CEP-2563

All of the cases of medullary thyroid carcinoma (MTC) express the RET receptor tyrosine kinase. In essentially all of the hereditary cases and approximately 40% of the sporadic cases of MTC, the RET kinase is constitutively activated by mutation. This suggests that RET may be an effective therapeutic target for treatment of MTC. We show that the indolocarbazole derivatives, CEP-701 and CEP-751, inhibit RET in MTC cells. These compounds effectively inhibit RET phosphorylation in a dose-dependent manner at concentrations <100 nM in 0.5% serum and at somewhat higher concentrations in the presence of 16% serum. They also blocked the growth of these MTC cells in culture. CEP-751 and its prodrug, CEP-2563, also inhibited tumor growth in MTC cell xenografts. These results show that inhibiting RET can block the growth of MTC cells and may have a therapeutic benefit in MTC.

Neuroblastoma is a common childhood tumor derived from the peripheral nervous system. Favorable neuroblastomas usually express TrkA, the receptor for nerve growth factor (NGF), whereas unfavorable, MYCN-amplified neuroblastomas usually express TrkB and its ligand, brain-derived neurotrophic factor (BDNF). Here, we provide evidence that the TrkB-BDNF pathway is associated with enhanced survival and resistance to chemotherapy in neuroblastoma. We transfected the neuroblastoma line SH-SY5Y, which has endogenous expression of BDNF, with a full-length TrkB expression vector, and obtained clones with moderate or high levels of expression. Cells were exposed in vitro to chemotherapy agents used to treat neuroblastomas: doxorubicin, etoposide (VP16), and cisplatin. Chemoresistance was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for cell survival and by ELISA for cell death. In all cases, the TrkB-expressing subclones were more resistant to treatment than the parent line. Furthermore, when the TrkB tyrosine kinase was blocked with the Trk-specific inhibitor CEP-2563, or by neutralizing antibody to BDNF, sensitivity to chemotherapy was significantly increased. We also found constitutive phosphorylation of AKT at the Ser-473 site in TrkB transfectants, whereas there was only a minimal level of constitutive phosphorylation of AKT in SY5Y cells. These results show that the TrkB-BDNF pathway provides a survival advantage when exposed to DNA-damaging reagents, and, therefore, this autocrine pathway may play an important role in mediating the drug-resistant phenotype associated with TrkB-expressing neuroblastomas. Activation of PI3K/AKT survival pathway may contribute to the increased drug resistance in TrkB-expressing neuroblastomas.