Anne Berberich1,2, Lara-Marie Schmitt1,2, Stefan Pusch3,4, Thomas Hielscher5, Petra Rübmann1, Nanina Hucke1,2, Pauline Latzer1, Bernd Heßling6, Dieter Lemke1,2, Tobias Kessler1,2, Michael Platten2,7,8, Wolfgang Wick9,10. 1. Clinical Cooperation Unit Neurooncology, German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany. 2. Department of Neurology and Neurooncology Program, National Center for Tumor Diseases, Heidelberg University Hospital, Heidelberg, Germany. 3. Clinical Cooperation Unit Neuropathology, German Cancer Research Center (DKFZ), Heidelberg, Germany. 4. Department of Neuropathology, Heidelberg University Hospital, Heidelberg, Germany. 5. Division of Biostatistics, German Cancer Research Center (DKFZ), Heidelberg, Germany. 6. Genomics and Proteomics Core Facility, German Cancer Research Center (DKFZ), Heidelberg, Germany. 7. Clinical Cooperation Unit, Neuroimmunology and Brain Tumor Immunology, German Cancer Research Center (DKFZ), Heidelberg, Germany. 8. Department of Neurology, Medical Faculty Mannheim, Heidelberg University, Mannheim, Germany. 9. Clinical Cooperation Unit Neurooncology, German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany. wolfgang.wick@med.uni-heidelberg.de. 10. Department of Neurology and Neurooncology Program, National Center for Tumor Diseases, Heidelberg University Hospital, Heidelberg, Germany. wolfgang.wick@med.uni-heidelberg.de.
Abstract
BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.
BACKGROUND: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. METHODS: 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. RESULTS: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. CONCLUSION: 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.
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