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Proteomics of BRAFi-treated endothelial cells

ABSTRACT: Targeted therapies against mutant BRAF are effectively used in combination with MEK inhibitors (MEKi) to treat advanced melanoma. However, treatment success is affected by resistance and adverse events (AEs). Approved BRAF inhibitors (BRAFi) show high levels of target promiscuity, which can contribute to these effects. Blood vessels are in direct contact with high plasma concentrations of BRAFi, but effects of the inhibitors in this cell type are unknown. Hence, we aimed to characterize responses to clinically relevant BRAF kinase inhibitors in the vascular endothelium. A mass spectrometry-based analysis of the phosphoproteome of dermal microvascular endothelial cells revealed distinct sets of off-targets of the used BRAFi. Meanwhile, there were only negligible changes to the proteome after 1 h of treatment. Together with our published functional data, these findings provide insights on the surprisingly distinct side effects of BRAFi on endothelial signaling and functionality.

INSTRUMENT(S): Orbitrap Eclipse

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Skin, Endothelial Cell

DISEASE(S): Melanoma

SUBMITTER: Sophie Bromberger

LAB HEAD: Klaudia Schossleitner

PROVIDER: PXD052251 | Pride | 2024-06-01

REPOSITORIES: Pride

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			Action	DRS
	20220426-0228_ECLIP_1002083_MRL_SB_E_20220425_NMod_F1b.raw	Raw
	20220426-0518_ECLIP_1002084_MRL_SB_E_20220425_NMod_F2.raw	Raw
	20220426-0808_ECLIP_1002085_MRL_SB_E_20220425_NMod_F3.raw	Raw
	20220426-1059_ECLIP_1002086_MRL_SB_E_20220425_NMod_F4.raw	Raw
	20220426-1349_ECLIP_1002087_MRL_SB_E_20220425_NMod_F5.raw	Raw

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Project description:Background Main drawback of BRAF/MEK inhibitors (BRAF/MEKi)-based targeted therapy in the management of BRAF- mutated cutaneous metastatic melanoma (MM) is the development of therapeutic resistance. We aimed to define in this context the specific role of mTORC2, a signaling complex defined by the presence of the essential RICTOR subunit and regarded as an oncogenic driver in several tumor types, including MM. Methods After analyzing the TCGA MM patients’ database to explore both overall survival and molecular signatures as a function of intra-tumor RICTOR levels, we investigated the effects of RICTOR downregulation in BRAF V600E MM cell lines on their response to BRAF/MEKi in vitro. We performed a proteomic screening to identify proteins modulated by changes in RICTOR expression and Seahorse analysis to evaluate the effects of RICTOR depletion on mitochondrial respiration. The combination of BRAFi with drugs targeting proteins and processes emerged in the proteomic screening was carried out on RICTOR-deficient cells in vitro and in a xenograft setting in vivo. Results We found that low RICTOR levels in MM correlate with an overall worse clinical outcome. GSEA of low- RICTOR tumors revealed a gene expression signature suggestive of activation of the mitochondrial Electron Transport Chain (ETC) energy producing pathway. RICTOR-deficient BRAF V600E cells are intrinsically tolerant to BRAFi/MEKi and anticipate the onset of resistance to BRAFi upon prolonged drug exposure. In RICTOR- depleted cells, both mitochondrial respiration and expression of nicotinamide phosphoribosyltransferase (NAMPT) are enhanced, while their pharmacological inhibition restores sensitivity to BRAFi. Conclusions Our work unveils a novel tumor suppressing role for mTORC2 in the responses of BRAF V600E melanoma cells to targeted therapy and identifies the NAMPT-ETC axis as a potential therapeutic vulnerability of low- RICTOR tumors. Importantly, our findings support the concept that the evaluation of intra-tumor RICTOR levels in MM has a prognostic value, and may help predicting the response of patients to targeted therapy.

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Proteomics of BRAFi-treated endothelial cells

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