Article | Published:

Bruton’s tyrosine kinase potentiates ALK signaling and serves as a potential therapeutic target of neuroblastoma

Oncogenevolume 37pages61806194 (2018) | Download Citation


Aberrant activation of anaplastic lymphoma kinase (ALK) can cause sporadic and familial neuroblastoma. Using a proteomics approach, we identified Bruton’s tyrosine kinase (BTK) as a novel ALK interaction partner, and the physical interaction was confirmed by co-immunoprecipitation. BTK is expressed in neuroblastoma cell lines and tumor tissues. Its high expression correlates with poor relapse-free survival probability of neuroblastoma patients. Mechanistically, we demonstrated that BTK potentiates ALK-mediated signaling in neuroblastoma, and increases ALK stability by reducing ALK ubiquitination. Both ALKWT and ALKF1174L can induce BTK phosphorylation and higher capacity of ALKF1174L is observed. Furthermore, the BTK inhibitor ibrutinib can effectively inhibit the growth of neuroblastoma xenograft in nude mice, and the combination of ibrutinib and the ALK inhibitor crizotinib further enhances the inhibition. Our study provides strong rationale for clinical trial of ALK-positive neuroblastoma using ibrutinib or the combination of ibrutinib and ALK inhibitors.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    Park JR, Eggert A, Caron H. Neuroblastoma: biology, prognosis, and treatment. Hematol Oncol Clin North Am. 2010;24:65–86.

  2. 2.

    Louis CU, Shohet JM. Neuroblastoma: molecular pathogenesis and therapy. Annu Rev Med. 2015;66:49–63.

  3. 3.

    Tabbo F, Barreca A, Piva R, Inghirami G, European TCLSG. ALK signaling and target therapy in anaplastic large cell lymphoma. Front Oncol. 2012;2:41.

  4. 4.

    Tan SL, Liao C, Lucas MC, Stevenson C, DeMartino JA. Targeting the SYK-BTK axis for the treatment of immunological and hematological disorders: recent progress and therapeutic perspectives. Pharmacol Ther. 2013;138:294–309.

  5. 5.

    Cheung NK, Dyer MA. Neuroblastoma: developmental biology, cancer genomics and immunotherapy. Nat Rev Cancer. 2013;13:397–411.

  6. 6.

    Maris JM. Recent advances in neuroblastoma. N Engl J Med. 2010;362:2202–11.

  7. 7.

    Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature. 2007;448:561–6.

  8. 8.

    Asati V, Mahapatra DK, Bharti SK. PI3K/Akt/mTOR and Ras/Raf/MEK/ERK signaling pathways inhibitors as anticancer agents: Structural and pharmacological perspectives. Eur J Med Chem. 2016;109:314–41.

  9. 9.

    Khozin S, Blumenthal GM, Zhang L, Tang S, Brower M, Fox E, et al. FDA approval: ceritinib for the treatment of metastatic anaplastic lymphoma kinase-positive non-small cell lung cancer. Clin Cancer Res. 2015;21:2436–9.

  10. 10.

    Larkins E, Blumenthal GM, Chen H, He K, Agarwal R, Gieser G, et al. FDA approval: Alectinib for the treatment of metastatic, alk-positive non-small cell lung cancer following crizotinib. Clin Cancer Res. 2016;22:5171–6.

  11. 11.

    Isozaki H, Ichihara E, Takigawa N, Ohashi K, Ochi N, Yasugi M, et al. Non-small cell lung cancer cells acquire resistance to the ALK inhibitor alectinib by activating alternative receptor tyrosine kinases. Cancer Res. 2016;76:1506–16.

  12. 12.

    Kodityal S, Elvin JA, Squillace R, Agarwal N, Miller VA, Ali SM, et al. A novel acquired ALK F1245C mutation confers resistance to crizotinib in ALK-positive NSCLC but is sensitive to ceritinib. Lung Cancer. 2016;92:19–21.

  13. 13.

    Geoerger B, Schulte J, Zwaan CM, Casanova M, Fischer M, Moreno L et al. Phase I study of ceritinib in pediatric patients (Pts) with malignancies harboring a genetic alteration in ALK (ALK plus): Safety, pharmacokinetic (PK), and efficacy results. J Clin Oncol. 2015; 33.

  14. 14.

    Mosse YP, Lim MS, Voss SD, Wilner K, Ruffner K, Laliberte J, et al. Safety and activity of crizotinib for paediatric patients with refractory solid tumours or anaplastic large-cell lymphoma: a Children’s Oncology Group phase 1 consortium study. Lancet Oncol. 2013;14:472–80.

  15. 15.

    Wang HQ, Halilovic E, Li X, Liang J, Cao Y, Rakiec DP, et al. Combined ALK and MDM2 inhibition increases antitumor activity and overcomes resistance in human ALK mutant neuroblastoma cell lines and xenograft models. eLife. 2017;6:e17137.

  16. 16.

    Chakraborty R, Kapoor P, Ansell SM, Gertz MA. Ibrutinib for the treatment of Waldenstrom macroglobulinemia. Expert Rev Hematol. 2015;8:569–79.

  17. 17.

    Guo W, Liu R, Bhardwaj G, Yang JC, Changou C, Ma AH, et al. Targeting Btk/Etk of prostate cancer cells by a novel dual inhibitor. Cell Death Dis. 2014;5:e1409.

  18. 18.

    Grassilli E, Pisano F, Cialdella A, Bonomo S, Missaglia C, Cerrito MG, et al. A novel oncogenic BTK isoform is overexpressed in colon cancers and required for RAS-mediated transformation. Oncogene. 2016;35:4368–78.

  19. 19.

    George RE, Sanda T, Hanna M, Frohling S, Luther W 2nd, Zhang J, et al. Activating mutations in ALK provide a therapeutic target in neuroblastoma. Nature. 2008;455:975–8.

  20. 20.

    Sun J, Mohlin S, Lundby A, Kazi JU, Hellman U, Pahlman S, et al. The PI3-kinase isoform p110delta is essential for cell transformation induced by the D816V mutant of c-Kit in a lipid-kinase-independent manner. Oncogene. 2014;33:5360–9.

  21. 21.

    Honigberg LA, Smith AM, Sirisawad M, Verner E, Loury D, Chang B, et al. The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy. Proc Natl Acad Sci USA. 2010;107:13075–80.

  22. 22.

    Motegi A, Fujimoto J, Kotani M, Sakuraba H, Yamamoto T. ALK receptor tyrosine kinase promotes cell growth and neurite outgrowth. J Cell Sci. 2004;117:3319–29.

  23. 23.

    Mosse YP, Laudenslager M, Longo L, Cole KA, Wood A, Attiyeh EF, et al. Identification of ALK as a major familial neuroblastoma predisposition gene. Nature. 2008;455:930–5.

  24. 24.

    MacGurn JA, Hsu PC, Emr SD. Ubiquitin and membrane protein turnover: from cradle to grave. Annu Rev Biochem. 2012;81:231–59.

  25. 25.

    Buggy JJ, Elias L. Bruton tyrosine kinase (BTK) and its role in B-cell malignancy. Int Rev Immunol. 2012;31:119–32.

  26. 26.

    Mendoza MC, Er EE, Blenis J. The Ras-ERK and PI3K-mTOR pathways: cross-talk and compensation. Trends Biochem Sci. 2011;36:320–8.

  27. 27.

    Ding N, Li X, Shi Y, Ping L, Wu L, Fu K, et al. Irreversible dual inhibitory mode: the novel Btk inhibitor PLS-123 demonstrates promising anti-tumor activity in human B-cell lymphoma. Oncotarget. 2015;6:15122–36.

  28. 28.

    Herman SE, Mustafa RZ, Gyamfi JA, Pittaluga S, Chang S, Chang B, et al. Ibrutinib inhibits BCR and NF-kappaB signaling and reduces tumor proliferation in tissue-resident cells of patients with CLL. Blood. 2014;123:3286–95.

  29. 29.

    Braun FK, Mathur R, Sehgal L, Wilkie-Grantham R, Chandra J, Berkova Z, et al. Inhibition of methyltransferases accelerates degradation of cFLIP and sensitizes B-cell lymphoma cells to TRAIL-induced apoptosis. PLoS ONE. 2015;10:e0117994.

  30. 30.

    Metro G, Tazza M, Matocci R, Chiari R, Crino L. Optimal management of ALK-positive NSCLC progressing on crizotinib. Lung Cancer. 2017;106:58–66.

  31. 31.

    Sasaki T, Okuda K, Zheng W, Butrynski J, Capelletti M, Wang L, et al. The neuroblastoma-associated F1174L ALK mutation causes resistance to an ALK kinase inhibitor in ALK-translocated cancers. Cancer Res. 2010;70:10038–43.

  32. 32.

    Roskoski R Jr. Ibrutinib inhibition of Bruton protein-tyrosine kinase (BTK) in the treatment of B cell neoplasms. Pharmacol Res. 2016;113:395–408.

  33. 33.

    Holla VR, Elamin YY, Bailey AM, Johnson AM, Litzenburger BC, Khotskaya YB, et al. ALK: a tyrosine kinase target for cancer therapy. Cold Spring Harb Mol Case Stud. 2017;3:a001115.

  34. 34.

    Yau NK, Fong AY, Leung HF, Verhoeft KR, Lim QY, Lam WY, et al. A Pan-review of ALK mutations: Implications for carcinogenesis and therapy. Curr Cancer Drug Targets. 2015;15:327–36.

  35. 35.

    Kwak EL, Bang YJ, Camidge DR, Shaw AT, Solomon B, Maki RG, et al. Anaplastic lymphoma kinase inhibition in non-small-cell lung cancer. N Engl J Med. 2010;363:1693–703.

  36. 36.

    Smith CI. From identification of the BTK kinase to effective management of leukemia. Oncogene. 2016;36:2045–53.

  37. 37.

    Tucker DL, Rule SA. Ibrutinib for mantle cell lymphoma. Future Oncol. 2016;12:477–91.

  38. 38.

    Vela CM, McBride A, Jaglowski SM, Andritsos LA. Ibrutinib for treatment of chronic lymphocytic leukemia. Am J Health-Syst Pharm: AJHP: Off J Am Soc Health-Syst Pharm. 2016;73:367–75.

  39. 39.

    de Claro RA, McGinn KM, Verdun N, Lee SL, Chiu HJ, Saber H, et al. FDA approval: Ibrutinib for patients with previously treated mantle cell lymphoma and previously treated chronic lymphocytic leukemia. Clin Cancer Res. 2015;21:3586–90.

  40. 40.

    Souttou B, Carvalho NB, Raulais D, Vigny M. Activation of anaplastic lymphoma kinase receptor tyrosine kinase induces neuronal differentiation through the mitogen-activated protein kinase pathway. J Biol Chem. 2001;276:9526–31.

  41. 41.

    Wang C, Kam RK, Shi W, Xia Y, Chen X, Cao Y, et al. The proto-oncogene transcription factor Ets1 regulates neural crest development through histone deacetylase 1 to mediate output of bone morphogenetic protein signaling. J Biol Chem. 2015;290:21925–38.

  42. 42.

    Jin J, Smith FD, Stark C, Wells CD, Fawcett JP, Kulkarni S, et al. Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins involved in cytoskeletal regulation and cellular organization. Curr Biol: CB. 2004;14:1436–50.

  43. 43.

    Li Y, Bouchlaka MN, Wolff J, Grindle KM, Lu L, Qian S, et al. FBXO10 deficiency and BTK activation upregulate BCL2 expression in mantle cell lymphoma. Oncogene. 2016;35:6223–34.

Download references


We thank Dr. Marc Vigny for kindly providing the ALKWT and ALKF1174L constructs. This work is supported by the Research Grants Council of Hong Kong (CUHK24100414, CUHK14167017) to HZ, the grants from Guangdong Natural Science of Foundation (2017A030313209) and Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research (2017B030301018) and Shenzhen Key Laboratory of Cell Microenvironment (ZDSYS20140509142721429) to YD, the National Natural Science Foundation of China (81660473) and West China Top Class Discipline Project (NXYLXK2017B07) in Basic Medical Sciences of Ningxia Medical University to JS, One-off Funding for KIZ-CUHK Joint Lab/Research Collaboration from CUHK to WYC, the National Natural Science Foundation of China (31471367, 31671519) to YC, and TL is supported by the Graduate Studentships from CUHK. We thank colleagues in our laboratories for the helpful discussion.

Author information

Author notes

  1. These authors contributed equally: Tianfeng Li, Yi Deng.


  1. Key Laboratory for Regenerative Medicine, Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China

    • Tianfeng Li
    • , Bo Feng
    • , Sun On Chan
    • , Wai Yee Chan
    •  & Hui Zhao
  2. Department of Pathogen Biology and Immunology, School of Basic Medical Sciences, Ningxia Medical University, No. 1160 Shengli Street, Yinchuan, 750004, China

    • Jianmin Sun
  3. Department of Biology, Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, and Shenzhen Key Laboratory of Cell Microenvironment, Southern University of Science and Technology, Shenzhen, Guangdong, 51805, China

    • Yi Deng
    •  & Yonglong Chen
  4. Department of Clinical Laboratory, Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Pediatrics, Chongqing Key Laboratory of Translational Medical Research in Cognitive Development and Learning and Memory Disorders, Children’s Hospital of Chongqing Medical University, Chongqing, 400014, China

    • Yu Shi
  5. Department of Chemistry, Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, and Shenzhen Key Laboratory of Cell Microenvironment, South University of Science and Technology of China, Shenzhen, Guangdong, 518055, China

    • Ruijun Tian
  6. Center for Clinical Molecular Medicine, Children’s Hospital, Chongqing Medical University, Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing, 400014, China

    • Lin Zou
  7. Division of Translational Cancer Research, Department of Laboratory Medicine, Lund University, Lund, Sweden

    • Julhash U. Kazi
    • , Lars Rönnstrand
    •  & Jianmin Sun
  8. Kunming Institute of Zoology Chinese Academy of Sciences, The Chinese University of Hong Kong Joint Laboratory of Bioresources and Molecular Research of Common Diseases, Hong Kong SAR, China

    • Wai Yee Chan
    •  & Hui Zhao
  9. Lund Stem Cell Center, Department of Laboratory Medicine, Lund University, Lund, Sweden

    • Lars Rönnstrand
  10. Division of Oncology, Skåne University Hospital, Lund, Sweden

    • Lars Rönnstrand


  1. Search for Tianfeng Li in:

  2. Search for Yi Deng in:

  3. Search for Yu Shi in:

  4. Search for Ruijun Tian in:

  5. Search for Yonglong Chen in:

  6. Search for Lin Zou in:

  7. Search for Julhash U. Kazi in:

  8. Search for Lars Rönnstrand in:

  9. Search for Bo Feng in:

  10. Search for Sun On Chan in:

  11. Search for Wai Yee Chan in:

  12. Search for Jianmin Sun in:

  13. Search for Hui Zhao in:

Conflict of interest

The authors declare that they have no conflict of interest.

Corresponding authors

Correspondence to Jianmin Sun or Hui Zhao.

Electronic supplementary material

About this article

Publication history





Issue Date