Effect of CEP-751 (KT-6587) on neuroblastoma xenografts expressing TrkB.
BACKGROUND
The compound CEP-751 (KT-6587), a potent and selective inhibitor of the Trk family of tyrosine kinases, has been shown to inhibit the growth of human neuroblastoma (NB) xenografts in nude mice [1].


METHODS
To address its mechanism of action, we studied SY5Y, a human NB cell line with no detectable Trk expression, and two subclones transfected with TrkB. The transfected clones, SY5Y (G8) and SY5Y (G12), expressed moderate and high levels, respectively, of TrkB mRNA and protein. These TrkB-expressing subclones and the parental line were then grown as xenografts in nude mice, and CEP-751 was used to inhibit TrkB tyrosine kinase activity in these xenografts. Animals were treated twice a day with CEP-751 (21 mg/kg), or with the carrier vehicle as a control. TrkB expression in the resultant tumors was examined by quantitative RT-PCR. The effect of CEP-751 on TrkB activation by BDNF was examined in G12 cells in culture by immunoprecipitation with antipan Trk antiserum, followed by Western blot analysis using antiphosphotyrosine antibodies. To determine if CEP-751 was causing apoptosis, the TUNEL assay was used.


RESULTS
CEP-751 had little effect on the growth of SY5Y tumors, but did slow the growth rate of the C8 and G12 tumors. The daily growth rate of the treated tumors was 0.16, 0.13, and 0.10 cm3, respectively, for the SY5Y, G8, and G12 tumors. RT PCR analysis confirmed the expression of TrkB in G8 and G12, but not in SY5Y tumors. Activation of TrkB by BDNF in G12 cells was inhibited by CEP-751 in a dose dependent fashion. The treated tumors showed marked evidence of apoptosis.


CONCLUSIONS
These data suggest that the effect of CEP-751 is due, at least in part, to its inhibition of TrkB kinase, and that CEP-751 may become a useful therapeutic tool for the treatment of aggressive neuroblastomas, which often express TrkB.
Evans
A E
AE
Division of Oncology, Children's Hospital of Philadelphia, Pennsylvania 19104, USA. evansa@email.chop.edu
Kisselbach
K D
KD
Liu
X
X
Eggert
A
A
Ikegaki
N
N
Camoratto
A M
AM
Dionne
C
C
Brodeur
G M
GM
D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use*; D017209:Apoptosis / Q000187:drug effects; D019208:Brain-Derived Neurotrophic Factor / Q000494:pharmacology; D002227:Carbazoles / Q000627:therapeutic use*; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000627:therapeutic use*; D005260:Female; D006801:Humans; D020287:In Situ Nick-End Labeling; D051379:Mice; D008819:Mice, Nude; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics; D009447:Neuroblastoma / Q000188:drug therapy* / Q000201:enzymology; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D020813:Receptor, trkB / Q000037:antagonists & inhibitors* / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000637:transplantation; D023041:Xenograft Model Antitumor Assays
D000970:Antineoplastic Agents; D019208:Brain-Derived Neurotrophic Factor; C107558:CEP 751; D002227:Carbazoles; D004791:Enzyme Inhibitors; D009363:Neoplasm Proteins; D020813:Receptor, trkB