Cooperative targeting of melanoma heterogeneity with an AXL antibody-drug conjugate and BRAF/MEK inhibitors

Abstract

Intratumor heterogeneity is a key factor contributing to therapeutic failure and, hence, cancer lethality. Heterogeneous tumors show partial therapy responses, allowing for the emergence of drug-resistant clones that often express high levels of the receptor tyrosine kinase AXL. In melanoma, AXL-high cells are resistant to MAPK pathway inhibitors, whereas AXL-low cells are sensitive to these inhibitors, rationalizing a differential therapeutic approach. We developed an antibody-drug conjugate, AXL-107-MMAE, comprising a human AXL antibody linked to the microtubule-disrupting agent monomethyl auristatin E. We found that AXL-107-MMAE, as a single agent, displayed potent in vivo anti-tumor activity in patient-derived xenografts, including melanoma, lung, pancreas and cervical cancer. By eliminating distinct populations in heterogeneous melanoma cell pools, AXL-107-MMAE and MAPK pathway inhibitors cooperatively inhibited tumor growth. Furthermore, by inducing AXL transcription, BRAF/MEK inhibitors potentiated the efficacy of AXL-107-MMAE. These findings provide proof of concept for the premise that rationalized combinatorial targeting of distinct populations in heterogeneous tumors may improve therapeutic effect, and merit clinical validation of AXL-107-MMAE in both treatment-naive and drug-resistant cancers in mono- or combination therapy.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: AXL-107-MMAE induces cytotoxicity in vitro and in vivo.
Figure 2: Elimination of distinct tumor populations by AXL-107-MMAE and MAPK pathway inhibition.
Figure 3: AXL-positive cells reside in pre-treatment melanomas and rapidly outgrow bulk AXL-negative cells following therapeutic pressure.
Figure 4: AXL-107-MMAE and MAPK inhibitors mutually potentiate cytotoxicity.
Figure 5: AXL-107-MMAE and MAPK pathway inhibitors cooperatively inhibit melanoma PDX growth.
Figure 6: Eliminating heterogeneous melanoma by targeting distinct populations with AXL-107-MMAE and MAPK pathway inhibitors.

References

  1. 1

    McGranahan, N. & Swanton, C. Clonal heterogeneity and tumor evolution: past, present, and the future. Cell 168, 613–628 (2017).

    CAS  PubMed  Google Scholar 

  2. 2

    Aparicio, S. & Caldas, C. The implications of clonal genome evolution for cancer medicine. N. Engl. J. Med. 368, 842–851 (2013).

    CAS  PubMed  Google Scholar 

  3. 3

    Sharma, P., Hu-Lieskovan, S., Wargo, J.A. & Ribas, A. Primary, adaptive, and acquired resistance to cancer immunotherapy. Cell 168, 707–723 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4

    Diaz, L.A. Jr. et al. The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers. Nature 486, 537–540 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5

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

    PubMed  Google Scholar 

  6. 6

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

    PubMed  Google Scholar 

  7. 7

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

    PubMed  Google Scholar 

  8. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9

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

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Long, G.V. et al. Factors predictive of response, disease progression, and overall survival after dabrafenib and trametinib combination treatment: a pooled analysis of individual patient data from randomised trials. Lancet Oncol. 17, 1743–1754 (2016).

    CAS  PubMed  Google Scholar 

  11. 11

    Smith, M.P. et al. The immune microenvironment confers resistance to MAPK pathway inhibitors through macrophage-derived TNFα. Cancer Discov. 4, 1214–1229 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12

    Nazarian, R. et al. Melanomas acquire resistance to B-RAFV600E inhibition by RTK or N-RAS upregulation. Nature 468, 973–977 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  13. 13

    Girotti, M.R., Saturno, G., Lorigan, P. & Marais, R. No longer an untreatable disease: how targeted and immunotherapies have changed the management of melanoma patients. Mol. Oncol. 8, 1140–1158 (2014).

    PubMed  PubMed Central  Google Scholar 

  14. 14

    Davies, M.A. & Samuels, Y. Analysis of the genome to personalize therapy for melanoma. Oncogene 29, 5545–5555 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

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

    CAS  PubMed  Google Scholar 

  16. 16

    Kemper, K. et al. Intra- and inter-tumor heterogeneity in a vemurafenib-resistant melanoma patient and derived xenografts. EMBO Mol. Med. 7, 1104–1118 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17

    Müller, J. et al. Low MITF/AXL ratio predicts early resistance to multiple targeted drugs in melanoma. Nat. Commun. 5, 5712 (2014).

    PubMed  PubMed Central  Google Scholar 

  18. 18

    Konieczkowski, D.J. et al. A melanoma cell state distinction influences sensitivity to MAPK pathway inhibitors. Cancer Discov. 4, 816–827 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19

    Leucci, E., Close, P. & Marine, J.-C. Translation rewiring at the heart of phenotype switching in melanoma. Pigment Cell Melanoma Res. 30, 282–283 (2017).

    PubMed  Google Scholar 

  20. 20

    Hoek, K.S. et al. Metastatic potential of melanomas defined by specific gene expression profiles with no BRAF signature. Pigment Cell Res. 19, 290–302 (2006).

    CAS  PubMed  Google Scholar 

  21. 21

    Hoek, K.S. et al. In vivo switching of human melanoma cells between proliferative and invasive states. Cancer Res. 68, 650–656 (2008).

    CAS  PubMed  Google Scholar 

  22. 22

    Elkabets, M. et al. AXL mediates resistance to PI3Kα inhibition by activating the EGFR/PKC/mTOR axis in head and neck and esophageal squamous cell carcinomas. Cancer Cell 27, 533–546 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23

    Reichl, P. et al. Axl activates autocrine transforming growth factor-β signaling in hepatocellular carcinoma. Hepatology 61, 930–941 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. 24

    Bansal, N., Mishra, P.J., Stein, M., DiPaola, R.S. & Bertino, J.R. Axl receptor tyrosine kinase is up-regulated in metformin resistant prostate cancer cells. Oncotarget 6, 15321–15331 (2015).

    PubMed  PubMed Central  Google Scholar 

  25. 25

    Byers, L.A. et al. An epithelial-mesenchymal transition gene signature predicts resistance to EGFR and PI3K inhibitors and identifies Axl as a therapeutic target for overcoming EGFR inhibitor resistance. Clin. Cancer Res. 19, 279–290 (2013).

    CAS  PubMed  Google Scholar 

  26. 26

    Zhang, Z. et al. Activation of the AXL kinase causes resistance to EGFR-targeted therapy in lung cancer. Nat. Genet. 44, 852–860 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 27

    Creedon, H. et al. Exploring mechanisms of acquired resistance to HER2 (human epidermal growth factor receptor 2)-targeted therapies in breast cancer. Biochem. Soc. Trans. 42, 822–830 (2014).

    CAS  PubMed  Google Scholar 

  28. 28

    Zhou, L. et al. Targeting MET and AXL overcomes resistance to sunitinib therapy in renal cell carcinoma. Oncogene 35, 2687–2697 (2016).

    CAS  PubMed  Google Scholar 

  29. 29

    Debruyne, D.N. et al. ALK inhibitor resistance in ALKF1174L-driven neuroblastoma is associated with AXL activation and induction of EMT. Oncogene 35, 3681–3691 (2016).

    CAS  PubMed  Google Scholar 

  30. 30

    Wang, C. et al. Gas6/Axl axis contributes to chemoresistance and metastasis in breast cancer through Akt/GSK-3β/β-catenin signaling. Theranostics 6, 1205–1219 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  31. 31

    Kurokawa, M., Ise, N., Omi, K., Goishi, K. & Higashiyama, S. Cisplatin influences acquisition of resistance to molecular-targeted agents through epithelial-mesenchymal transition-like changes. Cancer Sci. 104, 904–911 (2013).

    CAS  PubMed  Google Scholar 

  32. 32

    Hong, C.-C. et al. Receptor tyrosine kinase AXL is induced by chemotherapy drugs and overexpression of AXL confers drug resistance in acute myeloid leukemia. Cancer Lett. 268, 314–324 (2008).

    CAS  PubMed  Google Scholar 

  33. 33

    Aguilera, T.A. et al. Reprogramming the immunological microenvironment through radiation and targeting Axl. Nat. Commun. 7, 13898 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34

    Bhang, H.-E.C. et al. Studying clonal dynamics in response to cancer therapy using high-complexity barcoding. Nat. Med. 21, 440–448 (2015).

    CAS  PubMed  Google Scholar 

  35. 35

    Hata, A.N. et al. Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition. Nat. Med. 22, 262–269 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36

    Sharma, S.V. et al. A chromatin-mediated reversible drug-tolerant state in cancer cell subpopulations. Cell 141, 69–80 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37

    Turke, A.B. et al. Preexistence and clonal selection of MET amplification in EGFR mutant NSCLC. Cancer Cell 17, 77–88 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38

    Cichoń, M.A. et al. The receptor tyrosine kinase Axl regulates cell-cell adhesion and stemness in cutaneous squamous cell carcinoma. Oncogene 33, 4185–4192 (2014).

    PubMed  Google Scholar 

  39. 39

    Mak, M.P. et al. A patient-derived, pan-cancer EMT signature identifies global molecular alterations and immune target enrichment following epithelial-to-mesenchymal transition. Clin. Cancer Res. 22, 609–620 (2016).

    CAS  PubMed  Google Scholar 

  40. 40

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

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41

    Loges, S. et al. Malignant cells fuel tumor growth by educating infiltrating leukocytes to produce the mitogen Gas6. Blood 115, 2264–2273 (2010).

    CAS  PubMed  Google Scholar 

  42. 42

    Adams, G.P. et al. High affinity restricts the localization and tumor penetration of single-chain fv antibody molecules. Cancer Res. 61, 4750–4755 (2001).

    CAS  PubMed  Google Scholar 

  43. 43

    Donaghy, H. Effects of antibody, drug and linker on the preclinical and clinical toxicities of antibody-drug conjugates. MAbs 8, 659–671 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44

    Chen, R. et al. CD30 downregulation, MMAE resistance, and MDR1 upregulation are all associated with resistance to brentuximab vedotin. Mol. Cancer Ther. 14, 1376–1384 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45

    Szakács, G., Paterson, J.K., Ludwig, J.A., Booth-Genthe, C. & Gottesman, M.M. Targeting multidrug resistance in cancer. Nat. Rev. Drug Discov. 5, 219–234 (2006).

    PubMed  Google Scholar 

  46. 46

    Holderfield, M., Deuker, M.M., McCormick, F. & McMahon, M. Targeting RAF kinases for cancer therapy: BRAF-mutated melanoma and beyond. Nat. Rev. Cancer 14, 455–467 (2014).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47

    Kemper, K. et al. BRAFV600E kinase domain duplication identified in therapy-refractory melanoma patient-derived xenografts. Cell Rep. 16, 263–277 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  48. 48

    de Goeij, B.E. & Lambert, J.M. New developments for antibody-drug conjugate–based therapeutic approaches. Curr. Opin. Immunol. 40, 14–23 (2016).

    CAS  PubMed  Google Scholar 

  49. 49

    Thomas, A., Teicher, B.A. & Hassan, R. Antibody-drug conjugates for cancer therapy. Lancet Oncol. 17, e254–e262 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50

    Asundi, J. et al. MAPK pathway inhibition enhances the efficacy of an anti–endothelin B receptor drug conjugate by inducing target expression in melanoma. Mol. Cancer Ther. 13, 1599–1610 (2014).

    CAS  PubMed  Google Scholar 

  51. 51

    Breij, E.C.W. et al. An antibody-drug conjugate that targets tissue factor exhibits potent therapeutic activity against a broad range of solid tumors. Cancer Res. 74, 1214–1226 (2014).

    CAS  PubMed  Google Scholar 

  52. 52

    Doronina, S.O. et al. Development of potent monoclonal antibody auristatin conjugates for cancer therapy. Nat. Biotechnol. 21, 778–784 (2003).

    CAS  PubMed  Google Scholar 

  53. 53

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

    CAS  PubMed  Google Scholar 

  54. 54

    Manchado, E. et al. A combinatorial strategy for treating KRAS-mutant lung cancer. Nature 534, 647–651 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55

    Miller, M.A. et al. Reduced proteolytic shedding of receptor tyrosine kinases is a post-translational mechanism of kinase inhibitor resistance. Cancer Discov. 6, 382–399 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 56

    Creedon, H. et al. Identification of novel pathways linking epithelial-to-mesenchymal transition with resistance to HER2-targeted therapy. Oncotarget 7, 11539–11552 (2016).

    PubMed  PubMed Central  Google Scholar 

  57. 57

    Xu, W. et al. Up-regulation of the Hippo pathway effector TAZ renders lung adenocarcinoma cells harboring EGFR-T790M mutation resistant to gefitinib. Cell Biosci. 5, 7 (2015).

    PubMed  PubMed Central  Google Scholar 

  58. 58

    Tirosh, I. et al. Dissecting the multicellular ecosystem of metastatic melanoma by single-cell RNA-seq. Science 352, 189–196 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 59

    Shaffer, S.M. et al. Rare cell variability and drug-induced reprogramming as a mode of cancer drug resistance. Nature 546, 431–435 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60

    Fishwild, D.M. et al. High-avidity human IgG κ monoclonal antibodies from a novel strain of minilocus transgenic mice. Nat. Biotechnol. 14, 845–851 (1996).

    CAS  PubMed  Google Scholar 

  61. 61

    Vink, T., Oudshoorn-Dickmann, M., Roza, M., Reitsma, J.-J. & de Jong, R.N. A simple, robust and highly efficient transient expression system for producing antibodies. Methods 65, 5–10 (2014).

    CAS  PubMed  Google Scholar 

  62. 62

    Burton, D.R. et al. Efficient neutralization of primary isolates of HIV-1 by a recombinant human monoclonal antibody. Science 266, 1024–1027 (1994).

    CAS  Google Scholar 

  63. 63

    Colaprico, A. et al. TCGAbiolinks: an R/Bioconductor package for integrative analysis of TCGA data. Nucleic Acids Res. 44, e71 (2016).

    PubMed  Google Scholar 

Download references

Acknowledgements

We thank all the members of the Peeper and Blank laboratories for their valuable input and the FACS facility and animal facility at the NKI for their support. We would like to acknowledge the NKI-AVL Core Facility Molecular Pathology & Biobanking (CFMPB) for supplying NKI-AVL Biobank material and lab support. We thank M. van der Ven and the intervention unit (NKI) for their help with animal experiments, M. Mertz and E. Gielen for their help with confocal microscopy, and H. Witteveen, M. Houtkamp, G. Rigter and T. Kroes for help with experiments. The research leading to these results has been funded by a grant from Genmab, by the European Research Council under the European Union′s Seventh Framework Programme (FP7/2007-2013)/ERC synergy grant agreement n° 319661 COMBATCANCER and grants NKI 2014-7241, NKI 2013-5799 and NKI 2017-10425 from the Dutch Cancer Society (KWF).

Author information

Affiliations

Authors

Contributions

J.B., L.A.K., E.C.W.B., M.L.J., D.S., D.S.P. and P.W.H.I.P. designed the study, analyzed the data and wrote the manuscript. J.B., M.A.L., D.W.V., N.P. and E.G.-v.d.H. performed the experiments. O.K. performed the bioinformatics analyses. K.K. and D.S.P. developed the melanoma PDX-platform and engineered PDX-derived cell lines. A.S. and J.B. performed in vivo melanoma experiments. J.-Y.S. analyzed IHC from in vivo experiments. T.K. and C.U.B. gave critical input. J.T.M. directed toxicological analyses. M.G.F. and E.A.R. provided paired human melanoma samples. D.S.P. and P.W.H.I.P. supervised the study.

Corresponding authors

Correspondence to Esther C W Breij or Daniel S Peeper.

Ethics declarations

Competing interests

L.K., E.B., E.G.-v.d.H., N.P., M.J. and D.S. are full-time Genmab employees and have warrants and/or stock. P.P. is a Genmab shareholder. C.U.B. receives grants and/or research support from Novartis and BMS, and has received honoraria or consultation fees for MSD, BMS, Roche, Novartis, GSK, Pfizer and Lilly, and is a stock shareholder of Verastem.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–12 and Supplementary Tables 1–4 (PDF 16466 kb)

Life Sciences Reporting Summary (PDF 157 kb)

Source data

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Boshuizen, J., Koopman, L., Krijgsman, O. et al. Cooperative targeting of melanoma heterogeneity with an AXL antibody-drug conjugate and BRAF/MEK inhibitors. Nat Med 24, 203–212 (2018). https://doi.org/10.1038/nm.4472

Download citation

Further reading

Search

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