43AURKA connects NSD1 and SETD2 in an epigenetic axis governing mitotic fidelity in ccRCC

Abstract Background Chromothripsis-driven 3p deletion and 5q amplification are early, clonal events in clear cell renal cell carcinoma (ccRCC). These lesions respectively target the epigenetic regulators SETD2 and NSD1. Paradoxically, although NSD1 is amplified on 5q, it is frequently hypermethylated and transcriptionally silenced, suggesting functional inactivation is selected for. The mechanistic implications of NSD1 and SETD2 loss in mitotic control remain poorly understood. Methods We employed a multifaceted approach integrating in vitro kinase and methyltransferase assays, mass spectrometry, and molecular modeling to investigate the regulatory relationship between NSD1, AURKA, and SETD2. CRISPR/Cas9-engineered cell lines were generated to model genetic loss of NSD1, while pharmacologic inhibitors were used to perturb NSD1 and AURKA activity. Protein-protein interactions and post-translational modifications were characterized via co-immunoprecipitation, immunoblotting, fluorescence microscopy, and quantitative mass spectrometry. Functional outcomes were assessed in cell-based systems, and the impact on tumor growth was evaluated in xenograft models examining AURKA-mediated regulation of SETD2 in vivo. Results We identified NSD1 as a methyltransferase that directly methylates AURKA, serving as a negative regulator of its kinase activity and subcellular dynamics during mitosis. Loss of NSD1, through genetic deletion or pharmacologic inhibition, led to AURKA hyperactivation, defective spindle architecture, chromosome mis-segregation, and increased micronuclei formation. Unexpectedly, we found that AURKA phosphorylates SETD2, functionally linking these two epigenetic regulators. This phosphorylation selectively regulated SETD2’s cytoskeletal activity without affecting its chromatin-associated roles. Disruption of this modification—via AURKA inhibition or mutation of the phosphorylation site—compromised mitotic fidelity and enhanced genomic instability. Importantly, phosphorylation-deficient SETD2 mutants were incapable of sustaining tumor growth in xenograft models, underscoring the oncogenic relevance of this post-translational modification. Moreover, SETD2 loss sensitized ccRCC cells to AURKA inhibition, revealing a potential therapeutic vulnerability. Conclusions Our findings reveal a novel regulatory pathway in which NSD1-mediated methylation suppresses AURKA, while AURKA phosphorylation regulates SETD2 activity on the cytoskeleton, linking these two tumor suppressors altered in early ccRCC. Disruption of this NSD1-AURKA-SETD2 axis creates a state of mitotic vulnerability, opening the door for therapeutic intervention using AURKA inhibitors in genetically defined subsets of ccRCC.