Rapid activation of ARF6 after RAF inhibition augments BRAFV600E and promotes therapy resistance.

The intrinsic ability of cancer cells to evade death underpins tumorigenesis, progression, metastasis, and the survival of drug-tolerant persister (DTP) cells. Herein, we discovered that the small GTPase ARF6 plays a central role in tumor survival by fortifying RAF oncoprotein levels. ARF6 activation was sufficient to increase BRAFV600E, ARAF, and CRAF proteins through a post-transcriptional mechanism, while sustained inhibition of ARF6 eventually led to decay. In a genetically engineered model of aggressive melanoma, tumor-specific Arf6 deletion attenuated BRAFV600E protein expression and MAPK signaling and prevented rapid tumor progression. In human melanoma cells, pharmacologic inhibitors of BRAFV600E uniformly induced swift activation of ARF6, driving a positive feedback loop that restored MAPK-driven anti-apoptotic signaling and supported drug-tolerant survival and growth. Furthermore, in patient-derived melanoma xenografts with innate or clinically acquired resistance to MAPK inhibitors, ARF6 silencing alone significantly suppressed tumor growth in vivo. When combined with BRAF and MEK targeted therapy in vitro, inhibition of ARF6 markedly reduced survival and drug-tolerant growth. Collectively, these findings reveal a previously unknown mechanism of maintaining BRAFV600E protein expression that preserves the MAPK pathway during targeted therapy. This ARF6-dependent mechanism may be exploited in BRAFV600E driven cancers as a therapeutic vulnerability.