Fred Hutchinson Cancer Research Center (FHCRC-1): Identification of drug targets for combination therapy with Retinoic Acid in Neuroblastoma
Retinoic Acid (RA) is employed in the clinic during the “consolidation” phase of treatment regimens for high-risk neuroblastoma. While the addition of RA has greatly increased the survival of children with neuroblastoma, there is still a high frequency of relapse. With the goal of identifying novel drug combinations that would enhance the effect of RA on neuroblastoma, an siRNA screen in the presence or absence of sub-lethal concentrations of RA was carried out. For this project, three neuroblastoma cell lines were selected based on their sensitivity to RA: 1) SY-5Y, a non-MYCN amplified line with high sensitivity to RA; 2) SK-N-BE2, intermediate sensitivity to RA; 3) SK-N-AS, a cell line insensitive to the effect of RA. The siRNA library was a kinase targeting library as described for the previous screen (0001_NB_kinome). The Hits from each cell line were determined based on their significance with respect to their differential activity in the presence or absence of RA within each cell line and also ranked on P-value, based the three replicates.
Cells and Retinoic Acid
SY-5Y, SK-N-BE2 and SK-N-AS were obtained from the American Tissue Type Collection (ATCC) and cultured in RPMI with 10% FBS and Penicillin and Streptomycin at the concentration recommended. 25mM HEPES was added during the screens to minimize variation of pH during handling of the plates. Retinoic Acid (RA) was used at 5μM, which is half the optimal concentration used in vitro to obtain differentiation of most NB cell lines, and which is comparable to half of the therapeutic concentration achieved in vivo.
High-throughput siRNA transfections
Transfection feasibility for each cell line was established using a factorial-based optimization scheme with variable parameters in cell density, liposomal, and siRNA concentration. All transfection conditions were statistically evaluated using a simple Z-factor score to determine differentials between mock-transfected cell (transfection reagent only) versus a positive control for growth inhibition (KIF11) and a negative control (non-targeting siRNA). Based on these preliminary experiments performed on each cell line, the optimal transfection condition was selected as the following for all the cell lines: cells were seeded at 500 cells/well; the next day 2.5 μl of Dharmafect I (6.25 μl/ml) and siRNA (1.25 pM/well) mix was added and viability with CellTiter-Glo was read at 96 hrs for SK-N-BE2 and SK-N-AS and 120 hr for SY-5Y cells, after plating. The siRNA library targeting the human kinome (~713 kinases) was tested in triplicates to establish experimental variability and statistical validity. A detailed description of the method is available in the supplementary information by Toyoshima et al.
A Kinome-wide siRNA library targeting 713 human (MISSION® siRNA Human Gene Family Set, Sigma) were tested with viability as the phenotypic endpoint. Cell viability was quantified using an Envision Multilabel detector/plate reader (PerkinElmer) with the CellTiter-Glo assay (Promega) that measures ATP concentrations in cell lysates. siRNA libraries utilized pools of 3 independent siRNAs targeting each gene, in a one gene per well approach. RNAi screens were performed in 384-well format in triplicate, in independent plates, utilizing robotics instrumentation available at the University of Washington - Quellos facility.
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