Reversible and adaptive resistance to BRAF(V600E) inhibition in melanoma

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Abstract

Treatment of BRAF(V600E) mutant melanoma by small molecule drugs that target the BRAF or MEK kinases can be effective, but resistance develops invariably1,2. In contrast, colon cancers that harbour the same BRAF(V600E) mutation are intrinsically resistant to BRAF inhibitors, due to feedback activation of the epidermal growth factor receptor (EGFR)3,4. Here we show that 6 out of 16 melanoma tumours analysed acquired EGFR expression after the development of resistance to BRAF or MEK inhibitors. Using a chromatin-regulator-focused short hairpin RNA (shRNA) library, we find that suppression of sex determining region Y-box 10 (SOX10) in melanoma causes activation of TGF-β signalling, thus leading to upregulation of EGFR and platelet-derived growth factor receptor-β (PDGFRB), which confer resistance to BRAF and MEK inhibitors. Expression of EGFR in melanoma or treatment with TGF-β results in a slow-growth phenotype with cells displaying hallmarks of oncogene-induced senescence. However, EGFR expression or exposure to TGF-β becomes beneficial for proliferation in the presence of BRAF or MEK inhibitors. In a heterogeneous population of melanoma cells having varying levels of SOX10 suppression, cells with low SOX10 and consequently high EGFR expression are rapidly enriched in the presence of drug, but this is reversed when the drug treatment is discontinued. We find evidence for SOX10 loss and/or activation of TGF-β signalling in 4 of the 6 EGFR-positive drug-resistant melanoma patient samples. Our findings provide a rationale for why some BRAF or MEK inhibitor-resistant melanoma patients may regain sensitivity to these drugs after a ‘drug holiday’ and identify patients with EGFR-positive melanoma as a group that may benefit from re-treatment after a drug holiday.

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Figure 1: Acquired EGFR expression in BRAF(V600E) mutant melanoma after vemurafenib resistance.
Figure 2: FACS-assisted shRNA genetic screen identifies SOX10 as a determinant of vemurafenib resistance and EGFR expression.
Figure 3: Activation of TGF-β signalling leads to increased EGFR and PDGFRB expression.
Figure 4: Inverse relationship between SOX10 and receptor tyrosine kinase expression in melanoma.

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Gene Expression Omnibus

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RNA sequencing data are available at Gene Expression Omnibus with accession code GSE50535.

References

  1. 1

    Chapman, P. B. et al. Improved survival with vemurafenib in melanoma with BRAF V600E mutation. N. Engl. J. Med. 364, 2507–2516 (2011)

    CAS  Article  PubMed  Google Scholar 

  2. 2

    Flaherty, K. T. et al. Improved survival with MEK inhibition in BRAF-mutated melanoma. N. Engl. J. Med. 367, 107–114 (2012)

    CAS  Article  Google Scholar 

  3. 3

    Prahallad, A. et al. Unresponsiveness of colon cancer to BRAF(V600E) inhibition through feedback activation of EGFR. Nature 483, 100–103 (2012)

    ADS  CAS  Article  Google Scholar 

  4. 4

    Corcoran, R. B. et al. EGFR-mediated re-activation of MAPK signaling contributes to insensitivity of BRAF mutant colorectal cancers to RAF inhibition with vemurafenib. Cancer Discov. 2, 227–235 (2012)

    CAS  Article  PubMed  Google Scholar 

  5. 5

    Davies, H. et al. Mutations of the BRAF gene in human cancer. Nature 417, 949–954 (2002)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Flaherty, K. T., Hodi, F. S. & Fisher, D. E. From genes to drugs: targeted strategies for melanoma. Nature Rev. Cancer 12, 349–361 (2012)

    CAS  Article  Google Scholar 

  7. 7

    Poulikakos, P. I. et al. RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAF(V600E). Nature 480, 387–390 (2011)

    ADS  CAS  Article  PubMed  Google Scholar 

  8. 8

    Johannessen, C. M. et al. COT drives resistance to RAF inhibition through MAP kinase pathway reactivation. Nature 468, 968–972 (2010)

    ADS  CAS  Article  PubMed  Google Scholar 

  9. 9

    Wagle, N. et al. Dissecting therapeutic resistance to RAF inhibition in melanoma by tumour genomic profiling. J. Clin. Oncol. 29, 3085–3096 (2011)

    CAS  Article  PubMed  Google Scholar 

  10. 10

    Shi, H. et al. Melanoma whole-exome sequencing identifies (V600E)B-RAF amplification-mediated acquired B-RAF inhibitor resistance. Nature Commun. 3, 724 (2012)

    ADS  Article  Google Scholar 

  11. 11

    Nazarian, R. et al. Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation. Nature 468, 973–977 (2010)

    ADS  CAS  Article  PubMed  Google Scholar 

  12. 12

    Girotti, M. R. et al. Inhibiting EGF receptor or SRC family kinase signaling overcomes BRAF inhibitor resistance in melanoma. Cancer Discov 3, 158–167 (2013)

    CAS  Article  Google Scholar 

  13. 13

    Villanueva, J. et al. Acquired resistance to BRAF inhibitors mediated by a RAF kinase switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K. Cancer Cell 18, 683–695 (2010)

    CAS  Article  PubMed  Google Scholar 

  14. 14

    Real, F. X. et al. Expression of epidermal growth factor receptor in human cultured cells and tissues: relationship to cell lineage and stage of differentiation. Cancer Res. 46, 4726–4731 (1986)

    CAS  Google Scholar 

  15. 15

    Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D. & Lowe, S. W. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88, 593–602 (1997)

    CAS  Article  Google Scholar 

  16. 16

    Michaloglou, C. et al. BRAFE600-associated senescence-like cell cycle arrest of human naevi. Nature 436, 720–724 (2005)

    ADS  CAS  Article  Google Scholar 

  17. 17

    Brummelkamp, T. R. & Bernards, R. New tools for functional mammalian cancer genetics. Nature Rev. Cancer 3, 781–789 (2003)

    CAS  Article  Google Scholar 

  18. 18

    Huang, S. et al. MED12 controls the response to multiple cancer drugs through regulation of TGF-β receptor signaling. Cell 151, 937–950 (2012)

    CAS  Article  PubMed  Google Scholar 

  19. 19

    Johnson, A. C. et al. Activator protein-1 mediates induced but not basal epidermal growth factor receptor gene expression. Mol. Med. 6, 17–27 (2000)

    CAS  Article  PubMed  Google Scholar 

  20. 20

    Zenz, R. et al. c-Jun regulates eyelid closure and skin tumor development through EGFR signaling. Dev. Cell 4, 879–889 (2003)

    CAS  Article  Google Scholar 

  21. 21

    Steller, E. J. et al. PDGFRB promotes liver metastasis formation of mesenchymal-like colorectal tumor cells. Neoplasia 15, 204–217 (2013)

    CAS  Article  PubMed  Google Scholar 

  22. 22

    Zhang, Y., Feng, X.-H. & Derynck, R. Smad3 and Smad4 cooperate with c-Jun/c-Fos to mediate TGF-β-induced transcription. Nature 394, 909–913 (1998)

    ADS  CAS  Article  Google Scholar 

  23. 23

    Mialon, A. et al. DNA topoisomerase I is a cofactor for c-Jun in the regulation of epidermal growth factor receptor expression and cancer cell proliferation. Mol. Cell. Biol. 25, 5040–5051 (2005)

    CAS  Article  PubMed  Google Scholar 

  24. 24

    Bondurand, N. et al. Interaction among SOX10, PAX3 and MITF, three genes altered in Waardenburg syndrome. Hum. Mol. Genet. 9, 1907–1917 (2000)

    CAS  Article  Google Scholar 

  25. 25

    Garnett, M. J. et al. Systematic identification of genomic markers of drug sensitivity in cancer cells. Nature 483, 570–575 (2012)

    ADS  CAS  Article  PubMed  Google Scholar 

  26. 26

    Seghers, A. C., Wilgenhof, S., Lebbe, C. & Neyns, B. Successful rechallenge in two patients with BRAF-V600-mutant melanoma who experienced previous progression during treatment with a selective BRAF inhibitor. Melanoma Res. 22, 466–472 (2012)

    Article  Google Scholar 

  27. 27

    Yang, X. et al. A public genome-scale lentiviral expression library of human ORFs. Nature Methods 8, 659–661 (2011)

    CAS  Article  PubMed  Google Scholar 

  28. 28

    Cronin, J. C. et al. Frequent mutations in the MITF pathway in melanoma. Pigment Cell Melanoma Res 22, 435–444 (2009)

    CAS  Article  PubMed  Google Scholar 

  29. 29

    Huang, S. et al. ZNF423 is critically required for retinoic acid-induced differentiation and is a marker of neuroblastoma outcome. Cancer Cell 15, 328–340 (2009)

    CAS  Article  PubMed  Google Scholar 

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Acknowledgements

We thank the NKI Core Facilities for Genomics and Molecular Pathology & Biobanking for tumour tissue and support in DNA sequencing. We thank S. Roy for collecting clinical data and N. Kamsu Kom for tissue preparation. This work was supported by grants from the European Research Council (ERC), the Dutch Cancer Society (KWF), the EU COLTHERES project and grants by the Netherlands Organization for Scientific Research (NWO) to Cancer Genomics Netherlands (CGC.NL). Additional support was provided by Fondazione Piemontese per la Ricerca sul Cancro—ONLUS grant ‘Farmacogenomica—5 per mille 2009 MIUR’ (F.D.N.); AIRC MFAG 11349 (F.D.N.); AIRC IG grant n. 12812 (A.B.); and Canadian Institutes of Health Research (CIHR) grant MOP-130540 (S.Hu).

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Contributions

R.B., A.B., F.D.N., L.F.A.W., C.R., R.L.B. and A.M.M.E. supervised all research. R.B. and C.S. wrote the manuscript. C.S., L.W., S.Hu., G.J.J.E.H., A.P., D.Z., S.Ho., P.K.B., C.L., C.M., S.V., J.W., W.G., I.H. and A.S. designed and performed experiments and J.H., C.B., C.R., S.M.W., S.V. and A.M.M.E. provided clinical samples and gave advice.

Corresponding author

Correspondence to Rene Bernards.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Ectopic expression of oncogenic version of EGFR effectors induces senescence at different levels.

Oncogenic BRAF(V600E), MEK (MED-DD), PIK3CA(H1047R), or AKT (Myr-AKT) were introduced to A375 cells by retroviral transduction. pBabe empty vector served as a control vector (Ctrl). Senescence was detected by staining of β-galactosidase activity. All experiments shown were performed independently at least three times.

Extended Data Figure 2 Effects of SOX10 suppression in melanoma.

a, Suppression of SOX10 strongly induces EGFR expression. Multiple independent shRNA vectors (5 vectors per gene) targeting the top 10 gene candidates were individually introduced to A375 cells by lentiviral transduction. The level of EGFR induction was determined by qRT–PCR analysis of the relative mRNA level of EGFR. pLKO.1 empty vector served as a control vector (Ctrl). b, Knockdown efficiency of the shRNA vectors targeting the top 10 gene candidates from the genetic screen. Multiple independent shRNA vectors targeting the top 10 candidate genes were individually introduced to A375 cells by lentiviral transduction. The knockdown efficiency of the shRNA vectors was determined by qRT–PCR analysis of the mRNA levels of the corresponding genes. Means of duplicate measurements are shown. c, SOX10 suppression leads to EGFR upregulation in a second BRAF(V600E) mutant melanoma cell line SK-MEL-28. Error bars represent s.d. of measurement replicates (n = 3). d, Two independent shRNAs targeting SOX10 confer vemurafenib resistance. A375 cells expressing shRNAs against SOX10 were seeded at the same density in 96-well plates and treated with vemurafenib at the indicated concentrations for 6 days. Cell viability was determined by CellTiter-Blue assay according to the manufacturer’s instruction. Relative survival is presented as the ratio of cell viability in the presence of vemurafenib to that in the absence of drug treatment. Error bars represent the s.d. of triplicate independent experiments. e, SOX10 suppression is a disadvantage for melanoma cell proliferation. shRNAs targeting SOX10 were introduced into A375 cells by lentiviral transduction. pLKO.1 empty vector served as a control vector (Ctrl). After puromycin selection, cells were seeded in 384-well and cell confluence was measured by IncuCyte imaging system. Error bars represent s.d. of triplicate independent experiments. f, SOX10 suppression induces senescence. Senescence was detected by staining of β-galactosidase activity. g, Western blot analysis of RB protein, CDK inhibitors CDKN1A (p21cip1) and CDKN1B (p27kip1) in SOX10 knockdown A375 cells. HSP90 served as a loading control. h, Vemurafenib treatment selects for cells that have higher level of EGFR and lower level of SOX10. A375 cells expressing shRNAs targeting SOX10 as described above were cultured in the absence or presence of 1 μM vemurafenib for 10 days before the sample collection for qRT–PCR analysis. Error bars represent s.d. of measurement replicates (n = 3). All experiments shown, except panels a and b, were performed independently at least three times.

Extended Data Figure 3 SOX10 knockdown and TGF-β activation induce multiple RTKs.

a, EGFR inhibition (gefinitib) is not sufficient to restore vemurafenib sensitivity of SOX10 knockdown cells. Targeting PI3K, a common downstream effector of RTKs, with a selective inhibitor (GDC0941) sensitizes SOX10 knockdown cells to vemurafenib. shRNAs targeting SOX10 were introduced into A375 cells by lentiviral transduction. pLKO.1 empty vector served as a control vector (Ctrl). Cells were seeded in 6-well plates at the same density in the presence or absence of drug(s) at the indicated concentration. Cells were cultured for 2 weeks in the absence of vemurafenib or 4 weeks in the presence of vemurafenib before fixing and staining. Figure 2e is shown again as a reference. b, Increased RTKs activation in SOX10 knockdown cells by long-term vemurafenib treatment. A375 cells infected by shSOX10-1 vector or the PLKO.1 empty vector (Ctrl) were cultured in the absence or presence of 1 μM vemurafenib for the indicated number of days and processed with Human Phospho-Receptor Tyrosine Kinase Array Kit (R&D) according to the manufacturer’s instructions. c, SOX10 knockdown upregulates both EGFR and PDGFRβ. Quantification of protein and mRNA were accomplished by western blot and qRT–PCR analysis. Error bars represent s.d. of measurement replicates (n = 3). d, Increased RTKs activation in A375 cells by long-term treatment with recombinant TGF-β (200 pg ml−1) and vemurafenib (1 μM). A375 cells were cultured in the presence of vemurafenib (1 μM), recombinant TGF-β (200 pg ml−1) or their combination for indicated number of days and processed with Human Phospho-Receptor Tyrosine Kinase Array Kit (R&D) according to the manufacturer’s instructions. All experiments shown except RTK array analysis were performed independently at least twice.

Extended Data Figure 4 SOX10 loss activates TGF-β signalling and induces senescence in WM266-4 cells.

a, SOX10 loss confers vemurafenib resistance in BRAF(V600D) melanoma cell line WM266-4. Cells expressing empty vector PLKO.1 (Ctrl) or shRNAs targeting SOX10 transduced by lentivirus were treated with increasing concentrations of vemurafenib for 6 days. Cell viability was determined by CellTiter-Blue assay according to the manufacturer’s instructions. Relative survival is represented as the ratio of cell viability in the presence of vemurafenib to that in the absence of drug treatment. Error bars represent s.d. of triplicate independent experiments. b, SOX10 downregulation leads to growth deficit in WM266-4 cells. Cells expressing the control vector pLKO.1 (Ctrl) or shRNAs against SOX10 were seeded at the same density in 96-well plates and cultured for 6 days. Cell viability was determined by CellTiter-Blue assay. Error bars represent s.d. of triplicate independent experiments. c, SOX10 suppression results in EGFR and PDGFRB upregulation in WM266-4 cells. Error bars represent s.d. of measurement replicates (n = 3). d, SOX10 loss upregulates TGF-β receptor and its bona fide target genes. Relative mRNA level of EGFR, PDGFRB, SOX10, ANGPTL4, TAGLN, CYR61, CTGF, TGFBR2 and JUN were determined by qRT–PCR analysis. pLKO.1 empty vector served as a control vector (Ctrl). Error bars represent s.d. of measurement replicates (n = 3). e, SOX10 suppression induces senescence in WM266-4 cells. Senescence was detected by staining of β-galactosidase activity. f, Western blot analysis of RB protein, p-RB (S780), and CDK inhibitor CDKN1B (p27kip1) in SOX10 knockdown cells. HSP90 served as a loading control. g, Vemurafenib treatment compromises oncogene induced senescence in SOX10 knockdown cells. WM266-4 cells expressing pLKO.1 (Ctrl) or shSOX10-1 were seeded at the same density in 6-well plates and cultured in the absence or presence of vemurafenib at indicated concentration for 72 h before the sample collection for western blot analysis. All experiments shown were performed independently at least three times.

Extended Data Figure 5 SOX10 loss activates TGF-β signalling and induces senescence in COLO679 cells.

a, SOX10 loss confers vemurafenib resistance in BRAF(V600E) melanoma cell line COLO679. Cells expressing empty vector pLKO.1 (Ctrl.) or shRNAs targeting SOX10 transduced by lentivirus were treated with increasing concentrations of vemurafenib for 6 days. Cell viability was determined using CellTiter-Blue according to the instruction of manufacturer. Relative survival is represented as the ratio of cell viability in the presence of vemurafenib to that in the absence of drug treatment. Error bars represent s.d. of triplicate independent experiments. b, SOX10 downregulation leads to growth deficit in COLO679 cells. Cells expressing the control vector pLKO.1 (Ctrl) or shRNAs targeting SOX10 were seeded at the same density in 96-well plates and cultured for 6 days. Cell viability was determined using CellTiter-Blue assay. Error bars represent s.d. of triplicate independent experiments. c, SOX10 suppression results in EGFR and PDGFRB upregulation in COLO679 cells. Error bars represent s.d. of measurement replicates (n = 3). d, SOX10 loss upregulates TGF-β receptor and its bona fide target genes in COLO679 cells. Relative mRNA level of EGFR, PDGFRB, SOX10, ANGPTL4, TAGLN, CYR61, CTGF, TGFBR2 and JUN were determined by qRT–PCR analysis. pLKO.1 empty vector served as a control vector (Ctrl). Error bars represent s.d. of measurement replicates (n = 3). e, SOX10 suppression induces senescence in COLO679 cells. Senescence was detected by staining of β-galactosidase activity. f, Western blot analysis of RB protein, p-RB (S780) and CDK inhibitor CDKN1B (p27kip1) in SOX10 knockdown cells. HSP90 served as a loading control. All experiments shown were performed independently at least three times.

Extended Data Figure 6 EGFR and SOX10 expression are inversely correlated in melanoma.

a, A375 cells infected by two independent non-overlapping shSOX10 vectors or the pLKO.1 empty vector (Ctrl) were cultured in the absence or presence of 1 μM vemurafenib for the indicated number of days. The last two samples (labelled in blue) were first treated with 1 μM vemurafenib for 10 days and subsequently cultured in the absence of vemurafenib for the indicated number of days. Means of duplicate measurements are shown. b, Inverse correlation between SOX10 and PDGFRB in panel of human BRAF mutant melanoma cell lines. Relative gene expression levels of SOX10 and PDGFRB were acquired from Cancer Cell Line Encyclopedia (CCLE). R stands for Pearson product-moment correlation coefficient. c, d, Ectopic expression of SOX10 suppresses TGF-β signalling and downregulates EGFR and PDGFRB in LOXIMVI cell line. SOX10 was introduced to LOXIMVI cells by lentiviral transduction (SOX10, pLX301-SOX10). pLX301-GFP served as a control vector (Ctrl). Protein levels were determined by western blot analysis and mRNA levels were determined by qRT–PCR analysis. Error bars represent s.d. of measurement replicates (n = 3). e, Ectopic expression of SOX10 sensitizes LOXIMVI cell to vemurafenib. Cells expressing GFP or SOX10 transduced by lentivirus were treated with increasing concentrations of vemurafenib for 6 days. Cell viability was determined using CellTiter-Blue assay. Relative survival is represented as the ratio of cell viability in the presence of vemurafenib to that in the absence of drug treatment. Error bars represent s.d. of triplicate independent experiments. f, SOX10, EGFR and PDGFRB expression levels in tumour biopsies from patient 3. g, EGFR expression levels in patient tumour samples (patient 2, 3 and 5), represented as percentage of EGFR transcript reads of the total number of transcript reads obtained through RNA-seq analysis. h, Gene expression level of TGF-β receptors and target genes in tumour biopsies from patient 3. fh, Total RNA was isolated from FFPE specimens derived from tumour biopsies of patient as indicated both before and after development of drug resistance (Fig. 1a, b). After reverse transcription, gene expression levels were determined by transcriptome sequencing. All experiments shown except the ones that involve clinical samples were performed independently at least twice.

Extended Data Figure 7 Role of BRAF and MITF in SOX10-induced drug resistance.

a, PCR analysis of a BRAF splicing variant in cDNA from patient 5. PCR primers flanking the junction of exon 3 and exon 9 was used to detect the 61 kDa BRAF variant identified by ref. 7. cDNA derived from C4 clone of SKMEL-239 cells served as a positive control. b, Differential gene expression of BRAF and neural cell markers in patient biopsies. Total RNA was isolated from FFPE specimens derived from tumour biopsies of patient 5 before and after development of drug resistance (Fig. 1b). After reverse transcription, gene expression levels were determined by transcriptome sequencing. c, SOX10 suppression leads to MITF downregulation. The mRNA levels of MITF and SOX10 were determined by qRT–PCR analysis. pLKO.1 empty vector served as a control vector (Ctrl). Error bars represent s.d. of measurement replicates (n = 3). d, Suppression of MITF does not induce EGFR or PDGFRB. shRNAs targeting MITF were introduced to A375 cells by lentiviral transduction. Relative mRNA level of SOX10, MITF, EGFR, PDGFRB and DCT were determined by qRT–PCR analysis. Error bars represent s.d. of measurement replicates (n = 3). e, MITF knockdown does not affect vemurafenib sensitivity. shRNAs targeting MITF were introduced to A375 cells by lentiviral transduction. Cells were seeded at the same density in 6-well plates and cultured in the absence or presence of vemurafenib (for 3 weeks) at the indicated concentrations. The cells were fixed, stained and photographed. All experiments shown except the ones that involve clinical samples were performed independently at least twice.

Supplementary information

Supplementary Table 1

This table contains patient information. (XLS 11 kb)

Supplementary Table 2

A list of genes in chromatin library. (XLS 56 kb)

Supplementary Table 3

Top hits from genetic screen. (XLS 38 kb)

Supplementary Table 4

RNAseq data from shSOX10 cells. (XLS 1680 kb)

Supplementary Table 5

Gene Set Enrichment Analysis of SOX10 regulated genes. (XLS 12 kb)

Supplementary Table 6

shRNA IDs and sequences. (XLS 59 kb)

Supplementary Table 7

List of QRT-PCR primers used. (XLS 41 kb)

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Sun, C., Wang, L., Huang, S. et al. Reversible and adaptive resistance to BRAF(V600E) inhibition in melanoma. Nature 508, 118–122 (2014). https://doi.org/10.1038/nature13121

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