cMyc and ERK activity are associated with resistance to ALK inhibitory treatment in glioblastoma

  • Anne Berberich
  • Lara-Marie Schmitt
  • Stefan Pusch
  • Thomas Hielscher
  • Petra Rübmann
  • Nanina Hucke
  • Pauline Latzer
  • Bernd Heßling
  • Dieter Lemke
  • Tobias Kessler
  • Michael Platten
  • Wolfgang WickEmail author
Laboratory Investigation



Anaplastic lymphoma kinase (ALK) is expressed in ~ 60% of glioblastomas and conveys tumorigenic functions. Therefore, ALK inhibitory strategies with alectinib are conceivable for patients with glioblastoma. The aims of this preclinical study were to investigate efficacy as well as to understand and potentially overcome primary and acquired resistance mechanisms of alectinib in glioblastoma.


Efficacy of alectinib was analyzed dependent on ALK expression in different glioblastoma initiating cells and after lentiviral knockdown of ALK. Alectinib resistant cells were generated by continuous treatment with increasing alectinib doses over 3 months. M-RNA, phospho-protein and protein regulation were analyzed to decipher relevant pathways associated to treatment or resistance and specifically inhibited to evaluate rational salvage therapies.


Alectinib reduced clonogenicity and proliferation and induced apoptosis in ALK expressing glioblastoma initiating cells, whereas cells without ALK expression or after ALK depletion via knockdown showed primary resistance against alectinib. High expression of cMyc and activation of the ERK1/2 pathway conferred resistance against alectinib in ALK expressing glioblastoma cells. Pharmacological inhibition of these pathways by cMyc inhibitor or MEK inhibitor, trametinib, overcame alectinib resistance and re-sensitized resistant cells to continued alectinib treatment. The combination of alectinib with radiotherapy demonstrated synergistic effects in inhibition of clonogenicity in non-resistant and alectinib resistant glioblastoma cells.


The data offer rationales for alectinib treatment in ALK expressing glioblastoma and for the use of ALK expression status as potential biomarker for alectinib treatment. In addition, the results propose MEK inhibition or radiotherapy as reasonable salvage treatments after acquired alectinib resistance.


Glioblastoma Anaplastic lymphoma kinase Alectinib Biomarker Treatment resistance 



L.S. was funded by the German Cancer Aid while performing this study (Funding No. 70112464). N2M2 is funded by the German Cancer Aid (7011980) and the resistance studies by DFG SFB 1389 TP A03 to W.W. and T.K.

Compliance with ethical standards

Conflicts of interest

The authors declare no potential conflicts of interest.

Supplementary material

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  1. 1.
    Kinoshita K, Asoh K, Furuichi N, Ito T, Kawada H, Hara S, Ohwada J, Miyagi T, Kobayashi T, Takanashi K, Tsukaguchi T, Sakamoto H, Tsukuda T, Oikawa N (2012) Design and synthesis of a highly selective, orally active and potent anaplastic lymphoma kinase inhibitor (CH5424802). Bioorg Med Chem 20(3):1271–1280. CrossRefPubMedGoogle Scholar
  2. 2.
    Peters S, Camidge DR, Shaw AT, Gadgeel S, Ahn JS, Kim DW, Ou SI, Perol M, Dziadziuszko R, Rosell R, Zeaiter A, Mitry E, Golding S, Balas B, Noe J, Morcos PN, Mok T, Investigators AT (2017) Alectinib versus crizotinib in untreated ALK-positive non-small-cell lung cancer. N Engl J Med 377(9):829–838. CrossRefPubMedGoogle Scholar
  3. 3.
    Hida T, Nokihara H, Kondo M, Kim YH, Azuma K, Seto T, Takiguchi Y, Nishio M, Yoshioka H, Imamura F, Hotta K, Watanabe S, Goto K, Satouchi M, Kozuki T, Shukuya T, Nakagawa K, Mitsudomi T, Yamamoto N, Asakawa T, Asabe R, Tanaka T, Tamura T (2017) Alectinib versus crizotinib in patients with ALK-positive non-small-cell lung cancer (J-ALEX): an open-label, randomised phase 3 trial. Lancet 390(10089):29–39. CrossRefPubMedGoogle Scholar
  4. 4.
    Gadgeel S, Peters S, Mok T, Shaw AT, Kim DW, Ou SI, Perol M, Wrona A, Novello S, Rosell R, Zeaiter A, Liu T, Nuesch E, Balas B, Camidge DR (2018) Alectinib versus crizotinib in treatment-naive anaplastic lymphoma kinase-positive (ALK+) non-small-cell lung cancer: CNS efficacy results from the ALEX study. Ann Oncol. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Gourd E (2018) Alectinib shows CNS efficacy in ALK-positive NSCLC. Lancet Oncol 19(10):e520. CrossRefPubMedGoogle Scholar
  6. 6.
    Gainor JF, Sherman CA, Willoughby K, Logan J, Kennedy E, Brastianos PK, Chi AS, Shaw AT (2015) Alectinib salvages CNS relapses in ALK-positive lung cancer patients previously treated with crizotinib and ceritinib. J Thorac Oncol 10(2):232–236. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, Fujiwara S, Watanabe H, Kurashina K, Hatanaka H, Bando M, Ohno S, Ishikawa Y, Aburatani H, Niki T, Sohara Y, Sugiyama Y, Mano H (2007) Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 448(7153):561–566. CrossRefPubMedGoogle Scholar
  8. 8.
    Morris SW, Kirstein MN, Valentine MB, Dittmer K, Shapiro DN, Look AT, Saltman DL (1995) Fusion of a kinase gene, ALK, to a nucleolar protein gene, NPM, in non-Hodgkin's lymphoma. Science 267(5196):316–317CrossRefGoogle Scholar
  9. 9.
    Chen Y, Takita J, Choi YL, Kato M, Ohira M, Sanada M, Wang L, Soda M, Kikuchi A, Igarashi T, Nakagawara A, Hayashi Y, Mano H, Ogawa S (2008) Oncogenic mutations of ALK kinase in neuroblastoma. Nature 455(7215):971–974. CrossRefPubMedGoogle Scholar
  10. 10.
    George RE, Sanda T, Hanna M, Frohling S, Luther W 2nd, Zhang J, Ahn Y, Zhou W, London WB, McGrady P, Xue L, Zozulya S, Gregor VE, Webb TR, Gray NS, Gilliland DG, Diller L, Greulich H, Morris SW, Meyerson M, Look AT (2008) Activating mutations in ALK provide a therapeutic target in neuroblastoma. Nature 455(7215):975–978. CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Osajima-Hakomori Y, Miyake I, Ohira M, Nakagawara A, Nakagawa A, Sakai R (2005) Biological role of anaplastic lymphoma kinase in neuroblastoma. Am J Pathol 167(1):213–222. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Hallberg B, Palmer RH (2013) Mechanistic insight into ALK receptor tyrosine kinase in human cancer biology. Nat Rev Cancer 13(10):685–700. CrossRefPubMedGoogle Scholar
  13. 13.
    Pulford K, Morris SW, Turturro F (2004) Anaplastic lymphoma kinase proteins in growth control and cancer. J Cell Physiol 199(3):330–358. CrossRefPubMedGoogle Scholar
  14. 14.
    Parsons DW, Jones S, Zhang X, Lin JC, Leary RJ, Angenendt P, Mankoo P, Carter H, Siu IM, Gallia GL, Olivi A, McLendon R, Rasheed BA, Keir S, Nikolskaya T, Nikolsky Y, Busam DA, Tekleab H, Diaz LA Jr, Hartigan J, Smith DR, Strausberg RL, Marie SK, Shinjo SM, Yan H, Riggins GJ, Bigner DD, Karchin R, Papadopoulos N, Parmigiani G, Vogelstein B, Velculescu VE, Kinzler KW (2008) An integrated genomic analysis of human glioblastoma multiforme. Science 321(5897):1807–1812. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Cancer Genome Atlas Research N (2008) Comprehensive genomic characterization defines human glioblastoma genes and core pathways. Nature 455(7216):1061–1068. CrossRefGoogle Scholar
  16. 16.
    Karagkounis G, Stranjalis G, Argyrakos T, Pantelaion V, Mastoris K, Rontogianni D, Komaitis S, Kalamatianos T, Sakas D, Tiniakos D (2017) Anaplastic lymphoma kinase expression and gene alterations in glioblastoma: correlations with clinical outcome. J Clin Pathol 70(7):593–599. CrossRefPubMedGoogle Scholar
  17. 17.
    Ferguson SD, Xiu J, Weathers SP, Zhou S, Kesari S, Weiss SE, Verhaak RG, Hohl RJ, Barger GR, Reddy SK, Heimberger AB (2016) GBM-associated mutations and altered protein expression are more common in young patients. Oncotarget 7(43):69466–69478. CrossRefPubMedGoogle Scholar
  18. 18.
    Wellstein A (2012) ALK receptor activation, ligands and therapeutic targeting in glioblastoma and in other cancers. Front Oncol 2:192. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Chiba R, Akiya M, Hashimura M, Oguri Y, Inukai M, Hara A, Saegusa M (2017) ALK signaling cascade confers multiple advantages to glioblastoma cells through neovascularization and cell proliferation. PLoS ONE 12(8):e0183516. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Stylianou DC, Auf der Maur A, Kodack DP, Henke RT, Hohn S, Toretsky JA, Riegel AT, Wellstein A (2009) Effect of single-chain antibody targeting of the ligand-binding domain in the anaplastic lymphoma kinase receptor. Oncogene 28(37):3296–3306. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Powers C, Aigner A, Stoica GE, McDonnell K, Wellstein A (2002) Pleiotrophin signaling through anaplastic lymphoma kinase is rate-limiting for glioblastoma growth. J Biol Chem 277(16):14153–14158. CrossRefPubMedGoogle Scholar
  22. 22.
    Grzelinski M, Steinberg F, Martens T, Czubayko F, Lamszus K, Aigner A (2009) Enhanced antitumorigenic effects in glioblastoma on double targeting of pleiotrophin and its receptor ALK. Neoplasia 11(2):145–156CrossRefGoogle Scholar
  23. 23.
    Koyama-Nasu R, Haruta R, Nasu-Nishimura Y, Taniue K, Katou Y, Shirahige K, Todo T, Ino Y, Mukasa A, Saito N, Matsui M, Takahashi R, Hoshino-Okubo A, Sugano H, Manabe E, Funato K, Akiyama T (2014) The pleiotrophin-ALK axis is required for tumorigenicity of glioblastoma stem cells. Oncogene 33(17):2236–2244. CrossRefPubMedGoogle Scholar
  24. 24.
    Le Rhun E, Chamberlain MC, Zairi F, Delmaire C, Idbaih A, Renaud F, Maurage CA, Gregoire V (2015) Patterns of response to crizotinib in recurrent glioblastoma according to ALK and MET molecular profile in two patients. CNS Oncol 4(6):381–386. CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Wick W, Dettmer S, Berberich A, Kessler T, Karapanagiotou-Schenkel I, Wick A, Winkler F, Pfaff E, Brors B, Debus J, Unterberg A, Bendszus M, Herold-Mende C, Eisenmenger A, von Deimling A, Jones DTW, Pfister SM, Sahm F, Platten M (2018) N2M2 (NOA20) phase I/II trial of molecularly matched targeted therapies plus radiotherapy in patients with newly diagnosed non-MGMT hypermethylated glioblastoma. Neuro Oncol. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Pfaff E, Kessler T, Balasubramanian GP, Berberich A, Schrimpf D, Wick A, Debus J, Unterberg A, Bendszus M, Herold-Mende C, Capper D, Schenkel I, Eisenmenger A, Dettmer S, Brors B, Platten M, Pfister SM, von Deimling A, Jones DTW, Wick W, Sahm F (2018) Feasibility of real-time molecular profiling for patients with newly diagnosed glioblastoma without MGMT promoter hypermethylation-the NCT Neuro Master Match (N2M2) pilot study. Neuro Oncol 20(6):826–837. CrossRefPubMedGoogle Scholar
  27. 27.
    Lemke D, Weiler M, Blaes J, Wiestler B, Jestaedt L, Klein AC, Low S, Eisele G, Radlwimmer B, Capper D, Schmieder K, Mittelbronn M, Combs SE, Bendszus M, Weller M, Platten M, Wick W (2014) Primary glioblastoma cultures: can profiling of stem cell markers predict radiotherapy sensitivity? J Neurochem 131(2):251–264. CrossRefPubMedGoogle Scholar
  28. 28.
    Campeau E, Ruhl VE, Rodier F, Smith CL, Rahmberg BL, Fuss JO, Campisi J, Yaswen P, Cooper PK, Kaufman PD (2009) A versatile viral system for expression and depletion of proteins in mammalian cells. PLoS ONE 4(8):e6529. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Berberich A, Kessler T, Thome CM, Pusch S, Hielscher T, Sahm F, Oezen I, Schmitt LM, Ciprut S, Hucke N, Rubmann P, Fischer M, Lemke D, Breckwoldt MO, von Deimling A, Bendszus M, Platten M, Wick W (2018) Targeting resistance against the MDM2 inhibitor RG7388 in glioblastoma cells by the MEK inhibitor trametinib. Clin Cancer Res. CrossRefPubMedGoogle Scholar
  30. 30.
    Hu Y, Smyth GK (2009) ELDA: extreme limiting dilution analysis for comparing depleted and enriched populations in stem cell and other assays. J Immunol Methods 347(1–2):70–78. CrossRefPubMedGoogle Scholar
  31. 31.
    Hertenstein A, Schumacher T, Litzenburger U, Opitz CA, Falk CS, Serafini T, Wick W, Platten M (2011) Suppression of human CD4+ T cell activation by 3,4-dimethoxycinnamonyl-anthranilic acid (tranilast) is mediated by CXCL9 and CXCL10. Biochem Pharmacol 82(6):632–641. CrossRefPubMedGoogle Scholar
  32. 32.
    Bliss CI (1939) The toxicity of poisons applied jointly. Ann Appl Biol 26:585–615CrossRefGoogle Scholar
  33. 33.
    Gainor JF, Dardaei L, Yoda S, Friboulet L, Leshchiner I, Katayama R, Dagogo-Jack I, Gadgeel S, Schultz K, Singh M, Chin E, Parks M, Lee D, DiCecca RH, Lockerman E, Huynh T, Logan J, Ritterhouse LL, Le LP, Muniappan A, Digumarthy S, Channick C, Keyes C, Getz G, Dias-Santagata D, Heist RS, Lennerz J, Sequist LV, Benes CH, Iafrate AJ, Mino-Kenudson M, Engelman JA, Shaw AT (2016) Molecular mechanisms of resistance to first- and second-generation ALK inhibitors in ALK-rearranged lung cancer. Cancer Discov 6(10):1118–1133. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Golding B, Luu A, Jones R, Viloria-Petit AM (2018) The function and therapeutic targeting of anaplastic lymphoma kinase (ALK) in non-small cell lung cancer (NSCLC). Mol Cancer 17(1):52. CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Gadgeel SM, Gandhi L, Riely GJ, Chiappori AA, West HL, Azada MC, Morcos PN, Lee RM, Garcia L, Yu L, Boisserie F, Di Laurenzio L, Golding S, Sato J, Yokoyama S, Tanaka T, Ou SH (2014) Safety and activity of alectinib against systemic disease and brain metastases in patients with crizotinib-resistant ALK-rearranged non-small-cell lung cancer (AF-002JG): results from the dose-finding portion of a phase 1/2 study. Lancet Oncol 15(10):1119–1128. CrossRefPubMedGoogle Scholar
  36. 36.
    Kodama T, Hasegawa M, Takanashi K, Sakurai Y, Kondoh O, Sakamoto H (2014) Antitumor activity of the selective ALK inhibitor alectinib in models of intracranial metastases. Cancer Chemother Pharmacol 74(5):1023–1028. CrossRefPubMedGoogle Scholar
  37. 37.
    Iwahara T, Fujimoto J, Wen D, Cupples R, Bucay N, Arakawa T, Mori S, Ratzkin B, Yamamoto T (1997) Molecular characterization of ALK, a receptor tyrosine kinase expressed specifically in the nervous system. Oncogene 14(4):439–449. CrossRefPubMedGoogle Scholar
  38. 38.
    Rihawi K, Alfieri R, Fiorentino M, Fontana F, Capizzi E, Cavazzoni A, Terracciano M, La Monica S, Ferrarini A, Buson G, Petronini PG, Ardizzoni A (2018) MYC amplification as a potential mechanism of primary resistance to crizotinib in ALK-rearranged non-small cell lung cancer: a brief report. Transl Oncol 12(1):116–121. CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Pilling AB, Kim J, Estrada-Bernal A, Zhou Q, Le AT, Singleton KR, Heasley LE, Tan AC, DeGregori J, Doebele RC (2018) ALK is a critical regulator of the MYC-signaling axis in ALK positive lung cancer. Oncotarget 9(10):8823–8835. CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Chiarle R, Voena C, Ambrogio C, Piva R, Inghirami G (2008) The anaplastic lymphoma kinase in the pathogenesis of cancer. Nat Rev Cancer 8(1):11–23. CrossRefPubMedGoogle Scholar
  41. 41.
    Shaw AT, Friboulet L, Leshchiner I, Gainor JF, Bergqvist S, Brooun A, Burke BJ, Deng YL, Liu W, Dardaei L, Frias RL, Schultz KR, Logan J, James LP, Smeal T, Timofeevski S, Katayama R, Iafrate AJ, Le L, McTigue M, Getz G, Johnson TW, Engelman JA (2016) Resensitization to crizotinib by the lorlatinib ALK resistance mutation L1198F. N Engl J Med 374(1):54–61. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Anne Berberich
    • 1
    • 2
  • Lara-Marie Schmitt
    • 1
    • 2
  • Stefan Pusch
    • 3
    • 4
  • Thomas Hielscher
    • 5
  • Petra Rübmann
    • 1
  • Nanina Hucke
    • 1
    • 2
  • Pauline Latzer
    • 1
  • Bernd Heßling
    • 6
  • Dieter Lemke
    • 1
    • 2
  • Tobias Kessler
    • 1
    • 2
  • Michael Platten
    • 2
    • 7
    • 8
  • Wolfgang Wick
    • 1
    • 2
    Email author
  1. 1.Clinical Cooperation Unit Neurooncology, German Cancer Consortium (DKTK)German Cancer Research Center (DKFZ)HeidelbergGermany
  2. 2.Department of Neurology and Neurooncology Program, National Center for Tumor DiseasesHeidelberg University HospitalHeidelbergGermany
  3. 3.Clinical Cooperation Unit NeuropathologyGerman Cancer Research Center (DKFZ)HeidelbergGermany
  4. 4.Department of NeuropathologyHeidelberg University HospitalHeidelbergGermany
  5. 5.Division of BiostatisticsGerman Cancer Research Center (DKFZ)HeidelbergGermany
  6. 6.Genomics and Proteomics Core FacilityGerman Cancer Research Center (DKFZ)HeidelbergGermany
  7. 7.Clinical Cooperation Unit, Neuroimmunology and Brain Tumor ImmunologyGerman Cancer Research Center (DKFZ)HeidelbergGermany
  8. 8.Department of Neurology, Medical Faculty MannheimHeidelberg UniversityMannheimGermany

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