A Synthetic Lethality Druggable Genome RNAi Screen Identifies Genes Mediating Sensitivity To Lenalidomide In Multiple Myeloma Including RSK2

Immunomodulatory drugs (IMiDs) are widely used in the treatment of patients with Multiple Myeloma (MM) however, only 30% of relapsed MM patients respond to single agent therapy and most patients eventually develop drug resistance. The molecular target of IMiDs in MM is cereblon but other parallel pathways or downstream events which enhance or preclude drug responsiveness are unknown. We therefore conducted a genome scale small interfering RNA (siRNA) lethality study in MM in the presence of increasing concentrations of lenalidomide. Primary screening was performed in a single-siRNA-per-well format with the human druggable genome siRNA set V4 comprising four siRNAs targeting each of 6,992 genes (total 27968 siRNAs). Lenalidomide was added 24 hours post transfection and cell viability was measured by ATP-dependent luminescence at 144 hours after transfection. Primary screen data was rigorously evaluated for multiple quality control metrics and found to exceed all expected performance parameters with >98% global transfection efficiency, <0.25 CV values, and minimal plate-to-plate and set-to-set variations observed. Hit selection was performed by analysis of IC₅₀ value shift in the presence of each testing siRNA compared with three different control siRNA oligos. 160 candidate genes that enhance lenalidomide sensitivity upon silencing (sensitizers) were selected and re-screened with four siRNA oligos targeting each gene. 50 genes were identified as reproducible lenalidomide sensitizers including three Peroxisome (PEX) family proteins (PEX1, PEX10 and PEX7) and seven RAB family proteins (RAB17, RAB1A, RAB26, RAB30, RAB36, RAB4A and RAB8A). Four kinase genes were also identified in sensitizer hits and two of these, I-Kappa-B Kinase-Alpha (IKK1 or CHUK) and ribosomal protein S6 kinase (RPS6KA3 or RSK2), encode proteins that associate with significance together with a phosphorylation dependent transcription factor (CREB1) in Toll signaling pathways (p-value 0.0068). RSK2 is a serine/threonine-protein kinase that acts downstream of oncogenic FGFR3 mediated signaling and is phosphorylated by ERK (MAPK1/ERK2 and MAPK3/ERK1 signaling) during hematopoietic transformation. Phosphorylated RSK2 was previously reported to be frequently expressed in myeloma cell lines and primary myeloma cells. Using lentiviral shRNA expression, we demonstrated that knockdown of RSK2 in three genetically variable MM cell lines induced cyctocytoxiticy and consistently sensitized to lenalidomide. Two selective small molecular inhibitors of RSK2 (SL 0101-1 and BI-D1870) were then demonstrated to synergize with lenalidomide to induce myeloma cell cytotoxicixity. To further understand the mechanism underlying sensitization, immunoblotting analysis was performed to look at downstream changes after either RSK2 knockdown or RSK2 inhibition by BI-D1870. We found that both RSK2 knockdown and BI-D1870 treatment, mimicking lenalidomide treatment or cereblon inhibition, induced downregulation of both IRF4 and MYC in MM cells. The combination of lenalidomide and BI-D1870 not only produced a substantial synergistic effect inducing MM cytotoxicity, but also demonstrated a significant enhancement of downregulation of IRF4 and MYC. Forced overexpression of RSK2 attenuated the synergistic effects of lenalidomide and BI-D1870. In summary, our high throughput screen identified multiple gene targets that associate with increasing sensitivity to IMiDs in MM cells, of which, RSK2 was further validated by both shRNA silencing and specific inhibitors as an effective target to cooperate with IMiDs to induce myeloma cytotoxicity. Clinical studies of RSK2 inhibition in concert with IMiD (cereblon inhibitor) therapy would be appropriate.