New Strategies For Probing And Inhibiting Cdk4/Cyclin D Hyperactivity In Melanoma

Uncontrolled proliferation of melanocytes results in melanoma, one of the most aggressive forms of skin cancer, which further metastasizes into a deadly form of cancer. The p16‐cyclin D‐CDK4/6‐retinoblastoma protein pathway is dysregulated in 90% of melanomas and activation of this pathway potently cooperates with mutant BRAF or NRAS in transformation of melanocytes. CDK4/cyclin D kinase hyperactivation, associated with mutation of CDK4, amplification of cyclin D or loss of p16 INK4a leads to increased risk of developing melanoma. This kinase therefore constitutes a key biomarker and an emerging pharmacological target. The limited efficacy of current therapeutics for treatment of melanoma and the lack of diagnostic assays to detect the early onset of this disease call for new generations of targeted therapeutics and novel approaches to probe melanoma‐specific biomarkers. To this aim, we engineered a fluorescent biosensor which reports on CDK4 kinase hyperactivity in vitro and in living cells, which we employed to develop a diagnostic assay using human skin biopsies. We further designed biosensors for drug discovery purposes, which discriminate compounds that target the interface between CDK4 and cyclin D, or that interfere with conformational dynamics of CDK4 activation loop, which were successfully applied to screen for novel and potent classes of CDK4 inhibitors Our work highlights the potency of fluorescent biosensors for developing diagnostic strategies to monitor cancer‐associated alterations in CDK4 activity as well as drug discovery programmes.