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.

An approach to suppress the evolution of resistance in BRAFV600E-mutant cancer

Nature Medicine volume 23, pages 929–937 (2017)Cite this article

Subjects

Abstract

The principles that govern the evolution of tumors exposed to targeted therapy are poorly understood. Here we modeled the selection and propagation of an amplification in the BRAF oncogene (BRAFamp) in patient-derived tumor xenografts (PDXs) that were treated with a direct inhibitor of the kinase ERK, either alone or in combination with other ERK signaling inhibitors. Single-cell sequencing and multiplex fluorescence in situ hybridization analyses mapped the emergence of extra-chromosomal amplification in parallel evolutionary trajectories that arose in the same tumor shortly after treatment. The evolutionary selection of BRAFamp was determined by the fitness threshold, the barrier that subclonal populations need to overcome to regain fitness in the presence of therapy. This differed for inhibitors of ERK signaling, suggesting that sequential monotherapy is ineffective and selects for a progressively higher BRAF copy number. Concurrent targeting of the RAF, MEK and ERK kinases, however, imposed a sufficiently high fitness threshold to prevent the propagation of subclones with high-level BRAFamp. When administered on an intermittent schedule, this treatment inhibited tumor growth in 11/11 PDXs of lung cancer or melanoma without apparent toxicity in mice. Thus, gene amplification can be acquired and expanded through parallel evolution, enabling tumors to adapt while maintaining their intratumoral heterogeneity. Treatments that impose the highest fitness threshold will likely prevent the evolution of resistance-causing alterations and, thus, merit testing in patients.

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

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Learn more

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

Figure 1: ERK-inhibitor-resistant populations with extrachromosomal BRAF amplification.
Figure 2: BRAFamp emerges in parallel evolutionary tracts.
Figure 3: BRAFamp is sufficient to confer a selective advantage in the presence of ERKi treatment.
Figure 4: Fitness threshold model.
Figure 5: Identification of a treatment to suppress the evolution of BRAF-amplified clones.
Figure 6: An intermittent combination treatment inhibits tumor growth in BRAFV600E PDX models for lung cancer and melanoma.

References

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

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Planchard, D. et al. Dabrafenib in patients with BRAFV600E-positive advanced non-small-cell lung cancer: a single-arm, multicenter, open-label, phase 2 trial. Lancet Oncol. 17, 642–650 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Flaherty, K.T. et al. Combined BRAF and MEK inhibition in melanoma with BRAFV600 mutations. N. Engl. J. Med. 367, 1694–1703 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Larkin, J. et al. Combined vemurafenib and cobimetinib in BRAF-mutated melanoma. N. Engl. J. Med. 371, 1867–1876 (2014).

    Article  CAS  PubMed  Google Scholar 

  7. 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 

  8. Planchard, D. et al. Dabrafenib plus trametinib in patients with previously treated BRAFV600E-mutant metastatic non-small-cell lung cancer: an open-label, multicenter phase 2 trial. Lancet Oncol. 17, 984–993 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Poulikakos, P.I. et al. RAF inhibitor resistance is mediated by dimerization of aberrantly spliced BRAFV600E. Nature 480, 387–390 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Shi, H. et al. Melanoma whole-exome sequencing identifies V600EB-RAF amplification-mediated acquired B-RAF inhibitor resistance. Nat. Commun. 3, 724 (2012).

    Article  CAS  PubMed  Google Scholar 

  11. Das Thakur, M. et al. Modeling vemurafenib resistance in melanoma reveals a strategy to forestall drug resistance. Nature 494, 251–255 (2013).

    Article  CAS  PubMed  Google Scholar 

  12. Shi, H. et al. Acquired resistance and clonal evolution in melanoma during BRAF inhibitor therapy. Cancer Discov. 4, 80–93 (2014).

    Article  CAS  PubMed  Google Scholar 

  13. Lito, P., Rosen, N. & Solit, D.B. Tumor adaptation and resistance to RAF inhibitors. Nat. Med. 19, 1401–1409 (2013).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lito, P. et al. Relief of profound feedback inhibition of mitogenic signaling by RAF inhibitors attenuates their activity in BRAFV600E melanomas. Cancer Cell 22, 668–682 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Lipinski, K.A. et al. Cancer evolution and the limits of predictability in precision cancer medicine. Trends Cancer 2, 49–63 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  17. McGranahan, N. & Swanton, C. Biological and therapeutic impact of intratumor heterogeneity in cancer evolution. Cancer Cell 27, 15–26 (2015).

    Article  CAS  PubMed  Google Scholar 

  18. Merlo, L.M., Pepper, J.W., Reid, B.J. & Maley, C.C. Cancer as an evolutionary and ecological process. Nat. Rev. Cancer 6, 924–935 (2006).

    Article  CAS  PubMed  Google Scholar 

  19. Baslan, T. et al. Genome-wide copy number analysis of single cells. Nat. Protoc. 7, 1024–1041 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Garvin, T. et al. Interactive analysis and assessment of single-cell copy number variations. Nat. Methods 12, 1058–1060 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Navin, N. et al. Tumor evolution inferred by single-cell sequencing. Nature 472, 90–94 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Morris, E.J. et al. Discovery of a novel ERK inhibitor with activity in models of acquired resistance to BRAF and MEK inhibitors. Cancer Discov. 3, 742–750 (2013).

    Article  CAS  PubMed  Google Scholar 

  23. Wong, D.J. et al. Antitumor activity of the ERK inhibitor SCH772984 against BRAF-mutant, NRAS-mutant and wild-type melanoma. Mol. Cancer 13, 194 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  24. Baslan, T. et al. Optimizing sparse sequencing of single cells for highly multiplex copy number profiling. Genome Res. 25, 714–724 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Weir, B.A. et al. Characterizing the cancer genome in lung adenocarcinoma. Nature 450, 893–898 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Yao, Z. et al. BRAF mutants evade ERK-dependent feedback by different mechanisms that determine their sensitivity to pharmacologic inhibition. Cancer Cell 28, 370–383 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Joseph, E.W. et al. The RAF inhibitor PLX4032 inhibits ERK signaling and tumor cell proliferation in a V600E BRAF–selective manner. Proc. Natl. Acad. Sci. USA 107, 14903–14908 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  28. Kim, K.B. et al. Phase 2 study of the MEK1 and MEK2 inhibitor trametinib in patients with metastatic BRAF-mutant cutaneous melanoma previously treated with or without a BRAF inhibitor. J. Clin. Oncol. 31, 482–489 (2013).

    Article  CAS  PubMed  Google Scholar 

  29. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Prahallad, A. et al. Unresponsiveness of colon cancer to BRAFV600E inhibition through feedback activation of EGFR. Nature 483, 100–103 (2012).

    Article  CAS  PubMed  Google Scholar 

  31. Shi, H. et al. A novel AKT1 mutant amplifies an adaptive melanoma response to BRAF inhibition. Cancer Discov. 4, 69–79 (2014).

    Article  CAS  PubMed  Google Scholar 

  32. Sun, C. et al. Reversible and adaptive resistance to BRAFV600E inhibition in melanoma. Nature 508, 118–122 (2014).

    Article  CAS  PubMed  Google Scholar 

  33. Maertens, O. et al. Elucidating distinct roles for NF1 in melanomagenesis. Cancer Discov. 3, 338–349 (2013).

    Article  CAS  PubMed  Google Scholar 

  34. Johannessen, C.M. et al. A melanocyte lineage program confers resistance to MAP kinase pathway inhibition. Nature 504, 138–142 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Anderson, K. et al. Genetic variegation of clonal architecture and propagating cells in leukemia. Nature 469, 356–361 (2011).

    Article  CAS  PubMed  Google Scholar 

  36. Melchor, L. et al. Single-cell genetic analysis reveals the composition of initiating clones and phylogenetic patterns of branching and parallel evolution in myeloma. Leukemia 28, 1705–1715 (2014).

    Article  CAS  PubMed  Google Scholar 

  37. Campbell, P.J. et al. The patterns and dynamics of genomic instability in metastatic pancreatic cancer. Nature 467, 1109–1113 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Nguyen, A. et al. PKLR promotes colorectal cancer liver colonization through induction of glutathione synthesis. J. Clin. Invest. 126, 681–694 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  39. Poirier, J.T. et al. DNA methylation in small-cell lung cancer defines distinct disease subtypes and correlates with high expression of EZH2. Oncogene 34, 5869–5878 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Einarsdottir, B.O. et al. Melanoma-patient-derived xenografts accurately model the disease and develop fast enough to guide treatment decisions. Oncotarget 5, 9609–9618 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  41. Cheng, D.T. et al. Memorial Sloan Kettering–Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT): a hybridization-capture-based next-generation sequencing clinical assay for solid tumor molecular oncology. J. Mol. Diagn. 17, 251–264 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S.L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Li, H. & Durbin, R. Fast and accurate short-read alignment with Burrows–Wheeler transform. Bioinformatics 25, 1754–1760 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Gao, R. et al. Punctuated copy number evolution and clonal stasis in triple-negative breast cancer. Nat. Genet. 48, 1119–1130 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Wanjala, J. et al. Identifying actionable targets through integrative analyses of GEM model and human prostate cancer genomic profiling. Mol. Cancer Ther. 14, 278–288 (2015).

    Article  CAS  PubMed  Google Scholar 

  46. Hembrough, T. et al. Application of selected reaction monitoring for multiplex quantification of clinically validated biomarkers in formalin-fixed, paraffin-embedded tumor tissue. J. Mol. Diagn. 15, 454–465 (2013).

    Article  CAS  PubMed  Google Scholar 

  47. Catenacci, D.V. et al. Absolute quantitation of Met using mass spectrometry for clinical application: assay precision, stability and correlation with MET gene amplification in FFPE tumor tissue. PLoS One 9, e100586 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Lito, P., Solomon, M., Li, L.S., Hansen, R. & Rosen, N. Allele-specific inhibitors inactivate mutant KRASG12C by a trapping mechanism. Science 351, 604–608 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank D. Solit, N. Bouvier and the Center for Molecular Oncology at MSKCC for assistance with next-generation sequencing, Z. Yao (MSKCC) for providing the A375 doxycycline-inducible BRAFV600E cells, as well as C. Sawyers, C. Rudin, J. Poirier and M. Mroczkowski for reviewing the manuscript. This work was supported by the US National Institutes of Health (NIH) (grant K08 CA191082-01A1; P.L.), the Uniting Against Lung Cancer Foundation (P.L.), the Damon Runyon Clinical Investigator Award (P.L.), the Josie Robertson Investigator Program at MSKCC (P.L.), the Druckenmiller Center for Lung Cancer Center at MSKCC (P.L.) and a Medical Scientist Training Program grant from the National Institute of General Medical Sciences of the NIH under award number T32GM007739 to the Weill Cornell–Rockefeller–Sloan Kettering Tri-institutional MD–PhD Program (Y.X.). E.d.S. and S.W.L. are supported in part by the MSKCC Pilot Center for Precision Disease Modeling program (U54 OD020355). T.B. is supported by the William C. and Joyce C. O'Neil Charitable Trust, Memorial Sloan Kettering Single-Cell Sequencing Initiative. J.N. is supported by the Knut and Alice Wallenberg Foundation. The authors also acknowledge the MSKCC Support Grant–Core Grant program (P30 CA008748).

Author information

Author notes

  1. Luciano Martelotto

    Present address: School of Clinical Sciences, Monash University, Clayton, Victoria, Australia

  2. Yaohua Xue and Luciano Martelotto: These authors contributed equally to this work.

Authors and Affiliations

  1. Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center (MSKCC), New York, New York, USA

    Yaohua Xue, Alberto Vides, Martha Solomon, Trang Thi Mai, Neelam Chaudhary & Piro Lito

  2. Weill Cornell–Rockefeller–Sloan Kettering Tri-institutional MD–PhD Program, New York, New York, USA

    Yaohua Xue

  3. Department of Pathology, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Luciano Martelotto, Michael F Berger & Jorge S Reis-Filho

  4. Cancer Biology and Genetics Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Timour Baslan & Scott W Lowe

  5. Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Greg J Riely, Bob T Li & Piro Lito

  6. NantOmics, Rockville, Maryland, USA

    Kerry Scott, Fabiola Cechhi, Sarit Schwartz & Todd Hembrough

  7. Sahlgrenska Translational Melanoma Group, Sahlgrenska Cancer Center, University of Gothenburg, Gothenburg, Sweden

    Ulrika Stierner & Jonas Nilsson

  8. Molecular Cytogenetics Core Facility, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Kalyani Chadalavada & Gouri Nanjangud

  9. Antitumor Assessment Core Facility, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Elisa de Stanchina

  10. Weill Cornell Medical College, Cornell University, New York, New York, USA

    Michael F Berger & Piro Lito

  11. Molecular Pharmacology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Neal Rosen

Authors
  1. Yaohua Xue
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Luciano Martelotto
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Timour Baslan
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Alberto Vides
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Martha Solomon
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Trang Thi Mai
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Neelam Chaudhary
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Greg J Riely
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Bob T Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Kerry Scott
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Fabiola Cechhi
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Ulrika Stierner
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Kalyani Chadalavada
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Elisa de Stanchina
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Sarit Schwartz
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Todd Hembrough
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Gouri Nanjangud
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Michael F Berger
    View author publications

    You can also search for this author in PubMed Google Scholar

  19. Jonas Nilsson
    View author publications

    You can also search for this author in PubMed Google Scholar

  20. Scott W Lowe
    View author publications

    You can also search for this author in PubMed Google Scholar

  21. Jorge S Reis-Filho
    View author publications

    You can also search for this author in PubMed Google Scholar

  22. Neal Rosen
    View author publications

    You can also search for this author in PubMed Google Scholar

  23. Piro Lito
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Y.X., L.M., A.V., M.S., T.T.M. and N.C. performed experiments; L.M., T.B. and J.S.R.-F. performed, analyzed and helped interpret the single-cell sequencing experiments; M.F.B. analyzed the bulk-sequencing data; G.J.R. and B.T.L. provided the patient samples; J.N. and U.S. provided the PDX models; E.d.S. performed the animal experiments; K.S., F.C., T.H. and S.S. performed the mass spectrometry experiments; K.C. performed the FISH experiments, and G.N. analyzed the results. N.R. and S.W.L. provided key scientific insights and reagents; P.L. conceived and supervised the study, designed and performed experiments, and interpreted data; Y.X. and P.L. were the principal writers of the manuscript; and all of the authors reviewed the manuscript and contributed in writing.

Corresponding author

Correspondence to Piro Lito.

Ethics declarations

Competing interests

P.L. is listed as an inventor on a patent application filed by MSKCC that incorporates discoveries described in the manuscript. N.R. is on the scientific advisory board, and has received grant support from, Chugai Pharmaceutical and is on the SAB of Astra-Zeneca, Beigene and Kura. S.S., T.H., K.S. and F.C. are employees of NantOmics.

Supplementary information

Supplementary Tables and Figures

Supplementary Tables 1 and 2 and Supplementary Figures 1–7 (PDF 11679 kb)

Supplementary Dataset (XLSX 2434 kb)

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Xue, Y., Martelotto, L., Baslan, T. et al. An approach to suppress the evolution of resistance in BRAFV600E-mutant cancer. Nat Med 23, 929–937 (2017). https://doi.org/10.1038/nm.4369

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nm.4369

This article is cited by

Search

Advanced search

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