Dual targeting of ALK and RET: establishing a novel basis for the treatment of neuroblastoma

Background: Neuroblastoma (NB) is a pediatric cancer of the developing sympathetic nervous system for which survival rates for high-risk patients are still unsatisfactory. Moreover, current treatment is very harsh and toxic, causing severe short and longterm side effects. Therefore, the search for more effective and less toxic targeted drugs remains at the forefront of NB research. Activating mutations in the tyrosine kinase domain of the ALK transmembrane receptor are found in the majority of hereditary NB and occur as somatic defects in 7U+201310% of sporadic cases. Recently we showed that ALK mutations emerge or are selected for in relapsed NB. Small molecule inhibitors are available for targeted therapy in mutant ALK positive NB patients and open new possibilities for understanding the functional pathways through which ALK exerts its oncogenicity. With the aim of selecting novel nodes for therapy intervention in NB, we have generated a 77-gene signature list reminiscent of mutant ALK activation in NB cells. Cross species genomics analysis of MYCN and ALKF1174 driven tumors and targeted ALK inhibition studies in vitro indicated that ALKF1174L leads to upregulation of the oncogenic tyrosine kinase RET in mouse models of NB. Inhibition of mutant ALK in NB cells results in robust downregulation of RET, indicating a strong functional relationship between these two oncogenes. We have further determined the efficacy of dual versus single targeting of mutant ALK and RET in NB cell lines in vitro and in vivo. Material and Methods: Single and combinatorial targeting of mutant ALK and RET was performed on a panel of wild type, ALKF1174L and ALKR1275Q NB cell lines. Cell proliferation and apoptosis were assessed using Cell Titer Glow and Caspase Glo assays (Promega). Targeting compounds were selected based on their clinical applicability. Crizotinib (ALK-inhibitor) was combined with vandetanib, cabozantinib or sorafenib (RET-inhibitors). The combination index method by Chou and Talalay was used to determine an additive or synergistic effect between the drug combinations. In vivo experiments were performed using xenografted ALKR1275Q NB cells, carrying luciferase and GFP as reporter genes (CLBGA/Luc-GFP). Tumor response to drug treatment was determined by daily caliper measurements and bioluminescence imagining. Results: Our preliminary results indicate that dual RET and ALK inhibition by crizotinib and vandetanib in vitro has a very strong anti-proliferative effect on cells compared to either drug alone. We have established the growth parameters and dose response curve to crizotinib of the xenografted NB cell line CLBGA/Luc-GFP in immunocompromised mice. We show that targeting ALKR1275Q in vivo results in initial regression of the tumor, followed by regrowth upon discontinuation of the treatment. Further experiments are ongoing to determine the activation status of RET in the resistant cells and to establish the appropriate experimental parameters for combinatorial ALK and RET inhibition in vivo. Conclusions: Single compound treated tumors typically relapse following an initial response as is also the case for mutant ALK cells, either through acquisition of novel ALK mutations or mutations in downstream effectors of the ALK or other interfering signaling pathways. While higher affinity ALK inhibitors are underway, targeting the RET signaling pathway emerges as an important option for novel combination therapy which may render higher efficacy to future treatment in primary and relapsed NB patients.