Characterizing the SPOP-BRAF-mTORC1 signaling axis in melanoma pathogenesis

BRAF-targeted therapy has achieved great success in clinics for the treatment of BRAF V600 mutated melanomas. Its combination with MEK inhibition and anti-PD-1 immune checkpoint blockade is now the first-line option for treating non-resectable metastatic melanoma harboring the BRAF V600E mutation. However, the therapeutic options are still limited for more than half of the melanoma patients harboring wild-type (WT) or non- V600-mutated BRAF mutations. Different from V600-mutated and other Class I mutated BRAF proteins, which are hyperactive and refractory to many layers of negative regulations, WT and Class II/III BRAF proteins are subjected to multi-layered post-translational regulations. To this end, we and others have previously reported several mechanisms modulating BRAF oncogenic function in melanoma cells, including proteolytic and non- proteolytic polyubiquitination, as well as lysine-acetylation. Our recent data unveiled that the Cullin 3 ubiquitin E3 ligase SPOP catalyzed BRAF poly-ubiquitination and subsequent degradation. Notably, SPOP mutation is found in a small population of melanoma patients and could be subjected to AURKA-mediated phosphorylation and destabilization. SPOP inactivation in melanoma cells could unleash the oncogenic function of BRAF in both treatment-naïve and BRAF-inhibitor resistance melanomas. Moreover, results from ours and previous reports demonstrate that although MEK inhibitors are effective in suppressing ERK kinase activity in WT and non-V600- mutated BRAF melanoma, their efficacy in harnessing melanoma cell growth is marginal. This suggests a non- catalytic role of BRAF in these melanoma cells. Indeed, our preliminary results support a BRAF kinase activity- independent activation of mTORC1, which fuels melanoma cell protein translation and anabolism to facilitate tumorigenesis. These strong preliminary results laid a strong basis for our hypothesis that in WT and non-V600- mutated BRAF melanoma cells, BRAF activates mTORC1 independently of its kinase activity to drive protein translation and anabolism, while SPOP restrains BRAF oncogenic function via destabilizing BRAF and perturbing BRAF-dependent mTORC1 activation. In this proposal, we aim to: 1) characterize a tumor suppressor role of SPOP in melanoma cells in part through restraining BRAF oncogenic functions; 2) define a kinase-independent function of BRAF in activating mTORC1 and its downstream anabolic pathways; 3) assess if the SPOP-BRAF- mTORC1 signaling axis is a novel therapeutic vulnerability in melanoma cells. The successful completion of the proposed studies could, for the first time, characterize a tumor suppressor function for the SPOP E3 ligase in melanoma. The proposed studies will also fill the knowledge gap of how WT-BRAF, kinase-impaired Class III BRAF, or the BRAF protein in BRAF/MEK inhibitor-resistant melanoma cells, sustain melanoma cell proliferation with the absence of ERK/MEK pathway activation. We believe that these findings will provide new opportunities to target WT and non-V600-mutated BRAF melanomas, and eventually translate into future clinical trials. Project Number: 1R01CA299223-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Cancer Institute (NCI) | Principal Investigator: Lixin Wan (+1 co-PI) | Institution: H. LEE MOFFITT CANCER CTR & RES INST, TAMPA, FL | Award Amount: $682,495 | Activity Code: R01 | Study Section: Cancer Cell Biology Study Section[CCB] View on NIH RePORTER: https://reporter.nih.gov/project-details/11290533