ABSTRACT
Mutations in the ALK tyrosine kinase receptor gene represent important therapeutic targets in neuroblastoma, yet their clinical translation has been challenging. The ALK(F1174L) mutation is sensitive to the ALK inhibitor crizotinib only at high doses and mediates acquired resistance to crizotinib in ALK-translocated cancers. We have shown that the combination of crizotinib and an inhibitor of downstream signaling induces a favorable response in transgenic mice bearing ALK(F1174L)/MYCN-positive neuroblastoma. Here, we investigated the molecular basis of this effect and assessed whether a similar strategy would be effective in ALK-mutated tumors lacking MYCN overexpression. We show that in ALK-mutated, MYCN-amplified neuroblastoma cells, crizotinib alone does not affect mTORC1 activity as indicated by persistent RPS6 phosphorylation. Combined treatment with crizotinib and an ATP-competitive mTOR inhibitor abrogated RPS6 phosphorylation, leading to reduced tumor growth and prolonged survival in ALK(F1174L)/MYCN-positive models compared to single agent treatment. By contrast, this combination, while inducing mTORC1 downregulation, caused reciprocal upregulation of PI3K activity in ALK-mutated cells expressing wild-type MYCN. Here, an inhibitor with potency against both mTOR and PI3K was more effective in promoting cytotoxicity when combined with crizotinib. Our findings should enable a more precise selection of molecularly targeted agents for patients with ALK-mutated tumors.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/pharmacology , Mutation , Neuroblastoma/drug therapy , Phosphoinositide-3 Kinase Inhibitors , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , TOR Serine-Threonine Kinases/antagonists & inhibitors , Anaplastic Lymphoma Kinase , Animals , Cell Line, Tumor , Crizotinib , Dose-Response Relationship, Drug , Drug Resistance, Neoplasm , Gene Amplification , Humans , Mechanistic Target of Rapamycin Complex 1 , Mice, Inbred NOD , Mice, SCID , Molecular Targeted Therapy , Multiprotein Complexes/antagonists & inhibitors , Multiprotein Complexes/metabolism , N-Myc Proto-Oncogene Protein , Neuroblastoma/enzymology , Neuroblastoma/genetics , Neuroblastoma/pathology , Nuclear Proteins/genetics , Oncogene Proteins/genetics , Phosphatidylinositol 3-Kinase/metabolism , Phosphorylation , Protein Kinase Inhibitors/administration & dosage , Proto-Oncogene Proteins c-akt/metabolism , Pyrazoles/administration & dosage , Pyridines/administration & dosage , RNA Interference , Receptor Protein-Tyrosine Kinases/genetics , Receptor Protein-Tyrosine Kinases/metabolism , Ribosomal Protein S6/metabolism , Signal Transduction/drug effects , TOR Serine-Threonine Kinases/metabolism , Time Factors , Transfection , Xenograft Model Antitumor AssaysABSTRACT
The ALK(F1174L) mutation is associated with intrinsic and acquired resistance to crizotinib and cosegregates with MYCN in neuroblastoma. In this study, we generated a mouse model overexpressing ALK(F1174L) in the neural crest. Compared to ALK(F1174L) and MYCN alone, co-expression of these two oncogenes led to the development of neuroblastomas with earlier onset, higher penetrance, and enhanced lethality. ALK(F1174L)/MYCN tumors exhibited increased MYCN dosage due to ALK(F1174L)-induced activation of the PI3K/AKT/mTOR and MAPK pathways, coupled with suppression of MYCN pro-apoptotic effects. Combined treatment with the ATP-competitive mTOR inhibitor Torin2 overcame the resistance of ALK(F1174L)/MYCN tumors to crizotinib. Our findings demonstrate a pathogenic role for ALK(F1174L) in neuroblastomas overexpressing MYCN and suggest a strategy for improving targeted therapy for ALK-positive neuroblastoma.
Subject(s)
Mutation , Neuroblastoma/genetics , Oncogenes , Proto-Oncogene Proteins/physiology , Receptor Protein-Tyrosine Kinases/genetics , Anaplastic Lymphoma Kinase , Animals , Disease Models, Animal , Mice , Mice, Transgenic , N-Myc Proto-Oncogene Protein , Neuroblastoma/pathology , Proto-Oncogene Proteins/genetics , RNA, Messenger/genetics , Signal TransductionABSTRACT
Butyrate is an inhibitor of histone deacetylase (HDAC) and has been extensively evaluated as a chemoprevention agent for colon cancer. We recently showed that mutations in the adenomatous polyposis coli (APC) gene confer resistance to HDAC inhibitor-induced apoptosis in colon cancers. Here, we show that APC mutation rendered colon cancer cells resistant to butyrate-induced apoptosis due to the failure of butyrate to down-regulate survivin in these cells. Another cancer-preventive agent, 3,3'-diindolylmethane (DIM), was identified to be able to down-regulate survivin in colon cancers expressing mutant APC. DIM inhibited survivin mRNA expression and promoted survivin protein degradation through inhibition of p34(cdc2)-cyclin B1-mediated survivin Thr(34) phosphorylation. Pretreatment with DIM enhanced butyrate-induced apoptosis in colon cancer cells expressing mutant APC. DIM/butyrate combination treatment induced the expression of proapoptotic Bax and Bak proteins, triggered Bax dimerization/activation, and caused release of cytochrome c and Smac proteins from mitochondria. Whereas overexpression of survivin blocked DIM/butyrate-induced apoptosis, knocking down of survivin by small interfering RNA increased butyrate-induced apoptosis in colon cancer cells. We further showed that DIM was able to down-regulate survivin and enhance the effects of butyrate in apoptosis induction and prevention of familial adenomatous polyposis in APC(min/+) mice. Thus, the combination of DIM and butyrate is potentially an effective strategy for the prevention of colon cancer.
