Subtype-selective prenylated isoflavonoids disrupt regulatory drivers of MYCN-amplified cancers

Transcription factors have proven difficult to target with small molecule inhibitors because they often lack deep pockets necessary for potent binding. Alternatively, disruption of protein expression can suppress difficult targets and enable therapeutic intervention. To this end, we have developed a drug discovery workflow that incorporates cell line-selective screening and high-throughput expression profiling followed by regulatory network analysis to identify compounds that suppress regulatory drivers of disease. Applying this approach to neuroblastoma (NBL), we screened bioactive molecules in cell lines representing the MYC-dependent (MYCNA) and mesenchymal (MES) subtypes to identify selective compounds, followed by PLATESeq profiling of treated cells. This revealed compounds that disrupt a network of MYCNA-specific regulatory proteins, resulting in MYCN degradation in vivo. The top hit was isopomiferin, a prenylated isoflavonoid that inhibited Casein Kinase 2 (CK2) in cells. Isopomiferin and its structural analogs inhibited MYC and MYCN in NBL and lung cancer cells, highlighting the general MYC-inhibiting potential of this unique scaffold. First Experiment (PLATESeq): To identify NBL MYCNA subtype-selective inhibitors, we systematically screened >5,000 compounds from three chemical libraries chosen for their enrichment of bioactive molecules, diversity in chemical structure, and for inclusion of compounds with known mechanisms of action. Compounds were initially tested at a single concentration and time-point (20 µM for 72 h) to eliminate those with no in vitro activity in NBL cell lines. Lethal compounds were rescreened across a five-point dilution series ranging from 20 µM to ~250 nM, to determine IC50 values in each of the four cell lines. By ranking compounds based on average IC50 values for each of the two subtypes, MYCNA or MES-selective compounds were identified. The top 90 compounds with the highest relative MYCNA-specific selectivity were then used to generate PLATESeq expression profiles following 24 hours of treatment in SK-N-Be2 cell lines at their respective IC20 concentration. Second Experiment (PLATESeq): top 15 compounds were screened with PLATESeq at 6 and 24 hours at multiple concentrations in three replicates Third Experiment (PLATESeq): In an effort to identify a potent analog of isopomiferin, we assembled a small collection of structurally-related analogs and known CK2 inhibitors, and tested them in SK-N-Be2 cells using PLATESeq TruSeq Experiment (RNASeq): To uncover kinase targets of isopomiferin and its functional analogs, we developed a novel algorithm, the Virtual-Inference of Kinase INhibiton by Gene regulatory networks (VIKING), to enable data-driven inference of drug targets based on activity dysregulation of protein-protein interactions (PPIs). VIKING was run using RNASeq (TruSeq) data on isopomiferin, pomiferin and its null analog, at multiple concentrations and timepoints.