Targeted BRAF inhibition (BRAFi) and combined BRAF and MEK inhibition (BRAFi and MEKi) therapies have markedly improved the clinical outcomes of patients with metastatic melanoma. Unfortunately, the efficacy of these treatments is often countered by the acquisition of drug resistance1,2,3,4,5,6. Here we investigated the molecular mechanisms that underlie acquired resistance to BRAFi and to the combined therapy. Consistent with previous studies, we show that resistance to BRAFi is mediated by ERK pathway reactivation. Resistance to the combined therapy, however, is mediated by mechanisms independent of reactivation of ERK in many resistant cell lines and clinical samples. p21-activated kinases (PAKs) become activated in cells with acquired drug resistance and have a pivotal role in mediating resistance. Our screening, using a reverse-phase protein array, revealed distinct mechanisms by which PAKs mediate resistance to BRAFi and the combined therapy. In BRAFi-resistant cells, PAKs phosphorylate CRAF and MEK to reactivate ERK. In cells that are resistant to the combined therapy, PAKs regulate JNK and β-catenin phosphorylation and mTOR pathway activation, and inhibit apoptosis, thereby bypassing ERK. Together, our results provide insights into the molecular mechanisms underlying acquired drug resistance to current targeted therapies, and may help to direct novel drug development efforts to overcome acquired drug resistance.
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We thank Pfizer, Inc. for providing PF-3758309, and Plexxikon, Inc. for providing PLX4720 and PD0325901. This work is supported by NIH grants R01-GM085146, U54-CA193417 and CA174523 pilot grant to W.G., CA114046, CA25874 and CA174523 to X.X., the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation and NCI CA025874, CA114046 and CA174523 to M.H, and CA142928 to J.C.
The authors declare no competing financial interests.
Reviewer Information Nature thanks A. A. Qutub, C. Wells and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Related to Fig. 1. a, b, IHC staining of p-ERK(T202/Y204) in paired pre- and post-treatment tumour biopsy specimens acquired from patients who relapsed on BRAFi (a) or combined BRAFi and MEKi (b) treatment. Note that some of the strongly positive-stained cells are macrophages rather than tumour cells. Scale bars, 50 μm. The tissues were stained with Nova Red. c, Western blotting analysis of the levels of p-ERKT202/Y204 and p-PAK1(S199/S204)/PAK2(S192/S197) in WM3939 CRPDX tumour samples. Tumours from mice treated with vehicle control or with BRAFi and MEKi are shown. WM9-CR was used for comparison. d, Western blotting of PAK phosphorylation in matching parental, BR and CR cells. e, Western blotting using a polyclonal antibody that recognizes both CDC42 and RAC1 in matching parental, BR and CR cells. CDC42 and RAC1 could not be separated by SDS–PAGE owing to their similar molecular weights. f, h, j, Heat maps of expression levels of PAKs, RAC1 and CDC42 in paired pre- and post-treatment tumour biopsy specimens acquired from patients with metastatic melanoma who progressed on MAPK inhibitors. Data were analysed using the LIMMA package in R. The fold change of expression levels in a paired post-treatment tumour biopsy specimen over the pre-treatment tumour biopsy specimen is shown in the heat map. Colour scale, the log2-transformed expression of each gene was normalized to the mean value of all samples. g, i, k, Heat maps of the enrichment scores of two PAK signalling-related gene sets in paired pre- and post-treatment tumour biopsy specimens acquired from patients with metastatic melanoma who relapsed on MAPK inhibitors. The value for each entry is the difference in enrichment score of post-treatment over pre-treatment specimens. Gene expression microarray or RNA-sequencing data were downloaded from EGAD00001001306, GSE65184, GSE65185 and GSE61992.
Extended Data Figure 2 CR cells resistant to the combination of PLX4720 and PD0325901 exhibit cross-resistance to other combinations of BRAF and MEK inhibitors, and are sensitive to PAK inhibitor PF-3758309.
Related to Fig. 2. a, Paired parental and CR cells were treated with a combination of three different sets of BRAF and MEK inhibitors, separately, for four days and then fixed and stained with Giemsa. b, Quantification of cell survival (n = 3 biologically independent samples). c, d, Relative survival of matching parental, BR and CR cells treated with increasing concentrations of PF-3758309 (n = 3 or 4 biologically independent samples, as indicated). All IC50 values are listed in Supplementary Table 4. Two-sided Student’s t-tests were used for statistical analyses of the IC50 values. e–h, Seven BR (e) and six CR cell lines (f) were treated with DMSO, 1 μM or 3 μM PF-3758309 for 72 h, and then fixed and stained with Giemsa. The data are shown in g for BR cells and in h for CR cells (n = 3 biologically independent samples). The cell density was measured using ImageJ software (NIH). The values after background subtraction were normalized to cells treated with DMSO. Two-sided Student’s t-tests were used for statistical analyses (b–d, g, h). Data are plotted as mean ± s.e.m. Source data
Extended Data Figure 3 Inhibition of PAKs by PF-3758309 decreased the viability of drug-resistant melanoma cells.
Related to Fig. 2. a, b, Relative survival of BR and CR cells treated with increasing concentrations of the PF-3758309 (PF), PLX4720 (PLX) and PD0325901 (PD) for 48 h. Cell viability was analysed by MTT assay. The data were normalized to cells treated with DMSO (n = 4 biologically independent samples). c, FACS analysis of BR cells and CR cells treated with PF-3758309. All cells were labelled with propidium iodide and PSVue 643, and analysed using a BD LSRII. d, Quantification of cell apoptosis. The percentage of apoptosis cells after PF-3758309 treatment was compared to cells treated with DMSO (n = 3 or 4 biologically independent samples as indicated). e, g, Giemsa staining of BRPDX cell lines WM3936 and WM3903 that were treated with DMSO, or different concentrations of PF-3758309 for three days. The quantification of the staining is shown in g (n = 3 biologically independent samples). f, h, Anchorage-independent growth assay of WM3936 cells. A total of 2,000 cells were seeded in medium with soft agar in six-well plates. Scale bar, 200 μm. The number of colonies in each field is quantified in h (n = 6 biologically independent samples). i, WM3936 cells were treated with DMSO or different concentrations of PF-3758309 for three days. All cells were labelled with propidium iodide and PSVue 643, and then analysed using a BD LSRII. j, Quantification of cell apoptosis. The percentage of apoptosis cells after PF-3758309 treatment was compared to cells treated with DMSO (n = 5 biologically independent samples). Two-sided Student’s t-tests (a, b, d, g, h, j) were used for statistical analyses. Data are plotted as mean ± s.e.m. Source data
Related to Fig. 2. a, Immunofluorescence staining of Ki-67 (red) in indicated cells, which were treated with DMSO or 1 μM PF-3758309. The nuclei were stained with DAPI (blue). b, Quantification of cells with Ki-67 staining (n > 70 cells per assay, three independent experiments). c, Flow cytometric analysis (10,000 cells were analysed per assay). Cells were fixed, stained with propidium iodide, and then analysed by a FACscan flow cytometer and ModFit LT (Verity Software) d, Histograms of propidium iodide staining (n = 3 biologically independent samples). Two-sided Student’s t-tests (b, d) were used for statistical analyses. Data are plotted as mean ± s.e.m. Source data
Extended Data Figure 5 Inhibition of PAKs by siRNA, kinase-dead dominant-negative PAK1K299R mutant or PAK inhibitor IPA-3 decreased the viability of drug-resistant melanoma cells.
Related to Fig. 2. a, b, Relative survival of BR or CR cells transfected with PAK1K299R or PAK1PID, or siRNA against PAK1 and PAK2. Cells were then cultured with PLX4720 or PLX4720 and PD0325901 at different concentrations for 48 h. Cell viability was analysed by MTT assays (n = 4 biologically independent samples). Two-sided Student’s t-tests (for IC50 values) were used for statistical analysis. c, d, PAK1 and actin levels in cells were analysed by western blotting. e, BR and CR cells were treated with DMSO, 10 μM or 20 μM IPA-3 for 72 h, and then processed for Giemsa staining. f, Quantification of the staining in e (n = 3 biologically independent samples). g, RT–PCR analysis of the expression of PAK4 in indicated cells. h, Giemsa staining of indicated cells. i, Quantification of the staining in h (n = 3 biologically independent samples). Two-sided Student’s t-tests (a, b, f, i) were used for statistical analysis. Data are plotted as mean ± s.e.m. Source data
Extended Data Figure 6 Combined inhibition of MAPK and PAK pathways significantly inhibited BR and CR tumour proliferation in mice and improved survival.
Related to Fig. 2. a, b, Tumour growth curves. Mice were injected with 1205Lu-BR (n = 9 mice per group) or WM9-BR (n = 9 mice per group) cells (a), WM9-CR (n = 5 mice per group) or A2058-CR (control n = 8, other n = 9 mice per group) cells (b), and proceeded for MAPK or PAK inhibition for the indicated number of days. c, Survival curves of mice bearing 1205Lu-BR and WM9-CR xenografts (n = 5 mice per group). All groups were compared to the PLX or PLX and PD group; no multiple comparisons. Two-way ANOVA (a, b) or log-rank test (c) were used for statistical analyses. Individual tumour volume data points can be found in the Source Data. Data are plotted as mean ± s.e.m. For mouse survival, the function survdiff from the survival R package was used.
Extended Data Figure 7 RPPA and immunoblotting analyses of signalling proteins in melanoma cells treated with MAPK or PAK inhibitor.
Related to Figs 3, 4. a, BR cell lines and BRPDX cell lines were treated with DMSO or PF-3758309 for 48 h. Protein lysates from these cells were then analysed by RPPA. Data were analysed using the LIMMA package in R. The levels of identified proteins (that displayed significant changes in at least four BR cell lines after PF-3758309 treatment versus DMSO, P < 0.01) are shown in the heat map. Colour scale, log2-transformed expression for each protein was normalized to the mean value of all samples. b, 1205Lu-CR, UACC903-CR and WM164-CR cells were treated with DMSO or PF-3758309 for 48 h. Cell lysates were analysed by RPPA. Data were analysed using the LIMMA package in R. The levels of identified proteins (that displayed significant changes in at least two CR cell lines after PF-3758309 treatment versus DMSO, P < 0.01) are shown in the heat map. Colour scale, log2-transformed expression (red, high; blue, low) for each protein was normalized to the mean value of all samples. c, 1205Lu and UACC903 parental and CR cells were treated as indicated (Fig. 4b). Protein levels were analysed in three independent assays, and the staining was measured by ImageJ. (n = 2 for p-ELK1(S383) and p-BAD(S112), n = 3 for all other proteins). To minimize variation caused by different exposure time in each independent assay, the staining was normalized to the mean of all samples from the same group before statistical analyses. Two-sided Student’s t-tests were used for statistical analyses. Data are plotted as mean ± s.e.m. d, 1205Lu cells stably expressing PAK1107F/423E or vector control were treated with 1 μM PLX4720 and 100 nM PD0325901 for 48 h. The levels of MAPK pathway-related proteins, cell-cycle-related proteins and apoptosis-related proteins were analysed by western blotting. Source data
Related to Fig. 4. a, Giemsa staining of 1205Lu and UACC903 parental, BR and CR cells that were treated with DMSO, the ERK inhibitor SCH772984 (3 μM), the JNK inhibitor SP600125 (3 μM) or the S6K inhibitor PF-4708671 (3 μM) for three days. b, Quantification of cell survival (n = 3 biologically independent samples). Cell density was quantified with ImageJ. The values were normalized to those of parental cells treated with 1 μM of the respective inhibitor. c, Giemsa staining of indicated cells that were infected with luciferase shRNA, JNK1 shRNA, JNK1/2 shRNA and two different β-catenin shRNA. The quantification of the staining is shown in d (n = 3 biologically independent samples). e, RT–PCR analysis of the expression of JNK1 and β-catenin in the indicated cells. GAPDH was used as a loading control. f, Schematic diagrams showing the molecular mechanisms by which PAKs mediate acquired drug resistance of BRAFV600E melanoma cells to BRAFi (left) and BRAFi and MEKi (right). Two-sided Student’s t-tests (b, d) were used for statistical analyses. Data are plotted as mean ± s.e.m. Source data
This file contains figures 1, 3 and 4 and extended data figures 1, 5 and 7. (PDF 2338 kb)
This table contains patient information in PAK MS. (XLSX 10 kb)
This table contains p-ERK staining of BRAFi MEKi patients. (XLSX 10 kb)
This table contains patients with PAK activation from 3 data base. (XLSX 10 kb)
This table contains IC50 for MTT. (XLSX 14 kb)
This table contains antibody information. (XLSX 14 kb)
This table contains animal experiments’ raw data. (XLSX 49 kb)
This table contains assay repeat times. (XLSX 12 kb)
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Lu, H., Liu, S., Zhang, G. et al. PAK signalling drives acquired drug resistance to MAPK inhibitors in BRAF-mutant melanomas. Nature 550, 133–136 (2017). https://doi.org/10.1038/nature24040
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