Abstract LB-126: The identification of synthetic lethal partners cooperating with MEK1/2 inhibition in neuroblastoma
Authors: Lori S. Hart, Pichai Raman, Grace Coggins, JulieAnn Rader, John M. Maris
Journal: Cancer Research (2016)

Abstract Background: Neuroblastoma, a pediatric malignancy of the developing sympathetic nervous system, is treated with aggressive multi-modal therapy, yet more than 50% of patients experience relapses for which there are limited treatment options. Improvements in the management of relapsed neuroblastoma require the identification of novel targeted drug combination strategies. While lesions in the canonical MAPK signaling pathway occur in only 3-5% of newly diagnosed neuroblastoma specimens, it is now clear that neuroblastoma genomes evolve extensively under the selective pressure of intensely cytotoxic therapy. We have recently shown that 78% of relapsed neuroblastomas harbor mutations predicted to hyperactivate RAS signaling, many of which were clonally enriched after chemoradiotherapy (Nat Genet, 2015). The inhibition of MEK1/2 in xenograft models of MAPK-hyperactive neuroblastoma results in significant tumor growth delay, but progressive disease ultimately prevails. We hypothesized that a protein kinase siRNA screen performed in conjunction with trametinib treatment would identify synthetic lethal partners for MEK1/2 inhibition, and therefore, novel combination treatments to pursue in relapsed neuroblastoma patients characterized by MAPK-hyperactivating mutations. Methods: Neuroblastoma cell lines (N = 4) were optimized for cell plating, siRNA transfection, and trametinib treatment in 96-well format with the endpoint readout of cell viability (CellTiter-Glo). We chose a human protein kinase siRNA library (ON-TARGETplus, Dharmacon) to knockdown more than 700 kinases. Each cell line was screened in duplicate under both vehicle and trametinib treatments. Luminescence readings were log transformed and normalized (min-max) by plate according to positive (siPLK1) and negative (non-targeting siRNA) cell death controls. Hits were defined as genes that cooperated with trametinib in each cell line and in duplicate experiments. Results: Initial screening of two cell lines (NB-EBc1, SK-N-SH) yielded a list of 6 genes (COL4A3BP, MAPK3, RPS6KA6, SRMS, STK23, and TYR03) identified as hits. Two of these genes, MAPK3 (ERK1) and RPS6KA6 (RSK4), code for proteins downstream of MEK1/2, suggesting they may contribute to resistance to trametinib by bypassing MEK1/2 and activating MAPK effectors. Conclusions: Initial results of a synthetic lethal screen with trametinib suggest that dual inhibition of the MAPK kinase pathway via MEK1/2 and downstream effectors, such as ERK/RSK may provide synergistic inhibition of neuroblastoma. We are currently completing the screen in two additional neuroblastoma cell lines (SK-N-BE(2)-C and NB-16) and validating the initial findings. We expect to identify synthetic lethal partners for MEK1/2 inhibition, and therefore, trametinib-resistance pathways to exploit with novel combination therapies in neuroblastoma. Citation Format: Lori S. Hart, Pichai Raman, Grace Coggins, JulieAnn Rader, John M. Maris. The identification of synthetic lethal partners cooperating with MEK1/2 inhibition in neuroblastoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-126.