Targeting endothelin receptor signalling overcomes heterogeneity driven therapy failure

Abstract Approaches to prolong responses to BRAF targeting drugs in melanoma patients are challenged by phenotype heterogeneity. Melanomas of a “MITF‐high” phenotype usually respond well to BRAF inhibitor therapy, but these melanomas also contain subpopulations of the de novo resistance “AXL‐high” phenotype. > 50% of melanomas progress with enriched “AXL‐high” populations, and because AXL is linked to de‐differentiation and invasiveness avoiding an “AXL‐high relapse” is desirable. We discovered that phenotype heterogeneity is supported during the response phase of BRAF inhibitor therapy due to MITF‐induced expression of endothelin 1 (EDN1). EDN1 expression is enhanced in tumours of patients on treatment and confers drug resistance through ERK re‐activation in a paracrine manner. Most importantly, EDN1 not only supports MITF‐high populations through the endothelin receptor B (EDNRB), but also AXL‐high populations through EDNRA, making it a master regulator of phenotype heterogeneity. Endothelin receptor antagonists suppress AXL‐high‐expressing cells and sensitize to BRAF inhibition, suggesting that targeting EDN1 signalling could improve BRAF inhibitor responses without selecting for AXL‐high cells.


Appendix Supplementary Methods
Cell lines A375 and WM266-4 cells were bought from the American Type Culture Collection and 501mel and 888mel cells were a gift from Steve Rosenberg (NCI, MD); all were obtained in 2008. The human melanoma cell line WM164 used in the single cell analysis was genotypically characterized (1,2), grown as described (3)

Cell culture reagents and treatments
Vemurafenib, AZD6244, PD184352, Go-6983, RAF265, dovotininb, bosentan and macitentan were from Selleck Chemicals (Newmarket, UK). BQ788 and BQ123 were from Tocris. siRNAs transfections were carried out using lipofectamine or transferrin. All siRNAs were from Dharmacon/Thermo Fisher and the sequences were: SC control: AAUAUAAUCACUAUCAGGUGC; MITF#1: GAACGAAGAAGAAGAUUUAUU; MITF#3: GACCUAACCUGUACAACAAUU, EDN1: SMARTpool L-016692-00. For drug-dose response curves cells were plated in 96-well plates at a density of 0.1x10 5 and treated with serial dilutions of the drugs as indicated. After treatment cells were fixed and stained with crystal violet and the concentration required to inhibit cell growth by 50% (GI50) was calculated using GraphPad Prism version 6.0a for Mac OS, GraphPad Software (San Diego California, USA).

Incucyte analysis
Apoptotic cells were detected by caspase 3/7 dependent cleavage of a florescent reagent (Essen BioScience Cat no 4440). Cells were imaged using an IncuCyte ZOOM (Essen BioScience) under 20× objective, under normal cell culture conditions. Phase contrast and fluorescence images were acquired every 20mins with four images per well. IncuCyte ZOOM software was utilized in real-time for the measurement of apoptosis, this data was further analysed in Prism 6 (GraphPad).

Co-culture analysis
Cells were plated at a density of 1x10 5 per 6 well trans-well insert with a pore size of 0.4µm (Corning, catalogue number: 3412), and left to adhere for 24 h before being transferred to six wells with already seeded melanoma cell lines. Cells were treated with inhibitors after 24 h of co-culturing for indicated times followed by either cell lysis or cell survival analysis. Cell numbers were assessed by crystal violet staining of cells fixed in 4% formaldehyde. Quantification was achieved via spectrophotometrical analysis of the optical density at 540nM (OD 540) of the solubilized dye.

Cell lysis and immunoblotting
Cells were lysed in SDS sample buffer and analysed by standard Western-blotting protocols. The primary antibodies used were: phospho-ERK (MAPK-YT) from Sigma, St Louis, MO, USA; ERK2 (C-14) and beta-tubulin (H235) from Santa Cruz Biotechnology, USA; MITF (C5) from Fisher Scientific, UK; EDN1 mouse monoclonal Antibody was from Abcam, UK. PKC substrate (cat # 6967) from Cell Signaling.

RNA analysis and qPCR primers
RNA from cell lines or frozen tumour tissue was isolated with TRIZOL® as described previously (4,5). Selected genes were amplified using SYBR green (Qiagen, Valencia, CA, USA

Immunohistochemistry and immunofluorescence
After de-paraffinisation, antigen retrieval was performed by the pressure cooker method using EDTA buffer, pH 8.0. Staining was performed using an automated system (Autostainer plus Dako). A monoclonal mouse antibody raised against a N-terminal fragment of MITF protein of human origin (Santa Cruz Biotechnology, sc-56433) was used at a 1:60 dilution. Sections were counterstained with hematoxylin. Additional anti-bodies are listed in the table below. For immunofluorescence tumour samples either derived from a patient treated with dabrafenib or taken from mice treated with either vemurafenib (25mg/kg) or selumetinib (10mg/kg) were fixed and cryo-sections were permeabilised in a solution of 0.1 % Trition-X100 and 1 % saponin in PBS for 15 min. Sections were blocked in 10 % BSA at 37°C for 30 min and incubated overnight at 4°C with primary MITF antibody ((D5) Neomarkers, Lab Vision, Runcorn, UK) in 10 % BSA PBS. Stained sections were washed in PBS and then incubated with Cy3-labelled secondary antibody for 2 hr at RT and mounted using DAPI containing vectashield. For cell staining, cells were fixed with 4 % formaldehyde in PBS for 15 min before permeabilization with 0.1 % Triton-X100 and the protocolled followed as described above using the indicated antibodies. A Zeiss Axioskop 2 plus equipped with a 40x Plan Neofluar® objective was used; images were taken by a Photometrics Cool Snap HQ CCD camera driven by Metamorph software (Universal Imaging).

Mass Spectrometry and data analysis
Cells were cultured in serum-free medium for 48-72h. The supernatant was collected, cooled on ice and spun at 2000g for 20 min to remove debris and subsequently filtered through a 0.22 filter. Exosomes were removed by spinning the supernatant further at 100000g for 1 h. The samples were additionally concentrated with spin cut-off membranes with a 3-5kDa cut-off. Samples were frozen and subsequently lysed by boiling them in 1% SDS, 0.1M DTT. The FASP protocol (6) was used to remove SDS, alkylate cysteine and digest the proteins. The peptides were analysed by mass spectrometry as previously described (7). Protein identification quantification was performed by label-free quantification with the MaxQuant software package (8).