New insights into the classification of the RAC1 P29S hotspot mutation in melanoma as an oncogene

The RAC1 ^P29S hotspot mutation, which is prevalent in melanoma, drives tumorigenesis by promoting the persistent activation of RAC1. This mutation enhances molecular interactions, and hyperactivates key signaling pathways, making RAC1 ^P29S a promising target for cancer therapy. This study provides a comprehensive biochemical and cell-based characterization of RAC1 ^P29S , as well as comparisons with wild-type RAC1 and the T17N and F28L mutants. The P29S substitution significantly impairs nucleotide binding while accelerating intrinsic nucleotide exchange. While it minimally affects regulation by guanosine dissociation inhibitor 1 (GDI1), RAC1 ^P29S exhibits reduced activation via DBL family guanine nucleotide exchange factors (GEFs) but retains effective activation by dedicator of cytokinesis 2 (DOCK2). Importantly, the P29S mutation severely impairs GTPase-activating protein-stimulated GTP hydrolysis, which most likely contributes to RAC1 ^P29S hyperactivation by prolonging its GTP-bound active form. This mutation displays a stronger binding affinity for the IQ motif-containing GTPase-activating protein 1 (IQGAP1) than for the p21-activated kinase 1 (PAK1), indicating altered effector interactions that modulate downstream signaling spatially. These biochemical findings are consistent with the fact that RAC1 ^P29S predominantly adopts an active GTP-bound state under serum-starved conditions. IGR1 human melanoma cells harboring endogenous RAC1 ^P29S exhibit persistent RAC1 ^P29S •GTP accumulation, even without upstream GEF activation. Furthermore, the pharmacological inhibition of DOCK2 with CPYPP significantly reduces RAC1 ^P29S activation in these cells, which confirms the pivotal role of DOCK2 in sustaining RAC1 ^P29S -driven signaling. Overexpression of RAC1 ^P29S activates key oncogenic pathways, including ERK1/2 and p38 MAPK, highlighting its role as a constitutively active driver mutation. Together, these results imply that targeting upstream regulators such as DOCK2 and downstream effectors, such as IQGAP1, could be effective therapeutic strategies for counteracting RAC1 ^P29S -mediated melanoma progression and resistance to targeted therapies.