Abstract B015: Chromothripsis driven loss of <i>NSD1</i> and <i>SETD2</i> disrupts an epigenetic-kinase circuit governing mitotic fidelity

Abstract Chromothripsis driven 3p deletion and 5q amplification are early, clonal events in clear cell renal cell carcinoma (ccRCC), targeting the epigenetic regulators SETD2 (Set-domain containing 2) and NSD1 (Nuclear receptor binding SET-domain protein 1). Paradoxically, although NSD1 is amplified on 5q, it is frequently hypermethylated and transcriptionally silenced, suggesting that functional inactivation is selected for. The mechanistic consequences of dual loss of NSD1 and SETD2 remain poorly understood. The emergence of SETD2’s dual function in methylating chromatin and microtubules of the cytoskeleton, led us to investigate if NSD1 similarly functioned at the mitotic spindle. Using an integrated approach combining in vitro biochemical assays, quantitative mass spectrometry, molecular modeling, and CRISPR/Cas9-engineered cell lines we have dissected the regulatory interplay between NSD1 and SETD2 at the mitotic spindle. We identified NSD1 as a methyltransferase that directly methylates AURKA, acting as a negative regulator of its kinase activity during mitosis. Loss of NSD1, via genetic depletion or pharmacologic inhibition, induced AURKA hyperactivation, spindle defects, chromosome mis-segregation, and micronuclei formation. Notably, we found that AURKA phosphorylated SETD2 to modulate its activity in a feedforward circuit where SETD2 also methylates AURKA. Disruption of phosphorylation on SETD2 by AURKA either through AURKA inhibition or SETD2 mutation (phospho-site) compromised mitotic fidelity and accelerated genomic instability. These findings reveal a previously unrecognized NSD1-AURKA-SETD2 epigenetic-kinase axis that integrates mitotic and epigenetic regulation. Chromothripsis-driven depletion of NSD1 and SETD2 creates a state of mitotic fragility, providing a rationale for AURKA-targeted therapy in genetically defined subsets of ccRCC. Citation Format: Ruhee Dere, Ashley Boice, Richard Han, Xiaoli Wang, Manga Motrapu, Pratim Chowdhury, Sung Jung, B.V. Venkata Prasad. Chromothripsis driven loss of NSD1 and SETD2 disrupts an epigenetic-kinase circuit governing mitotic fidelity [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Innovations in Kidney Cancer Research: From Molecular Insights to Therapeutic Breakthroughs; 2026 Mar 13-16; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2026;86(5_Suppl_2):Abstract nr B015.