Investigating the role of ARAF and KSR1 in NRAS-driven malignant melanoma

Malignant melanomas are characterised by activating mutations in the RAS-RAF-ERK pathway including BRAF (50%) and NRAS (30%). Alarmingly, Ireland’s rate of melanoma incidence and mortality are approximately 30% above the EU average and are increasing. Even worse, the frequency of drug responsive BRAF mutations in Irish patients is less than half than in other European patients, whereas NRAS mutations are almost twice as frequent. While treatment for BRAF driven melanomas exists in the form of RAF and MEK inhibitors, so far, no targeted therapies exist to treat NRAS driven melanomas. Our group’s data suggest that NRAS driven melanomas can be successfully targeted with synergistic RAF inhibitor combinations. However, our mechanistic models suggest that melanomas can compensate drug blockage by the third RAF member ARAF and the signalling scaffold KSR1 highlighting the prominent role of RAF and scaffold usage in NRAS-mutated melanoma. Besides their role in RAF signalling, ARAF and KSR1 seem to fine-tune other processes including migration and metabolism, but regulation in melanoma is not understood and the molecular mechanisms for this phenomenon are unknown. To study these effects, we applied CRISPR/Cas9 gene editing and generated ARAF or KSR1-deficient single cell clones in NRAS-driven melanoma cell lines. Both ARAF and KSR1 deficient cells were subjected to sophisticated multi-omics analysis, including metabolomic, phenotypic and interactome studies. The subsequent bioinformatics analysis and data integration suggest novel crucial signalling crosstalk of ARAF and KSR1 in malignant melanoma. We can demonstrate that ARAF regulates cell migration, invasion, and colony formation suggesting a potential role in cancer metastasis. Importantly, re-expression of ARAF reverses this phenotype. Interestingly, our data also indicate major adaptations in cell metabolism, while parental Mel-Juso cells show typical signs of the Warburg effect, ARAF depletion reactivates oxidative phosphorylation via adaptations in TCA cycle and mitochondrial output. In contrast to the ARAF data, we can demonstrate that KSR1 only regulates cell migration but not cell invasion and the clonogenic potential of cells. However, we confirmed a significant role of KSR1 in mitochondrial metabolism and further provide evidence that melanoma cells can adapt the loss of KSR1 by re-wiring signalling differently. Together, our data suggest crucial roles for ARAF and KSR1 in melanoma migration and metabolism. Consequently, both proteins might represent suitable, novel targets for tailored therapies to overcome intrinsic resistance in NRAS-mutated melanomas.