Elevated NR2F1 underlies the persistence of invasive disease after treatment of BRAF-mutant melanoma
Manoela Tiago, Timothy J. Purwin, Casey D. Stefanski, Renaira Oliveira da Silva, Mitchell E. Fane, Yash Chhabra, Jelan I. Haj, Jessica L.F. Teh, Rama Kadamb, Weijia Cai, Sheera R. Rosenbaum, Vivian Chua, Nir Hacohen, Michael A. Davies, Jessie Villanueva, Inna Chervoneva, Ashani T. Weeraratna, Dan A. Erkes, Claudia Capparelli, Julio A. Aguirre-Ghiso, Andrew E. Aplin
Manoela Tiago, Timothy J. Purwin, Casey D. Stefanski, Renaira Oliveira da Silva, Mitchell E. Fane, Yash Chhabra, Jelan I. Haj, Jessica L.F. Teh, Rama Kadamb, Weijia Cai, Sheera R. Rosenbaum, Vivian Chua, Nir Hacohen, Michael A. Davies, Jessie Villanueva, Inna Chervoneva, Ashani T. Weeraratna, Dan A. Erkes, Claudia Capparelli, Julio A. Aguirre-Ghiso, Andrew E. Aplin
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Research Article Oncology

Elevated NR2F1 underlies the persistence of invasive disease after treatment of BRAF-mutant melanoma

Abstract

Despite the success of targeted inhibitors in cutaneous melanoma, therapeutic responses are limited by the aged tumor microenvironment and drug-tolerant residual cells. Given the similarities between drug tolerance and cellular dormancy, we studied the dormancy marker, nuclear receptor subfamily 2 group F member 1 (NR2F1), in response to BRAF-V600E inhibitors (BRAFi) plus MEK inhibitors (MEKi) in BRAF-mutant melanoma models. Transcriptomic analysis of melanoma patient samples treated with BRAFi + MEKi showed increased NR2F1. NR2F1 was highly expressed in the drug-tolerant invasive cell state of minimal residual disease in patient-derived and mouse-derived xenografts on BRAFi + MEKi. NR2F1 over-expression was sufficient to reduce BRAFi + MEKi effects on tumor growth in vivo, and cell proliferation, death, and invasion in vitro. Effects were linked to genes involved in mTORC1 signaling. These cells were sensitive to the combination of BRAFi, MEKi plus rapamycin. Melanomas from aged mice, known to exhibit decreased responses to BRAFi + MEKi, displayed higher levels of NR2F1 compared to tumors from young mice. Depleting NR2F1 in an aged mouse melanomas improved the response to targeted therapy. These findings show high NR2F1 expression in ‘invasive-state’ residual cells and that targeting NR2F1-high cells with mTORC1 inhibitors may improve outcomes in patients with melanoma.

Authors

Manoela Tiago, Timothy J. Purwin, Casey D. Stefanski, Renaira Oliveira da Silva, Mitchell E. Fane, Yash Chhabra, Jelan I. Haj, Jessica L.F. Teh, Rama Kadamb, Weijia Cai, Sheera R. Rosenbaum, Vivian Chua, Nir Hacohen, Michael A. Davies, Jessie Villanueva, Inna Chervoneva, Ashani T. Weeraratna, Dan A. Erkes, Claudia Capparelli, Julio A. Aguirre-Ghiso, Andrew E. Aplin

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Figure 2

NR2F1 overexpression minimizes tumor inhibition effects by BRAFi and MEKi therapy.

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NR2F1 overexpression minimizes tumor inhibition effects by BRAFi and MEK...
(A) NR2F1 protein levels in human BRAF-mutant melanoma cell lines expressing DOX-inducible NR2F1, 1205LuTR-NR2F1, WM793TR-NR2F1, and A375TR-NR2F1 after 72 hours of treatment using BRAFi + MEKi + DOX (1 μmol/L PLX4720 + 35 nmol/L PD0325901 + 100 ng/mL DOX). (B) Colony assay for cell lines overexpressing DOX-inducible NR2F1 after 1 week of treatment using BRAFi + MEKi + DOX. Original magnification, ×20. (C) Detection of S-phase cell-cycle arrest following EdU staining for the BRAF-mutant human melanoma cell lines listed above after 72 hours of treatment with BRAFi + MEKi + DOX. *P < 0.05, by Tukey’s test. (D) PI uptake over cell confluence in the BRAF-mutant human melanoma cell lines listed above after 72 hours of treatment with BRAFi + MEKi + DOX as determined by IncuCyte analysis. *P < 0.05 and **P < 0.01, by Tukey’s test. (E) Representative images of 3D tumor spheroids of the human BRAF-mutant melanoma cell lines 1205LuTR-NR2F1, WM793TR-NR2F1, and A375TR-NR2F1 after 72 hours of treatment with BRAFi + MEKi + DOX (1 μmol/L PLX4720 + 35 nmol/L PD0325901 + 100 ng/mL DOX). 3D tumor spheroids were stained with calcein-AM (7 μmol/L) for cell viability evaluation. Scale bars: 100 μm. (F) Scheme of coculturing of tdTomato cells overexpressing DOX-inducible NR2F1 WT (94) and GFP cells overexpressing a DOX-inducible dominant-negative form of NR2F1 (38) harboring a C141S point mutation within its DNA-binding domain (C141S). Cells were mixed at a ratio of 1:1 and then cocultured for 72 hours with or without BRAFi + MEKi + DOX (1 μmol/L PLX4720 + 35 nmol/L PD0325901 + 100 ng/mL DOX). Cocultures from F were collected and analyzed by FACS for tdTomato and GFP positivity. (G) The percentage of tdTomato and GFP positivity was compared with DMSO. **P < 0.01 and ***P < 0.001, by 2-way ANOVA. Data are presented as the mean ± SD.