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 5

Rapamycin targets NR2F1-overexpressing drug-tolerant cells and delays tumor growth.

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Rapamycin targets NR2F1-overexpressing drug-tolerant cells and delays tu...
(A) Colony assay for cell lines overexpressing DOX-inducible NR2F1 after 1 week of treatment with BRAFi + MEKi + mTORC1i (either 1 μmol/L AZD2014 or 1 μmol/L rapamycin). Original magnification, ×20. (B) IncuCyte live-cell analysis for DOX-inducible cells overexpressing NR2F1 after 4 weeks of treatment using BRAFi + MEKi + DOX. Cell growth was analyzed for percentage of cell confluence on the plate (representative of three independent experiments). (C) Images show the expression of NR2F1 in the MRD state following BRAFi + MEKi + rapamycin treatment (4 mg/kg) in vivo. Results are for mice bearing 1205Lu-tdTomato–labeled xenografts following continuous BRAFi + MEKi chow (200 ppm PLX4720 + 7 ppm PD0325901) for 3 weeks and then either control chow + vehicle (control), control chow + 4 mg/kg rapamycin (rapamycin), BRAFi + MEKi chow + vehicle (BRAFi + MEKi), or BRAFi + MEKi chow + rapamycin (BRAFi + MEKi + rapamycin) for 1 week. Plot on the right shows the mean nuclear intensity of NR2F1 protein expression in MRD tumor xenografts by immunofluorescence compared with the control. Statistical significance was determined by 2-way ANOVA. (D) Tumor volume results for mice bearing 1205Lu-tdTomato–labeled xenografts following continuous treatment with BRAFi + MEKi chow (200 ppm PLX4720 + 7 ppm PD0325901) until the tumors entered a state of MRD for several weeks (day 52 after BRAFi + MEKi), and were then given either continuous BRAFi + MEKi chow + vehicle (BRAFi + MEKi) or continuous BRAFi + MEKi chow + 4 mg/kg rapamycin twice per week (BRAFi + MEKi + rapamycin) for the duration of the experiment (treatment start indicated with dotted line on x axis). Tumor growth of the treated mice is shown. An “X” on a tracing denotes an animal that was euthanized for nonexperimental reasons. Data indicate the mean ± SD.