Functional validation of driver mutation‐specific uveal melanoma biomarkers: role of COL9A3 in cancer cell plasticity

Abstract Uveal melanoma (UM) is a deadly ocular malignancy with well‐described genetic alterations that predict disease outcome. However, our current understanding of the biological underpinnings of high‐risk uveal melanoma progression remains relatively limited. Using RNA expression profiles from 250 patients with UM, we identified 12 novel biomarkers associated with high‐risk UM, with protein expression level validation of the top two candidates: the transcriptional regulator RBFOX2 and matrix protein COL9A3 . Moreover, we investigated the functional contribution of COL9A3 via its overexpression in EIF1AX mut and BAP1 mut UM cell lines. Our data suggest that COL9A3 enhances cell motility and cell proliferation and alters morphology specifically in BAP1 mut UM cells. Furthermore, zebrafish xenograft studies revealed increased cell dissemination in a EIF1AX mut UM cell line upon overexpression of COL9A3, whereas a BAP1 mut UM cell line remained unchanged under wild‐type conditions. Interestingly, RNA sequencing revealed a high‐stress profile by upregulated metabolic activity and loss of protein translation due to COL9A3 overexpression. BAP1 mut UM appears to adapt to this stress by upregulation of plasticity markers, such as CD44 , Nestin , EZH2 , ABCB5 , PAX3 and CD166 , as a coping mechanism. These markers are known as differentiation markers during the development of melanocyte biogenesis and are implicated in plasticity in other high‐risk cancers. The relationship between COL9A3 and plasticity was validated by multiplex immunohistochemistry of formalin‐fixed paraffin‐embedded (FFPE) tissue, which demonstrated colocalization of COL9A3, CD44 and Nestin. BAP1 mut ‐UM samples presented higher levels of COL9A3, CD44 and Nestin expression than EIF1AX mut or SF3B1 mut UM samples based on the mean fluorescent intensity of each marker. A positive correlation between COL9A3 and Nestin expression in triple‐positive cells, irrespective of the UM subtype, was observed. Collectively, our findings suggest that COL9A3 is a novel high‐risk biomarker that may contribute to UM cell plasticity and is most prevalent in BAP1 mut UM. © 2026 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.