Background. Our laboratory employs a functional genomic siRNA array platform to identify novel therapeutic targets. Using this approach we have interrogated the human kinome for novel targets for neuroblastoma (NB) therapeutics.
Goals. To identify novel kinases as future drug targets to be used as a combined therapy with Retinoic Acid in NB.
Methods. We employed high-throughput siRNA screening in three molecularly defined NB cell lines with different sensitivities to RA. Using a kinome-focused siRNA library, we identified 49 kinase encoding genes out of 713 genes which were selected for their statistical significance and for their biological relevance.
Results. We tested 40 genes across three cell lines +/-RA. Fourteen out of the 40 kinases were validated as sensitizers through multiple growth inhibition assays with independent siRNAs. In SKNAS, a MYCN non-amplified cell line, we identified kinases in the phosphatidylcholine biosynthesis and involved in neurite differentiation as well as others with important roles in non-homologous-end-joining repair of DNA damage pathway. In SKNBE2, a MYCN-amplified cell line, we also identified kinases involved in cell motility such as a bifunctional enzyme implicated in N-acetylmuramic acid biosynthesis, in differentiation such as tyrosine-kinase receptor involved in phosphatidylserine modulation and other kinases in the homologous recombination repair pathway. We confirmed that, for a total of fourteen genes, knock-down strongly decreased cell viability in combination with RA. Interestingly, several genes in common between the two cell lines were involved in the DNA damage repair pathway.
Conclusion. Our experiments identified novel therapeutic targets to use in combination with RA in high-risk NB. The detection of a concordance between different cell lines indicates DNA repair and differentiation-linked genes as promising pathways to be targeted for RA combinations. Furthermore, linkage of these kinase sensitivity with specific genomic features of NB will allow to tailor these combinations to individual patients.