PF581 FUNCTIONAL HIGH‐THROUGHPUT SCREENING FOR IDENTIFICATION OF NOTCH1 DOWNSTREAM EFFECTORS AS NOVEL THERAPEUTIC TARGETS IN MULTIPLE MYELOMA

Abstract

Background:Intrinsic and acquired drug resistance of multiple myeloma (MM) cells often cause disease progression in MM patients. Therefore, the identification of novel drug targets is needed to further improve treatment strategies. The deregulated Notch1 pathway is a master regulator of MM onset, progression and drug resistance. Inhibition of Notch through use of γ‐secretase inhibitors causes serious side effects and is not used in the clinic for treatment of MM patients.Aims:In this study, we identify novel druggable downstream effectors of the Notch1 pathway to overcome drug resistance of MM cells.Methods:We employ high‐throughput (HT) functional screening based on tailored Notch1 shRNA libraries under selective pressure of chemotherapeutic agents. First, we performed genome‐wide expression profiling through RNA sequencing (RNAseq) in Notch‐positive MM.1S cells after inhibition of the pathway. Strongly and consistently regulated genes (log2FC; padj <0.05) were chosen as targets for two tailored shRNA libraries. Functional screens are conducted using native U266 cells (positive selection screens) or U266 cells engineered to express the Notch1 variants NIC or NΔE (negative selection screens). NIC is the active and γ‐secretase‐independent form of Notch1. By using the membrane‐bound and γ‐secretase‐dependent NΔE variant, screening additionally comprises investigation of cooperation between Notch1 and cytoplasmic signaling complexes. Following transduction of U266 cells with the corresponding shRNA library, cells are cultured under selective pressure of Melphalan, Bortezomib or Lenalidomide. HT sequencing and bioinformatics analysis of changes in the representation of shRNAs will indicate critical Notch1 effectors. For in vitro validation of candidate genes, MM cell lines will be treated with target gene‐specific shRNAs or chemical inhibitors in combination with chemotherapeutic agents. Analyses of proliferation, cell cycle progression and apoptosis will reveal candidate genes, which contribute to growth and survival advantages. Most promising genes will be further tested in vivo using the MM mouse models BALB/c‐MOPC315.BM and VK∗MYC.Results:RNAseq analysis revealed 842 Notch1‐regulated genes. 40 downregulated and 28 upregulated genes were chosen as targets for the shRNA libraries. Specificity of the RNAseq approach was verified by quantitative PCR for known Notch1 target genes such as HEY1 and HES6. Interestingly, the group of Notch1‐upregulated genes further includes bone‐related genes supporting MM growth and the aggressive MM bone phenotype such as RUNX2, osteocalcin or cathepsin K (Trotter et. al., Blood, 2015).Expression of NIC and NΔE in engineered U266 cells was validated by flow cytometric and western blot analysis. Cell viability analysis by Annexin V/PI staining and CellTiter‐Glo® luminescent assay showed reduced drug sensitivity of Notch1‐expressing U266 cells compared to Notch1‐negative U266 cells, confirming the role of Notch1 as survival factor.Based on these results, the first functional screen was recently performed. Intermediate analysis of cell viability clearly showed a sensitizing effect of shRNAs, indicating promising screen hits. First outcomes of the screen will be presented at the meeting.Summary/Conclusion:ShRNA‐based functional HT screening is a potent tool for unveiling Notch1 downstream effectors as specific therapeutic targets in MM to improve treatment efficacy and reduce side effects.