Skip to main content

Thank you for visiting nature.com. 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.

  • Article
  • Published:

RAS-MAPK dependence underlies a rational polytherapy strategy in EML4-ALK–positive lung cancer

Nature Medicine volume 21, pages 1038–1047 (2015)Cite this article

Subjects

Abstract

One strategy for combating cancer-drug resistance is to deploy rational polytherapy up front that suppresses the survival and emergence of resistant tumor cells. Here we demonstrate in models of lung adenocarcinoma harboring the oncogenic fusion of ALK and EML4 that the GTPase RAS–mitogen-activated protein kinase (MAPK) pathway, but not other known ALK effectors, is required for tumor-cell survival. EML4-ALK activated RAS-MAPK signaling by engaging all three major RAS isoforms through the HELP domain of EML4. Reactivation of the MAPK pathway via either a gain in the number of copies of the gene encoding wild-type K-RAS (KRASWT) or decreased expression of the MAPK phosphatase DUSP6 promoted resistance to ALK inhibitors in vitro, and each was associated with resistance to ALK inhibitors in individuals with EML4-ALK–positive lung adenocarcinoma. Upfront inhibition of both ALK and the kinase MEK enhanced both the magnitude and duration of the initial response in preclinical models of EML4-ALK lung adenocarcinoma. Our findings identify RAS-MAPK dependence as a hallmark of EML4-ALK lung adenocarcinoma and provide a rationale for the upfront inhibition of both ALK and MEK to forestall resistance and improve patient outcomes.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: EML4-ALK (E13;A20, variant 1) lung adenocarcinoma cells are specifically dependent upon MAPK signaling.
Figure 2: Cells expressing EML4-ALK (E13:A20, variant 1) activate H-, N- and K-RAS to drive MAPK signaling, via the HELP domain of EML4.
Figure 3: Enhanced therapeutic effect of upfront co-treatment with an ALK inhibitor and a sub-maximal MEK inhibitor.
Figure 4: Reactivation of MAPK signaling by KRASWT copy-number gain promotes ALK-inhibitor resistance in EML4-ALK lung adenocarcinoma.
Figure 5: Reactivation of MAPK signaling by suppression of DUSP6 promotes ALK-inhibitor resistance in EML4-ALK lung adenocarcinoma.
Figure 6: Combined inhibition of ALK and MEK enhances response and eliminates resistance in EML4-ALK lung adenocarcinoma models, in vitro and in vivo.

Similar content being viewed by others

References

  1. Solomon, B.J. et al. First-line crizotinib versus chemotherapy in ALK-positive lung cancer. N. Engl. J. Med. 371, 2167–2177 (2014).

    Article  CAS  PubMed  Google Scholar 

  2. Shaw, A.T. et al. Ceritinib in ALK-rearranged non-small-cell lung cancer. N. Engl. J. Med. 370, 1189–1197 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Camidge, D.R. & Doebele, R.C. Treating ALK-positive lung cancer—early successes and future challenges. Nat. Rev. Clin. Oncol. 9, 268–277 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Shaw, A.T. et al. Crizotinib versus chemotherapy in advanced ALK-positive lung cancer. N. Engl. J. Med. 368, 2385–2394 (2013).

    Article  CAS  PubMed  Google Scholar 

  5. Robert, C. et al. Improved overall survival in melanoma with combined dabrafenib and trametinib. N. Engl. J. Med. 372, 30–39 (2015).

    Article  CAS  PubMed  Google Scholar 

  6. Chapman, P.B., Solit, D.B. & Rosen, N. Combination of RAF and MEK inhibition for the treatment of BRAF-mutated melanoma: feedback is not encouraged. Cancer Cell 26, 603–604 (2014).

    Article  CAS  PubMed  Google Scholar 

  7. Johnson, D.B. et al. Combined BRAF (Dabrafenib) and MEK inhibition (Trametinib) in patients with BRAFV600E-mutant melanoma experiencing progression with single-agent BRAF inhibitor. J. Clin. Oncol. 32, 3697–3704 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Flaherty, K.T. et al. Combined BRAF and MEK inhibition in melanoma with BRAF V600 mutations. N. Engl. J. Med. 367, 1694–1703 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Long, G.V. et al. Combined BRAF and MEK inhibition versus BRAF inhibition alone in melanoma. N. Engl. J. Med. 371, 1877–1888 (2014).

    Article  CAS  PubMed  Google Scholar 

  10. Lemmon, M.A. & Schlessinger, J. Cell signaling by receptor tyrosine kinases. Cell 141, 1117–1134 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. The Cancer Genome Atlas Research Network. Comprehensive molecular profiling of lung adenocarcinoma. Nature 511, 543–550 (2014).

  12. Lovly, C.M. et al. Rationale for co-targeting IGF-1R and ALK in ALK fusion- positive lung cancer. Nat. Med. 20, 1027–1034 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Alessandrini, A. et al. Mek1 phosphorylation site mutants activate Raf-1 in NIH 3T3 cells. J. Biol. Chem. 271, 31612–31618 (1996).

    Article  CAS  PubMed  Google Scholar 

  14. Takezawa, K. et al. Role of ERK-BIM and STAT3-survivin signaling pathways in ALK inhibitor-induced apoptosis in EML4-ALK-positive lung cancer. Clin. Cancer Res. 17, 2140–2148 (2011).

    Article  CAS  PubMed  Google Scholar 

  15. Hainsworth, J.D. et al. A phase II, open-label, randomized study to assess the efficacy and safety of AZD6244 (ARRY-142886) versus pemetrexed in patients with non-small cell lung cancer who have failed one or two prior chemotherapeutic regimens. J. Thorac. Oncol. 5, 1630–1636 (2010).

    Article  PubMed  Google Scholar 

  16. Bivona, T.G. et al. Phospholipase Cγ activates Ras on the Golgi apparatus by means of RasGRP1. Nature 424, 694–698 (2003).

    Article  CAS  PubMed  Google Scholar 

  17. Grabocka, E. et al. Wild-type H- and N-Ras promote mutant K-Ras-driven tumorigenesis by modulating the DNA damage response. Cancer Cell 25, 243–256 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Hancock, J.F. Ras proteins: different signals from different locations. Nat. Rev. Mol. Cell Biol. 4, 373–384 (2004).

    Article  CAS  Google Scholar 

  19. Bivona, T.G. & Philips, M.R. Ras pathway signaling on endomembranes. Curr. Opin. Cell Biol. 15, 136–142 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Mano, H. ALKoma: a cancer subtype with a shared target. Cancer Discov. 2, 495–502 (2012).

    Article  CAS  PubMed  Google Scholar 

  21. Shaw, A.T. & Solomon, B. Targeting anaplastic lymphoma kinase in lung cancer. Clin. Cancer Res. 17, 2081–2086 (2011).

    Article  CAS  PubMed  Google Scholar 

  22. Soda, M. et al. Identification of the transforming EML4-ALK fusion gene in non- small-cell lung cancer. Nature 448, 561–566 (2007).

    Article  CAS  PubMed  Google Scholar 

  23. Richards, M.W. et al. Crystal structure of EML1 reveals the basis for Hsp90 dependence of oncogenic EML4-ALK by disruption of an atypical B-propeller domain. Proc. Natl. Acad. Sci. USA 111, 5195–5200 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Pollmann, M. et al. Human EML4, a novel member of the EMAP family, is essential for microtubule formation. Exp. Cell Res. 312, 3241–3251 (2006).

    Article  CAS  PubMed  Google Scholar 

  25. Choi, Y.L. et al. Identification of novel isoforms of the EML4-ALK transforming gene in non-small cell lung cancer. Cancer Res. 68, 4971–4976 (2008).

    Article  CAS  PubMed  Google Scholar 

  26. Shimizu, T. et al. The clinical effect of the dual-targeting strategy involving PI3K/AKT/mTOR and RAS/MEK/ERK pathways in patients with advanced cancer. Clin. Cancer Res. 18, 2316–2325 (2012).

    Article  CAS  PubMed  Google Scholar 

  27. Zhao, Y.S. & Adjei, A.A. The clinical development of MEK inhibitors. Nat Rev Clin Oncol. 11, 385–400 (2014).

    Article  CAS  PubMed  Google Scholar 

  28. Katayama, R. et al. Mechanisms of acquired crizotinib resistance in ALK- rearranged lung cancers. Sci. Transl. Med. 4, 120ra117 (2012).

    Article  CAS  Google Scholar 

  29. Pfister, S. et al. BRAF gene duplication constitutes a mechanism of MAPK pathway activation in low-grade astrocytomas. J. Clin. Invest. 118, 1739–1749 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Graham, J.M. Jr. et al. Genomic duplication of PTPN11 is an uncommon cause of Noonan syndrome. Am. J. Med. Genet. 149A, 2122–2128 (2009).

    Article  CAS  PubMed  Google Scholar 

  31. Caunt, C.J. & Keyse, S.M. Dual-specificity MAP kinase phosphatase (MKPs): shaping the outcome of MAP kinase signaling. FEBS J. 280, 489–504 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ercan, D. et al. Reactivation of ERK signaling causes resistance to EGFR kinase inhibitors. Cancer Discov. 2, 934–947 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Wilson, T.R. et al. Widespread potential for growth factor-driven resistance to anticancer kinase inhibitors. Nature 487, 505–509 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Crystal, A.S. et al. Patient-derived models of acquired resistance can identify effective drug combinations for cancer. Science 346, 1480–1486 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Nissan, M.H. et al. Loss of NF1 in cutaneous melanoma is associated with RAS activation and MEK dependence. Cancer Res. 74, 430–2350 (2014).

    Article  CAS  Google Scholar 

  37. Esfahani, K., Agulnik, J.S. & Cohen, V. A systemic review of resistance mechanisms and ongoing clinical trials in ALK-rearranged non-small cell lung cancer. Front Oncol. 4, 174 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  38. Doebele, R.C. et al. Mechanisms of resistance to crizotinib in patients with ALK gene rearranged non-small cell lung cancer. Clin. Cancer Res. 18, 1472–1482 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Wilson, F.H. et al. A Functional landscape of resistance to ALK inhibition in lung cancer. Cancer Cell 27, 397–408 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Tanizaki, J. et al. Combined effect of ALK and MEK inhibitors in EML4-ALK-positive non-small-cell lung cancer cells. Br. J. Cancer 106, 763–767 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Lin, L. et al. Mapping the molecular determinants of BRAF oncogene dependence in human lung cancer. Proc. Natl. Acad. Sci. USA 111, E748–E757 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Camidge, D.R. et al. Optimizing the detection of lung cancer patients harboring anaplastice lymphoma kinase (ALK) gene rearrangements potentially suitable for ALK inhibitor treatment. Clin. Cancer Res. 16, 5581–5590 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

We thank M. McMahon, F. McCormick, K. Shannon and M. Von Zastrow (UCSF) for advice and discussions, and H. Mano (University of Tokyo) for EML4-ALK cDNA constructs and for advice. We acknowledge funding support from the following sources: a US National Institute of Health (NIH) Director's New Innovator Award, the Howard Hughes Medical Institute, the Doris Duke Charitable Foundation, the American Lung Association, the National Lung Cancer Partnership, the Sidney Kimmel Foundation for Cancer Research and the Searle Scholars Program (T.G.B.); the UCSF Clinical and Translational Science Institute (G.H.); the National Cancer Institute (NCI) of the NIH (R01CA121210 and P01CA129243), a Damon Runyon Clinical Investigator Award and a LUNGevity Career Development Award (C.M.L.); the NIH (NCI5K12CA086913) (R.C.D.); an NIH Paul Calabresi Cancer Development Award in Clinical Oncology (K12CA138464) (J.W.R.); the NIH (NCI-P30CA046934) and Lung Cancer SPORE (NCI-P50CA058187) (M.V.G.); and the La Caixa Foundation and Redes Temáticas de Investigación en Cáncer (RD12/0036/0072) (R.R.).

Author information

Authors and Affiliations

  1. Department of Medicine, University of California at San Francisco, San Francisco, California, USA

    Gorjan Hrustanovic, Victor Olivas, Evangelos Pazarentzos, Asmin Tulpule, Saurabh Asthana, Collin M Blakely, Ross A Okimoto, Luping Lin, Dana S Neel, Amit Sabnis, Jennifer Flanagan, Elton Chan, Eric A Collisson & Trever G Bivona

  2. Helen Diller Family Comprehensive Cancer Center, University of California at San Francisco, San Francisco, California, USA

    Gorjan Hrustanovic, Victor Olivas, Evangelos Pazarentzos, Asmin Tulpule, Saurabh Asthana, Collin M Blakely, Ross A Okimoto, Luping Lin, Dana S Neel, Amit Sabnis, Jennifer Flanagan, Elton Chan, Eric A Collisson & Trever G Bivona

  3. Department of Medicine, University of Colorado Anschutz Medical Campus, Denver, Colorado, USA

    Marileila Varella-Garcia, Aria Vaishnavi & Robert C Doebele

  4. Department of Pathology, University of Colorado Anschutz Medical Campus, Denver, Colorado, USA

    Marileila Varella-Garcia & Dara L Aisner

  5. Division of Hematology-Oncology, University of California Irvine School of Medicine, Orange, California, USA

    Sai-Hong I Ou

  6. Chao Family Comprehensive Cancer Center, University of California Irvine School of Medicine, Orange, California, USA

    Sai-Hong I Ou

  7. Department of Medicine, Vanderbilt University School of Medicine, Nashville, Tennessee, USA

    Eiki Ichihara & Christine M Lovly

  8. University of California Davis School of Medicine,

    Philip C Mack & Jonathan W Riess

  9. Comprehensive Cancer Center, Sacramento, California, USA

    Philip C Mack & Jonathan W Riess

  10. Cancer Biology and Precision Medicine Program Catalan Institute of Oncology Hospital Germans Trias i Pujol Badalona, Barcelona, Spain

    Niki Karachaliou & Rafael Rosell

Authors
  1. Gorjan Hrustanovic
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Victor Olivas
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Evangelos Pazarentzos
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Asmin Tulpule
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Saurabh Asthana
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Collin M Blakely
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Ross A Okimoto
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Luping Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Dana S Neel
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Amit Sabnis
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Jennifer Flanagan
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Elton Chan
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Marileila Varella-Garcia
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Dara L Aisner
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Aria Vaishnavi
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Sai-Hong I Ou
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Eric A Collisson
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Eiki Ichihara
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Philip C Mack
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Christine M Lovly
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Niki Karachaliou
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Rafael Rosell
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Jonathan W Riess
    View author publications

    You can also search for this author in PubMed Google Scholar

  24. Robert C Doebele
    View author publications

    You can also search for this author in PubMed Google Scholar

  25. Trever G Bivona
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

G.H. contributed to the design, conduct and interpretation of all experiments; V.O. contributed in vivo experiments; E.P., A.T., C.M.B., R.A.O., D.S.N., E.C., A.S. and A.V. contributed cell line experiments and aided in experimental design; S.A. contributed deep-sequencing analysis; L.L. contributed sequencing library preparation; J.F., M.V.-G., D.L.A. and R.C.D. contributed analysis of patient tumor data; S.-H.I.O., P.C.M., N.K., R.R., J.W.R. and R.C.D. contributed patient tumor samples and clinical data; E.A.C. contributed to experimental design and interpretation; E.I. and C.M.L. contributed patient-derived cell lines and conducted experiments; T.G.B. supervised the project and contributed to the design and interpretation of all experiments; and G.H. and T.G.B. wrote the manuscript with input from all co-authors.

Corresponding author

Correspondence to Trever G Bivona.

Ethics declarations

Competing interests

T.G.B. is a consultant to Driver Group, Novartis, Clovis Oncology, Natera and Genoptix and is the recipient of a research grant from Servier, all of which are each engaged in cancer diagnostics and/or treatment.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–10 (PDF 8873 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hrustanovic, G., Olivas, V., Pazarentzos, E. et al. RAS-MAPK dependence underlies a rational polytherapy strategy in EML4-ALK–positive lung cancer. Nat Med 21, 1038–1047 (2015). https://doi.org/10.1038/nm.3930

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.3930

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Cancer

Sign up for the Nature Briefing: Cancer newsletter — what matters in cancer research, free to your inbox weekly.

Get what matters in cancer research, free to your inbox weekly. Sign up for Nature Briefing: Cancer