title: Combating autophagy is a strategy to increase cytotoxic effects of novel ALK inhibitor entrectinib in neuroblastoma cells.
abstract: Neuroblastoma (NB) is a threatening childhood malignancy. Its prognosis is affected by several morphological, and biological characteristics, including the constitutive expression of ALK tyrosine kinase. In this study we examined the therapeutic potential of a novel ALK inhibitor, entrectinib, in obliterating NB tumor cells. Entrectinib showed the growth-inhibitory effects on NB cells with a 50% inhibitory concentration range of 0.03-5 μM. In the ALK-dependent cells, entrectinib mediated G1-arrest, which was associated with modified expression of multiple cell-cycle regulators. Down-regulation of Ki-67, and attenuated phosphorylation of ERK1/2, and STAT3, correlated with observed antiproliferative capacity of entrectinib. Initial cytostatic activity of entrectinib was followed by concentration-dependent apoptotic cell death, and Caspase-3 activation. However, we delineated a reduced sensitivity of ALK mutated NB cells to entrectinib, and demonstrated strong activation of autophagy in SH-SY5YF1174L NB cell line. Abrogation of autophagy by chloroquine increased significantly the toxicity of entrectinib, as confirmed by enhanced death rate, and PARP protein cleavage in SH-SY5YF1174L cells. In aggregate, our data show that entrectinib inhibits proliferation, and induces G1-arrest, and apoptosis in NB cells. We propose entrectinib for further consideration in treatment of NB, and recommend pharmacological inhibition of autophagy to be explored for a combined therapeutic approach in NB patients that might develop resistance to entrectinib.
authors.affiliation: Neuroblastoma Laboratory, Pediatric Research Institute, Città della Speranza, Padua, Italy.
authors.forename: Sanja
authors.initials: S
authors.lastname: Aveic
authors.affiliation: Neuroblastoma Laboratory, Pediatric Research Institute, Città della Speranza, Padua, Italy.
authors.forename: Marcella
authors.initials: M
authors.lastname: Pantile
authors.affiliation: Neuroblastoma Laboratory, Pediatric Research Institute, Città della Speranza, Padua, Italy.
authors.forename: Anke
authors.initials: A
authors.lastname: Seydel
authors.affiliation: Neuroblastoma Laboratory, Pediatric Research Institute, Città della Speranza, Padua, Italy.
authors.forename: Maria Rosaria
authors.initials: MR
authors.lastname: Esposito
authors.affiliation: Neuroblastoma Laboratory, Pediatric Research Institute, Città della Speranza, Padua, Italy.
authors.forename: Carlo
authors.initials: C
authors.lastname: Zanon
authors.affiliation: Ignyta Inc., San Diego, California, USA.
authors.forename: Gary
authors.initials: G
authors.lastname: Li
authors.affiliation: Neuroblastoma Laboratory, Pediatric Research Institute, Città della Speranza, Padua, Italy.
authors.forename: Gian Paolo
authors.initials: GP
authors.lastname: Tonini
mesh_terms: D000077548:Anaplastic Lymphoma Kinase; D017209:Apoptosis / Q000187:drug effects*; D001343:Autophagy / Q000187:drug effects*; D001549:Benzamides / Q000494:pharmacology*; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D005455:Fluorescent Antibody Technique; D006801:Humans; D007124:Immunoenzyme Techniques; D007191:Indazoles / Q000494:pharmacology*; D009447:Neuroblastoma / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology*; D010766:Phosphorylation / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020794:Receptor Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D014407:Tumor Cells, Cultured; D014945:Wound Healing / Q000187:drug effects
chemical_list: D001549:Benzamides; D007191:Indazoles; D012333:RNA, Messenger; C000626173:ALK protein, human; D000077548:Anaplastic Lymphoma Kinase; D020794:Receptor Protein-Tyrosine Kinases; C000607349:entrectinib
keywords: ALK inhibitors; autophagy; drug combination; neuroblastoma