETD2 to investigate the role of SETD2 in metastasis. In ccRCC cells, SETD2 mutation increased proliferation, migration, invasion, and spreading cell culture assay. Moreover, loss of SETD2 increased metastasis in xenograft model of 786-O cells, suggesting that SETD2 loss plays a role in promoting metastatic spread. Given the role of SETD2 as a microtubule methyltransferase, we showed that SETD2 mutation is accompanied by loss of α-TubK40me3, resulting in mitotic defects. By introducing mutations in the catalytic SET domain (R1625C) and SRI domain (R2510H) which binds tubulin, we showed that loss of tubulin methylation decreases the migratory and invasive behaviour of ccRCC cells but increases cell spreading, suggesting that α-TubK40me3 may play a role in tumor invasion. Moreover, we demonstrated that SETD2 mutation reduces the percentage and length of cilia in ccRCC cells. Additionally, cilia formation is reduced by mutations in the SET (R1625C) and SRI (R2510H) domains, indicating a potential role for α-TubK40me3 in cilia development. Since the phospho-serine and -threonine sites of SETD2 are known to be targeted by Aurora A Kinase (AURKA), we showed that cilia formation in ccRCC cells is reduced after treatment with the AURKA kinase inhibitor Alisertib, implying that AURKA may have an effect on SETD2’s catalytic function. We also showed that EPZ-719, a novel SETD2 inhibitor, decreases SETD2 catalytic activity in ccRCC cells, inhibits cilia formation and affects cell spreading. Finally, we showed that AURKA inhibition affects ccRCC cell proliferation and migration, suggesting that AURKA may affect SETD2 catalytic activity towards cytoskeletal substrates, which alters cellular mobility.