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NAMPT and NNMT released via extracellular vesicles and as soluble mediators are distinguished traits of BRAF inhibitor resistance of melanoma cells impacting on the tumor microenvironment

ABSTRACT: Drugs targeting mutant oncogenes are effective, even though resistance rapidly develops. This complex picture includes acquired intrinsic tumor and tumor microenvironmental -mediated mechanisms. Here we showed that melanoma cells resistant to BRAF inhibitors (BRAFi) overexpressed the rate limiting enzymes involved in nicotinamide (NAM) metabolism nicotinamide phosphoribosyltransferase (NAMPT) and nicotinamide N-methyltransferase (NNMT). Importantly, NAMPT and NNMT are released by these cells, both in the free-form or loaded in extracellular vesicles (EVs). NAMPT is emerging as mediator of BRAFi resistance in melanoma, but to date mainly associated with its role as main NAD-biosynthetic enzyme. It had been previously identified as soluble factor, but never in EVs released from melanoma cells, which highlights an impact on the tumor microenvironment (TME). NNMT was revealed increased in melanoma compared to benign nevi, however we showed for the first time its overexpression in resistant cells at intracellular and extracellular levels (present in secretome and in EVs). NNMT increased in BRAF-mutated patients linking its expression with the BRAF oncogenic signaling and correlates positively with pro-inflammatory signaling, immune cell migration and chemokine-mediated signaling pathways opening to a future deeper exploration of its functional role. Lastly, we proposed a tetrameric NNMT:TLR4 binding model offering a plausible structural and mechanistic basis for their association. Overall, the identification of NAMPT and, surprisingly also NNMT, included in EVs and abundantly released from resistant melanoma cells supports the impact of these moonlighting proteins involved in nicotinamide metabolism as mediators of BRAF/MEK inhibitors resistance with tumor intrinsic and potentially tumor microenvironment-mediated mechanisms. Interfering with nicotinamide metabolism could be a valid strategy to counteract drug resistance acting on the multifactorial tumor-host interactions.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Romina Belli

LAB HEAD: Alessandro Provenzani

PROVIDER: PXD063060 | Pride | 2025-07-28

REPOSITORIES: Pride

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NAMPT and NNMT released via extracellular vesicles and as soluble mediators are distinguished traits of BRAF inhibitor resistance of melanoma cells impacting on the tumor microenvironment.

Ghezzi Beatrice B Fiorilla Irene I Carreira Ágata Á Recco Francesco F Sorci Leonardo L Avalle Lidia L Ponzano Alessia A Mazzola Francesca F Todesco Alberto Maria AM Tommasi Nicoletta N Gasparrini Massimiliano M D'Agostino Vito Giuseppe VG Mignone Flavio F Provenzani Alessandro A Audrito Valentina V

Cell communication and signaling : CCS 20250721 1

PMID: 40691620

Similar Datasets

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.

Project description:Nearly 50% of cutaneous melanomas carry activating mutations on the BRAF oncogene, and the combination of BRAF- and MEK-inhibitors (BRAF/MEKi) is frequently used for their clinical management. One major drawback of BRAF/MEKi targeted therapy is the rapid development of therapeutic resistance, which can occur via multiple mechanisms, including the metabolic rewiring of cancer cells that often involves the upregulation of mitochondrial bionergetics and NAD+ biosynthetic pathways. mTORC2 is a signaling complex that requires the presence of its essential RICTOR subunit to play its regulatory functions in cell growth and metabolism. mTORC2 is believed to play mostly pro-oncogenic roles in several tumor types, including melanoma. However, bioinformatics analysis of TCGA melanoma patients’ database revealed that low RICTOR levels in tumors correlate with an overall worse clinical outcome. GSEA analysis of low-RICTOR tumors evidenced also a gene expression signature suggestive of activation of mitochondrial energy producing pathways. On these bases, we have hypothesized that inhibition of mTORC2 activity may render BRAFV600E melanoma cells resistant to BRAFi/MEKi. We show here that RICTOR/mTORC2-deficient cells are intrinsically tolerant to BRAFi/MEKi, and anticipate the onset of resistance to BRAFi after sustained drug exposure both in vitro and in vivo, indicating that mTORC2 activity normally opposes the acquisition of targeted therapy resistance in BRAFV600E melanomas. Mechanistically, RICTOR-deficient cells show an enhanced mitochondrial respiratory potential and increased expression of nicotinamide phosphoribosyltransferase (NAMPT) protein, the rate-limiting enzyme of NAD+ salvage pathway, and pharmacological inhibition of these processes in RICTOR-deficient cells is sufficient to restore sensitivity to BRAFi. Thus, our work identifies a novel role for mTORC2 in favoring the responses of BRAF-mutated melanoma cells to targeted therapy, and suggest that the evaluation of the intratumor level of RICTOR may help to predict the responses of melanoma patients to these treatments.

Project description:Despite the clinical success of targeted therapy inhibitors, tumor responses are transient, and a subpopulation of residual drug-tolerant cells may seed resistance. Understanding minimal residual disease (MRD) mechanisms and the connection with dormancy-like traits may guide strategies to target drug-tolerant persister cells and optimize target therapies. Here, we studied the relationship between persister cell tolerance to BRAF and MEK inhibitors (BRAFi + MEKi) and expression of nuclear receptor subfamily 2 group F member 1 (NR2F1), which has been linked to tumor dormancy in the context of cutaneous melanoma. In previously published RNA-seq datasets, NR2F1 expression was enriched in melanoma samples following BRAF inhibitor and MEK inhibitor (BRAFi + MEKi) treatment compared to pre-treatment patient-matched samples. We also found that NR2F1 is highly expressed in the residual drug-tolerant cell state following BRAFi + MEKi treatment in patient-derived and cell line-derived melanoma xenografts. Additionally, xenografts overexpressing NR2F1 displayed higher tumor growth and poorer animal survival following BRAFi + MEKi treatment compared to control xenografts expressing basal levels of NR2F1. In vitro, NR2F1-overexpressing cells showed increased tumor proliferation and higher expression of cell cycle and survival-related proteins on targeted therapy, such as phosphorylation of both RB and S6K proteins. In addition, NR2F1, highly expressed in invasive melanoma cells, was sufficient to promote invasiveness following BRAFi + MEKi in 3D tumor spheroid assays. Furthermore, when compared to young mice, NR2F1 was overexpressed in tumor xenografts in an old microenvironment. The use of shRNA NR2F1 in older mice led to slower tumor growth and the highest survival in animals on BRAFi + MEKi treatment. We also tested a pharmacological approach to target drug-tolerant persister cells that overexpress NR2F1 on BRAFi + MEKi treatment. The inhibition of mTORC1 delayed NR2F1-overexpressing cell proliferation compared to non-NR2F1-overexpressing cells on targeted therapy. Altogether, our findings suggest that NR2F1 plays a role in the persistence of MRD in melanoma, indicating that targeting NR2F1 expression could improve targeted therapy outcomes in melanoma patients.

Project description:BRAF oncogene is mutated in ~50% of human cutaneous melanomas. The BRAF V600E mutation leads to constitutive activation of the mitogen-activated protein kinase (MAPK) pathway fuelling cancer growth. The inhibitors of BRAF V600E (BRAFi), lead to massive and high response rate. However, BRAFi-resistant cells that operate as a cellular reservoir for relapses severely limits the duration of the clinical response. The recent depiction of these resistant cells did not identify druggable targets to ensure long-term survival under BRAFi. Here, we identify the aryl hydrocarbon receptor (AhR) as a target to eradicate resistant cells. We show that BRAFi bind to AhR on a new site, named beta-pocket, and reprogram gene expression independently of its partner ARNT. beta-pocket activation induces a pigmentation signature, which is associated to BRAFi-induced cell death of sensitive BRAF V600E melanoma cells and tumour shrinkage. Intriguingly, in resistant cells, BRAFi does not induced a pigmentation signature since these cells display another AhR program; AhR-ARNT dependant. By this way, AhR directs several key BRAFi-resistant genes. At single cell level, this constitutive activation of AhR-ARNT is identified in rare cells before BRAFi-treatment of melanoma tumours and an enrichment of these alpha-cells is observed under BRAFi. Our data strongly suggest that an endogenous AhR ligand activates AhR-ARNT via the canonical AhR pocket (alpha-pocket), thus favouring BRAFi-resistant gene expression. Importantly, we identify the clinically compatible AhR antagonist, the resveratrol (RSV), able to abrogate the deleterious constitutive activation of AhR and to reduce the cellular reservoir for the relapse. Taken together, this work reveals that constitutive AhR signalling drives BRAFi resistance and constitutes a therapeutic target to achieve long-term patient survival under BRAFi. More broadly, the constitutive activation of AhR by endogenous ligands is in line with the ability of UV radiations to generate potent AhR ligands and to favour melanoma onset.

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