A new ALK isoform transported by extracellular vesicles confers drug resistance to melanoma cells

Abstract

Background: Drug resistance remains an unsolved clinical issue in oncology. Despite promising initial responses obtained with BRAF and MEK kinase inhibitors, resistance to treatment develops within months in virtually all melanoma patients.

Methods: Microarray analyses were performed in BRAF inhibitor-sensitive and resistant cell lines to identify changes in the transcriptome that might play a role in resistance. siRNA approaches and kinase inhibitors were used to assess the involvement of the identified Anaplastic Lymphoma Kinase (ALK) in drug resistance. The capability of extracellular vesicles (EVs) to transfer drug resistant properties was investigated in co-culture assays.

Results: Here, we report a new mechanism of acquired drug resistance involving the activation of a novel truncated form of ALK. Knock down or inhibition of ALK re-sensitised resistant cells to BRAF inhibition and induced apoptosis. Interestingly, truncated ALK was also secreted into EVs and we show that EVs were the vehicle for transferring drug resistance.

Conclusions: To our knowledge, this is the first report demonstrating the functional involvement of EVs in melanoma drug resistance by transporting a truncated but functional form of ALK, able to activate the MAPK signalling pathway in target cells. Combined inhibition of ALK and BRAF dramatically reduced tumour growth in vivo. These findings make ALK a promising clinical target in melanoma patients.

Keywords: ALK; Drug resistance; Extracellular vesicles; Kinase inhibitors; Melanoma.

Fig. 1

Characterisation of resistant melanoma cells. (a) Vemurafenib dose-response analysis in sensitive A375 (black) and resistant A375X1 cells (grey). (b) Growth comparison between untreated sensitive cells versus resistant cells under constant PLX4032 treatment (1 μM). (c) Western blot analysis of A375 and A375X1 in absence or presence of PLX4032 (3 h). Before PLX4032 treatment cells were starved for 16 h. α-Tubulin was used as a loading control; representative blots of three biological replicates are shown. (d) Vulcano plot showing differentially expressed genes in resistant compared to sensitive melanoma cells (FDR < 0.01, at least 1.5-log fold change). (e) Top differentially expressed mRNAs in resistant cells. (f) Western blot analysis detecting ALK^RES only in resistant A375X1 cells. α-Tubulin was used as a loading control; representative blots of three biological replicates are shown

Fig. 2

Knock down of ALK^RES re-sensitises resistant cells to BRAF inhibition. A375X1 cells were transfected with three different siRNAs against ALK or a scrambled control (100 nM) for 72 h. 48 h prior to collection, the cells were incubated with either PLX4032 (1 μM) (a) or Trametinib (3 nM) (b) or MK2206 (1 μM) (c). α-Tubulin was used as a loading control and one representative of three biological replicates is shown. (a-c) Corresponding growth assays on the right. The plates were imaged every 3 h using an IncuCyte ZOOM live cell microscope (Essen BioScience) and images were taken for a total of 90 h. Results are shown for one representative of three biological replicates

Fig. 3

The combination of ALK and PLX4032 inhibitors is efficient in resistant melanoma cells. (a) ALK inhibitors (Crizotinib, Ceritinib and ASP3026) dose-response in resistant A375X1 cells cultured in the absence or presence of 1 μM of PLX4032. (b) PLX4032 dose-response in resistant cells cultured with or without 1 μM of ALK inhibitors. (c) Western blot analysis of resistant A375X1 cells treated with PLX4032 for the indicated time points in the presence of absence of ALK inhibitors. α-Tubulin was used as a loading control and one representative of three biological replicates is shown. (d) Apoptosis assays showing the activity of caspase-3 in resistant and sensitive cells treated either with single inhibitors or with a combination of ALK and BRAF inhibitors, normalised to the untreated control. Error bars represent the standard deviation of three technical replicates of three biological replicates. Statistical significance was determined with a one-way ANOVA coupled with Tukey’s multiple comparison tests. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

ALK is detected in melanoma samples. (a) Immunohistochemistry and corresponding Hematoxylin and Eosin staining of FFPE slides of melanoma patient samples. ALK immunohistochemistry reveals a minor population of moderate immunopositive cells scattered throughout the tumour (Patients 1–4). Patient 5 is representative for ALK-negative sample. Magnification: 40X. (b) Table summarizing patient information. (c) Combination treatments with BRAF and ALK inhibitors strongly reduce melanoma tumour volumes. NSG mice were injected subcutaneously with 2 million A375-X1 cells. After 10 days, treatment was initiated by daily gavage (arrow). Tumour growth was followed over time (left panel) and weight of extracted tumours were measured (right panel). Data are presented as means of tumour volumes (mm³) ± SEM and means of tumour weights (mg) ± SEM, *p < 0.05, **p < 0.01, compared to vehicle-treated tumours (left panel); ***p < 0.001 between groups as indicated (right panel)

Fig. 5

EVs can transfer functional properties. (a) Sensitive A375 melanoma cells were co-cultured with both EV-A375 and EV-A375X1 (10 μg/ml). After 24 h, vemurafenib dose-response analysis was performed to calculate the IC₅₀. Representative dose-response curves of sensitive A375 (black), sensitive A375 plus EV-A375 (grey) and sensitive A375 plus EV-A375X1 (dotted line). (b) PLX4032 IC₅₀ values of sensitive A375 (black), sensitive A375 plus EV-A375 (grey) and sensitive A375 plus EV-A375X1 (white). Error bars represent the standard deviation of three biological replicates. Statistical significance was determined using paired Student’s t-tests. *p < 0.05, **p < 0.01, ***p < 0.001. (c) Venn diagram showing unique and shared proteins identified by mass spectrometry in EVs isolated from both sensitive A375 and resistant A375X1 cells. (d) ALK consensus sequence in which the highlighted peptides are the ones detected by MS in the resistant EVs. (e) ALK western blot analysis of sensitive and resistant cells and corresponding EVs. Results are shown for one representative of three biological replicates

Fig. 6

Functional ALK^RES is transferred to sensitive cells via EVs. (a) Sensitive A375 melanoma cells were co-cultured with 10 μg of both EV-A375 and EV-A375X1. After 24 h, untreated A375 cells, resistant A375X1 cells and A375 co-cultured with both types of EVs were fixed and stained for ALK. Images were captured by fluorescence confocal microscopy. Representative images of two biological replicates. Scale bar, 20 μm. Blue: nucleus; green: ALK. (b) Sensitive A375 cells were treated with 1 μM of PLX4032. After 1 h, increasing concentrations of resistant EVs were added to the cells for additional 6 h. α-Tubulin was used as a loading control; representative blots of three biological replicates are shown. (c) Quantification of pERK levels, normalised to the untreated control. Error bars represent the standard deviation of three biological replicates. Statistical significance was determined using paired Student’s t-tests. *p < 0.05, **p < 0.01, ***p < 0.001