NSD1-AURKA-SETD2 axis creates novel mitotic vulnerabilities in clear cell renal cell carcinoma

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 integrated multi-pronged approaches using in vitro kinase and methyltransferase assays, mass spectrometry, molecular modeling, and CRISPR-engineered models to dissect the regulatory relationship between NSD1, AURKA, and SETD2. Protein-protein interactions and post-translational modifications were characterized via co-immunoprecipitation, immunoblotting, fluorescence microscopy, and quantitative proteomics. 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. 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 renal cell carcinoma (RCC). 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. Our data present a rationale for loss of NSD1 and SETD2 in the setting of RCC aimed at dismantling this regulatory axis to drive tumor evolution. This dual loss creates a state of dependency on AURKA activity, providing a rationale for AURKA-based inhibition in a subset of RCC patients with dual loss of NSD1 and SETD2 .