Neuroblastomas demonstrate clinical heterogeneity, from spontaneous regression to relentless progression. Data from our laboratory and others suggests that Trk receptors play an important role in these disparate clinical behaviors (4
). TrkA is expressed in favorable tumors, and these tumors are likely to differentiate or regress, depending on the presence or absence of NGF in their microenvironment (9
). Conversely, aggressive tumors, especially those with MYCN
amplification, express TrkB and its ligand, BDNF (11
). The coexpression of TrkB and BDNF comprise an autocrine survival pathway that promotes invasion, metastasis, angiogenesis and drug resistance (17
). Therefore, targeted therapy aimed at inhibiting the Trk receptor pathways could be a useful adjunct to conventional therapy in these tumors.
Despite improvements in the overall cure rate of neuroblastomas, the progress in curing patients with high-risk disease (stage 4, over 18 months of age; or with MYCN amplification) has been modest. Therapy for these patients includes surgery, local radiation therapy, intensive chemotherapy and one or two autologous stem cell transplantations. These therapies have reached the maximum tolerated intensity, so improvements in the cure rates of these patients will likely require more biologically targeted therapy, and/or therapy that enhances the effectiveness of current therapy without substantially increasing side effects.
Given the important role that Trk receptors play in the behavior of favorable and unfavorable neuroblastomas, we wanted to develop an approach that targeted these receptors. CEP-751 (KT-6587) is a Trk-selective inhibitor provided by Cephalon, Inc. (38
). This compound inhibits Trk family kinases at nanomolar concentrations, whereas most other tyrosine kinases are only inhibited at micromolar concentrations. Lestaurtinib (CEP-701, KT-5555) is an active metabolite of CEP-751 that can be administered orally, making it more suitable for clinical trials (23
). Previously, we have tested the efficacy of CEP-751 to inhibit Trk-expressing neuroblastomas in vitro
and in vivo
). In this report we examined the efficacy of Lestaurtinib alone and in combination with conventional and biologically targeted therapies in a mouse xenograft model.
We showed that Lestaurtinib dramatically inhibited the autophosphorylation of TrkB (after BDNF exposure) in SY5Y-TrkB cells. Maximal inhibition was seen at concentrations of 100-200 nM (), which is well within the range of what is achievable clinically. These results are also comparable to those we obtained previously with CEP-751 (27
). Then, we tested the ability of Lestaurtinib to inhibit the growth of SY5Y-TrkB cells growing in vivo as xenografts in athymic nu/nu mice. The biological effect on phosphorylation of TrkB and signaling intermediates in the xenografts was modest, probably due to the low level of steady-state activation by the autocrine survival pathway. However, significant inhibition of tumor growth was seen with Lestaurtinib treatment compared to a vehicle control (, ), and this was achieved without apparent toxicity.
Next we tested the ability of Lestaurtinib to enhance the efficacy of conventional chemotherapy. We tested the ability of Cyclo and/or Lestaurtinib to inhibit the growth of SY5Y-TrkB xenografts. We demonstrated significantly greater inhibition of tumor growth in tumors treated with Lestaurtinib plus Cyclo compared to either alone (). We also tested the antitumor efficacy of Cyclo-Topo with or without Lestaurtinib. Topo-Cyclo has proven to be an effective combination for recurrent or refractory neuroblastomas (), and it is currently being used in front-line therapy for high-risk neuroblastoma patients. The combination of Topo-Cyclo plus Lestaurtinib inhibited tumor growth more effectively than either Topo-Cyclo or Lestaurtinib alone. Furthermore, we tested the combination of Irino-Temo with or without Lestaurtinib. Irino-Temo is an effective combination that is currently in use for high-risk neuroblastoma patients with recurrent or refractory disease. Again, we saw significant inhibition of tumor growth with the combination compared to chemotherapy alone (). Together these results demonstrate that Lestaurtinib enhanced the efficacy of chemotherapy agents in current clinical use, either alone or in pairwise combinations.
Finally, we tested the combination of Lestaurtinib with biologically targeted agents that are in use to treat neuroblastomas. 13-cRA did not have a significant effect on neuroblastoma xenografts in our model system, but 4-HPR did cause a significant inhibition in tumor growth. Nevertheless, Lestaurtinib was more effective at inhibiting tumor growth as a single agent than either 13-cRA or 4-HPR in this system, and the combination was no more effective than Lestaurtinib alone (). The results with Bevacizumab plus Lestaurtinib were more striking. Although both were effective at inhibiting tumor growth as single agents, the combination resulted in very significant growth inhibition. However, this combination was also associated with substantial systemic toxicity in the combination group, which raises questions about using this particular therapeutic combination in clinical trials without further testing.
Lestaurtinib can inhibit neuroblastoma xenograft growth, and it substantially enhances the efficacy of single and pair-wise combinations of chemotherapy agents, without additional toxicities. Lestaurtinib also enhanced the efficacy of Bevacizumab, but no enhancement was seen for the retinoids 13-cRA or 4-HPR. Assuming that the mechanism of Lestaurtinib inhibition is by blocking an important autocrine survival pathway, this agent may have a more profound effect on the growth of neuroblastomas treated with conventional chemotherapy because the pathways affected by 13-cRA or 4-HPR are different. The reason for substantial systemic toxicity with the combination of Lestaurtinib plus Bevacizumab compared to either agent alone is unclear but presumably is a result of a synergistic or off-target effect with the combination.
Brown and colleagues (40
) reported that Lestaurtinib combined synergistically with other agents in the treatment of childhood acute lymphoblastic leukemias containing MLL
gene rearrangements and FLT3 kinase overexpression. Furthermore, they reported that this synergism was sequence dependent, with the greatest effect seen when chemotherapy was given first, followed by Lestaurtinib. We saw significant inhibition when given simultaneously with chemotherapy (e.g., Cyclo), but there was no added benefit when CEP-751, an analog of Lestaurtinib, was given first, followed by Cyclo (data not shown). We did not test the schedule of chemotherapy followed by Lestaurtinib, but our protocol of continuous administration did continue the Lestaurtinib for weeks after the chemotherapy was given, essentially mimicking the most effective schedule identified by Brown (40
Lestaurtinib is currently in Phase 3 clinical trials to treat patients with FLT3–positive acute myelogenous leukemia, in combination with conventional induction therapy, and it is in Phase 2 clinical trials to treat patients with myeloproliferative diseases and myelofibrosis (26
). Furthermore, Lestaurtinib has been tested in a Phase I clinical trials in neuroblastoma patients. Our preclinical studies would suggest that Lestaurtinib will be particularly effective in combination with conventional chemotherapy agents, and may be effective when combined with selective biological agents. However, caution should be used in testing Lestaurtinib plus Bevacizumab in clinical trials without further preclinical studies of this combination.