Clear cell renal cell carcinoma (ccRCC) is characterized by two major chromosome abnormalities, loss of 3p and gain of 5q, which occur nearly universally in this disease. Here we focus on the unique insights these defects provide into activities at the nexus of chromatin and cytoskeletal biology: coordinated activity of chromatin remodelers on spindle microtubules needed for the integrity of mitosis. Colocalized on chromosome 3p are the chromatin remodelers SETD2, PBRM1, and BAP1 and the E3 ligase VHL. Our groups together have pioneered the concept that SETD2 and PBRM1 have active roles on the cytoskeleton that regulate mitotic fidelity, with impacts on genomic integrity that are only now being revealed. SETD2 is a methyltransferase for both histones and spindle microtubules. Using mutant alleles, we isolated loss of microtubule methylation as underlying the genomic instability tied to SETD2 loss. We further discovered that PBRM1, a substrate recognition member of the PBAF chromatin remodeler, specifically recognizes the SETD2 methyl mark on microtubules, and like the mark it “reads”, is required for genomic stability. Finally, we identified the mitotic kinase AURKA as a new target for VHL-mediated degradation, linking this canonical protein (VHL) to complex regulation of mitotic spindle assembly in ccRCC. In Preliminary Data, we find in addition to VHL loss stabilizing AURKA, AURKA regulates SETD2 via phosphorylation on S2080, connecting VHL and SETD2 for the first time in a common oncogenic pathway. We have also discovered another chromatin remodeler, the chromosome 5q histone methyltransferase NSD1, is also acting at the spindle, and excitingly scores in a CRISPR synthetic lethality screen with SETD2 loss. Our Overarching Hypothesis for this application is that VHL and RCC-associated 3p and 5q chromatin remodelers coordinately regulate methylation of spindle microtubules to maintain genomic stability, which when defective, offers unique opportunities for therapeutic intervention. To address our Overarching Hypothesis, we offer three Specific Aims: We will 1) dissect the convergence on the mitotic spindle of 3p and 5q ccRCC chromatin remodelers, 2) define the features of their interactions at the molecular and biochemical levels that promote mitotic integrity or failure, and 3) mechanistically evaluate points of intervention that lend insight into the controls governing mitotic spindle integrity. To accomplish these aims, we will use innovative tools and strategies that precisely evaluate combinatorial mono- and bi-allelic loss of 3p and 5q genes that occur during progression of ccRCC, as well as a rich pipeline of primary ccRCC organoids. The classic genomic features of ccRCC provide new insights on how cytoskeletal activities of chromatin remodelers converge to maintain genomic stability, the principles of study are broadly applicable to many other cancers and have important ramifications for the development of targeted therapies for this dis...