RICTOR/mTORC2 downregulation in BRAFV600E melanoma cells promotes resistance to BRAF/MEK inhibition

Luca Ponzone; Valentina Audrito; Claudia Landi; Enrico Moiso; Chiara Levra Levron; Sara Ferrua; Aurora Savino; Nicoletta Vitale; Massimiliano Gasparrini; Lidia Avalle; Lorenza Vantaggiato; Enxhi Shaba; Beatrice Tassone; Stefania Saoncella; Francesca Orso; Daniele Viavattene; Eleonora Marina; Irene Fiorilla; Giulia Burrone; Youssef Abili; Fiorella Altruda; Luca Bini; Silvia Deaglio; Paola Defilippi; Alessio Menga; Valeria Poli; Paolo Ettore Porporato; Paolo Provero; Nadia Raffaelli; Chiara Riganti; Daniela Taverna; Federica Cavallo; Enzo Calautti

doi:10.1186/s12943-024-02010-1

RICTOR/mTORC2 downregulation in BRAF^V600E melanoma cells promotes resistance to BRAF/MEK inhibition

Mol Cancer. 2024 May 16;23(1):105. doi: 10.1186/s12943-024-02010-1.

Authors

Luca Ponzone^{1

2}, Valentina Audrito³, Claudia Landi⁴, Enrico Moiso⁵, Chiara Levra Levron^{1

6}, Sara Ferrua^{1

2}, Aurora Savino^{1

2}, Nicoletta Vitale^{1

2}, Massimiliano Gasparrini⁷, Lidia Avalle^{1

2

3}, Lorenza Vantaggiato⁴, Enxhi Shaba⁴, Beatrice Tassone^{1

2

8}, Stefania Saoncella^{1

2}, Francesca Orso^{1

2}, Daniele Viavattene^{1

2}, Eleonora Marina^{1

2}, Irene Fiorilla³, Giulia Burrone^{1

2

9}, Youssef Abili^{1

2

10}, Fiorella Altruda^{1

2}, Luca Bini⁴, Silvia Deaglio^{1

11}, Paola Defilippi^{1

2}, Alessio Menga^{1

2}, Valeria Poli^{1

2}, Paolo Ettore Porporato^{1

2}, Paolo Provero¹², Nadia Raffaelli⁷, Chiara Riganti^{1

13}, Daniela Taverna^{1

2}, Federica Cavallo^{1

2}, Enzo Calautti^{14

15}

Affiliations

¹ Molecular Biotechnology Center "Guido Tarone", University of Turin, Turin, 10126, Italy.
² Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, 10126, Italy.
³ Department of Science and Technological Innovation, University of Piemonte Orientale, Alessandria, 15121, Italy.
⁴ Functional Proteomic Section, Department of Life Sciences, University of Siena, Siena, 53100, Italy.
⁵ Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, USA.
⁶ Department of Life Sciences and Systems Biology, University of Turin, Turin, 10126, Italy.
⁷ Department of Agriculture, Food and Environmental Sciences, Polytechnic University of Marche, Ancona, 60131, Italy.
⁸ Department of Personal Care, dsm-firmenich, Kaiseraugst, 4303, Switzerland.
⁹ Department of Clinical and Biological Sciences, University of Turin, Turin, 10124, Italy.
¹⁰ GenomeUp, Rome, 00144, Italy.
¹¹ Department of Medical Sciences, University of Turin, Turin, 10124, Italy.
¹² Neuroscience Department "Rita Levi Montalcini", University of Turin, Turin, 10126, Italy.
¹³ Department of Oncology, University of Turin, Turin, 10124, Italy.
¹⁴ Molecular Biotechnology Center "Guido Tarone", University of Turin, Turin, 10126, Italy. vincenzo.calautti@unito.it.
¹⁵ Department of Molecular Biotechnology and Health Sciences, University of Turin, Turin, 10126, Italy. vincenzo.calautti@unito.it.

Abstract

Background: The 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 assess in this context the role of mTORC2, a signaling complex defined by the presence of the essential RICTOR subunit, regarded as an oncogenic driver in several tumor types, including MM.

Methods: After analyzing The Cancer Genome Atlas 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. We performed 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: Low RICTOR levels in BRAF-mutated MM correlate with a worse clinical outcome. Gene Set Enrichment Analysis of low-RICTOR tumors display gene signatures suggestive of activation of the mitochondrial Electron Transport Chain (ETC) energy production. RICTOR-deficient BRAF^V600E cells are intrinsically tolerant to BRAF/MEKi and anticipate the onset of resistance to BRAFi upon prolonged drug exposure. Moreover, in drug-naïve cells we observed a decline in RICTOR expression shortly after BRAFi exposure. In RICTOR-depleted cells, both mitochondrial respiration and expression of nicotinamide phosphoribosyltransferase (NAMPT) are enhanced, and their pharmacological inhibition restores sensitivity to BRAFi.

Conclusions: Our work unveils an unforeseen tumor-suppressing role for mTORC2 in the early adaptation phase 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 indicate that the evaluation of intra-tumor RICTOR levels has a prognostic value in metastatic melanoma and may help to guide therapeutic strategies in a personalized manner.

Keywords: BRAFV600E melanoma; Drug resistance; Mitochondrial metabolism; NAMPT; RICTOR; Targeted therapy; mTORC2.

MeSH terms

Animals
Cell Line, Tumor
Down-Regulation
Drug Resistance, Neoplasm* / genetics
Gene Expression Regulation, Neoplastic
Humans
Mechanistic Target of Rapamycin Complex 2* / genetics
Mechanistic Target of Rapamycin Complex 2* / metabolism
Melanoma* / drug therapy
Melanoma* / genetics
Melanoma* / metabolism
Melanoma* / pathology
Mice
Mutation
Protein Kinase Inhibitors* / pharmacology
Protein Kinase Inhibitors* / therapeutic use
Proteomics / methods
Proto-Oncogene Proteins B-raf* / genetics
Rapamycin-Insensitive Companion of mTOR Protein* / genetics
Rapamycin-Insensitive Companion of mTOR Protein* / metabolism
Xenograft Model Antitumor Assays

Substances

RICTOR protein, human
BRAF protein, human

Abstract

MeSH terms

Substances

Grants and funding