191 Cancer-associated KNSTRN mutations disrupt protein-protein interactions needed for SCC neoplasia

Abstract

Genome instability and sustained proliferation are cardinal features of cancer, yet our understanding of the molecular mechanisms that underlie these characteristics continues to grow. Mutations in the kinetochore gene KNSTRN occur in 19% of cutaneous squamous cell carcinomas (SCC) and interfere with the ability of the encoded protein to regulate spindle function during mitosis. Specifically, cancer-associated KNSTRN mutations disrupt sister chromatid cohesion, enhance tumorigenesis, and correspond to increased aneuploidy in primary SCC, suggesting that Kinastrin is required for genome stability and regulates cellular proliferation in vivo. To test whether Kinastrins protein-protein interactions might be important to its function, we performed proximity ligation assays (PLA) in mitotic primary human keratinocytes transduced to express wild type or cancer-associated mutant Kinastrin. Among known Kinastrin interactors tested, EB1 selectively bound to wild type Kinastrin with loss of this interaction in the context of the cancer-associated mutant protein. We further confirmed disruption of Kinastrin-EB1 binding in human SCC with mutant but not wild type KNSTRN, suggesting that loss of this interaction might contribute to the accelerated aneuploidy seen in this setting. Efforts are underway to identify potentially novel Kinastrin interactors by proximity-dependent biotin ligation assays in mitotic primary human keratinocytes. Both wild type and mutant Kinastrin localize to the spindle and kinetochore in this context, indicating that its temporospatial relationships are preserved and providing further support that loss of specific Kinastrin protein interactions may be required for neoplasia. These data identify previously unrecognized Kinastrin protein interactions that are disrupted in SCC and may provide the foundation for non-surgical therapeutics.