Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Melanomas acquire resistance to B-RAF(V600E) inhibition by RTK or N-RAS upregulation


Activating B-RAF(V600E) (also known as BRAF) kinase mutations occur in 7% of human malignancies and 60% of melanomas1. Early clinical experience with a novel class I RAF-selective inhibitor, PLX4032, demonstrated an unprecedented 80% anti-tumour response rate among patients with B-RAF(V600E)-positive melanomas, but acquired drug resistance frequently develops after initial responses2. Hypotheses for mechanisms of acquired resistance to B-RAF inhibition include secondary mutations in B-RAF(V600E), MAPK reactivation, and activation of alternative survival pathways3,4,5. Here we show that acquired resistance to PLX4032 develops by mutually exclusive PDGFRβ (also known as PDGFRB) upregulation or N-RAS (also known as NRAS) mutations but not through secondary mutations in B-RAF(V600E). We used PLX4032-resistant sub-lines artificially derived from B-RAF(V600E)-positive melanoma cell lines and validated key findings in PLX4032-resistant tumours and tumour-matched, short-term cultures from clinical trial patients. Induction of PDGFRβ RNA, protein and tyrosine phosphorylation emerged as a dominant feature of acquired PLX4032 resistance in a subset of melanoma sub-lines, patient-derived biopsies and short-term cultures. PDGFRβ-upregulated tumour cells have low activated RAS levels and, when treated with PLX4032, do not reactivate the MAPK pathway significantly. In another subset, high levels of activated N-RAS resulting from mutations lead to significant MAPK pathway reactivation upon PLX4032 treatment. Knockdown of PDGFRβ or N-RAS reduced growth of the respective PLX4032-resistant subsets. Overexpression of PDGFRβ or N-RAS(Q61K) conferred PLX4032 resistance to PLX4032-sensitive parental cell lines. Importantly, MAPK reactivation predicts MEK inhibitor sensitivity. Thus, melanomas escape B-RAF(V600E) targeting not through secondary B-RAF(V600E) mutations but via receptor tyrosine kinase (RTK)-mediated activation of alternative survival pathway(s) or activated RAS-mediated reactivation of the MAPK pathway, suggesting additional therapeutic strategies.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: In vitro models of PLX4032 acquired resistance display differential MAPK reactivation.
Figure 2: PDGFRβ upregulation is strongly correlated with PLX4032 acquired resistance.
Figure 3: N-RAS upregulation correlates with a distinct subset of PLX4032 acquired resistance.
Figure 4: PDGFRβ- and N-RAS-mediated growth and survival pathways differentially predict MEK inhibitor sensitivity.

Accession codes

Primary accessions

Gene Expression Omnibus

Data deposits

Gene expression and copy number data are deposited at Gene Expression Omnibus under accession numbers GSE24862 and GSE24890, respectively.


  1. 1

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

    ADS  CAS  Article  Google Scholar 

  2. 2

    Flaherty, K. T. et al. Inhibition of mutated, activated BRAF in metastatic melanoma. N. Engl. J. Med. 363, 809–819 (2010)

    CAS  Article  Google Scholar 

  3. 3

    Jänne, P. A., Gray, N. & Settleman, J. Factors underlying sensitivity of cancers to small-molecule kinase inhibitors. Nature Rev. Drug Discov. 8, 709–723 (2009)

    Article  Google Scholar 

  4. 4

    Montagut, C. et al. Elevated CRAF as a potential mechanism of acquired resistance to BRAF inhibition in melanoma. Cancer Res. 68, 4853–4861 (2008)

    CAS  Article  Google Scholar 

  5. 5

    Poulikakos, P. I., Zhang, C., Bollag, G., Shokat, K. M. & Rosen, N. RAF inhibitors transactivate RAF dimers and ERK signalling in cells with wild-type BRAF. Nature 464, 427–430 (2010)

    ADS  CAS  Article  Google Scholar 

  6. 6

    Søndergaard, J. N. et al. Differential sensitivity of melanoma cell lines with BRAFV600E mutation to the specific Raf inhibitor PLX4032. J. Transl. Med. 8, 39–50 (2010)

    Article  Google Scholar 

  7. 7

    Halaban, R. et al. PLX4032, a selective BRAFV600E kinase inhibitor, activates the ERK pathway and enhances cell migration and proliferation of BRAFWT melanoma cells. Pigment Cell Melanoma Res. 23, 190–200 (2010)

    CAS  Article  Google Scholar 

  8. 8

    Hatzivassiliou, G. et al. RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth. Nature 464, 431–435 (2010)

    ADS  CAS  Article  Google Scholar 

  9. 9

    Heidorn, S. J. et al. Kinase-dead BRAF and oncogenic RAS cooperate to drive tumor progression through CRAF. Cell 140, 209–221 (2010)

    CAS  Article  Google Scholar 

  10. 10

    Bollag, G. et al. Clinical efficacy of a RAF inhibitor needs broad target blockade in BRAF-mutant melanoma. Nature 467, 596–599 (2010)

    ADS  CAS  Article  Google Scholar 

  11. 11

    Tsai, J. et al. Discovery of a selective inhibitor of oncogenic B-Raf kinase with potent antimelanoma activity. Proc. Natl Acad. Sci. USA 105, 3041–3046 (2008)

    ADS  CAS  Article  Google Scholar 

  12. 12

    Whittaker, S. et al. Gatekeeper mutations mediate resistance to BRAF-targeted therapies. Sci. Transl. Med. 2, 35ra41 (2010)

    Article  Google Scholar 

  13. 13

    Pratilas, C. A. et al. V600EBRAF is associated with disabled feedback inhibition of RAF–MEK signaling and elevated transcriptional output of the pathway. Proc. Natl Acad. Sci. USA 106, 4519–4524 (2009)

    ADS  CAS  Article  Google Scholar 

  14. 14

    Packer, L. M., East, P., Reis-Filho, J. S. & Marais, R. Identification of direct transcriptional targets of BRAF/MEK signalling in melanoma. Pigment Cell Melanoma Res. 22, 785–798 (2009)

    CAS  Article  Google Scholar 

  15. 15

    Wu, E. et al. Comprehensive dissection of PDGF-PDGFR signaling pathways in PDGFR genetically defined cells. PLoS ONE 3, e3794 (2008)

    ADS  Article  Google Scholar 

  16. 16

    Smith, G. et al. Activating K-Ras mutations without ‘hotspot’ codons in sporadic colorectal tumours - implications for personalised cancer medicine. Br. J. Cancer 102, 693–703 (2010)

    CAS  Article  Google Scholar 

  17. 17

    Emery, C. M. et al. MEK1 mutations confer resistance to MEK and B-RAF inhibition. Proc. Natl Acad. Sci. USA 106, 20411–20416 (2009)

    ADS  CAS  Article  Google Scholar 

  18. 18

    Dumaz, N. et al. In melanoma, RAS mutations are accompanied by switching signaling from BRAF to CRAF and disrupted cyclic AMP signaling. Cancer Res. 66, 9483–9491 (2006)

    CAS  Article  Google Scholar 

Download references


We are grateful to G. Bollag and P. Lin (Plexxikon) for providing PLX4032, J. S. Economou for biopsies, B. Comin-Anduix for FACS assistance, S. Mok for assistance with virus production, C. Ng for tissue acquisition and culture establishment, R. Huang for patient tissue processing, N. Doan for immunohistochemistry, P. Mischel for discussion, B. Chmielowski for coordinated patient care, T.L. Toy for technical help with library generation for deep sequencing, and B. Harry for help with analysis of deep sequence data. R.S.L. acknowledges funding from the following: Dermatology Foundation, Burroughs Wellcome Fund, STOP CANCER Foundation, Margaret E. Early Medical Trust, Ian Copeland Memorial Melanoma Fund, V Foundation for Cancer Research, Melanoma Research Foundation, American Skin Association, Caltech-UCLA Joint Center for Translational Medicine, Wesley Coyle Memorial Fund, and Melanoma Research Alliance. R.N. is supported by a post-doctoral fellowship from the T32 Tumor Immunology Training Grant (S. Dubinett). A.R. is supported by the California Institute for Regenerative Medicine (CIRM), the Jonsson Cancer Center Foundation (JCCF), and Caltech-UCLA Joint Center for Translational Medicine. Array and sequence work were performed within the Jonsson Comprehensive Cancer Center Gene Expression Shared Resource. Patient-informed consent was obtained for the research performed in this study. We would like to thank all the patients that participated in this study.

Author information




R.N., H.S., Q.W., X.K., H.L., Z.C. designed and performed experiments and analysed data. M.L. helped analyse data. R.C.K., N.A., H.S., T.C., G.M., J.A.S., and A.R. provided reagents. S.F.N. helped design experiments and interpreted data. A.R. and R.S.L. designed research aims. H.L., S.F.N., G.M., J.A.S. and A.R. helped write the paper. R.S.L. designed and performed experiments, analysed data, provided reagents, and wrote the paper.

Corresponding author

Correspondence to Roger S. Lo.

Ethics declarations

Competing interests

A.R. and J.A.S. report receiving honorarium from Roche Pharmaceuticals. All other authors declare no competing financial interests.

Supplementary information

Supplementary Figures

The file contains Supplementary Figures 1-23 with legends. (PDF 19296 kb)

Supplementary Tables

The file contains Supplementary Tables 1-5. (PDF 468 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing