Identification of novel synergistic antiproliferative multiple myeloma drug combinations using cHTS. A select set of approved drugs, emerging therapeutics, and research probes were combined with the glucocorticoid dexamethasone using our high-throughput combination screening platform and antiproliferative activity was determined using the multiple myeloma cell lines RPMI-8226, MM.1S, and MM.1R. (A) A 6 × 6 dose matrix was generated for each combination. The matrix samples all mixtures of 2 serially diluted single-agent concentrations. Phenotypic assay measurements of inhibition over the test matrix are visualized using a color scale. Shown are the combination activities for dexamethasone combined with Chloro-IB-M ECA, an adenosine receptor agonist and papaverine, a PDE inhibitor. (B) For comparative purposes, a small sampling of single agents and combinations are graphed at concentrations where combination activities are more than additive (synergistic). Single-agent and combination activities used are from the matrix shown in panel A and are highlighted by white boxes. Comparison of panels A and B demonstrate how the dose-matrix format captures the breadth of combination activities at multiple ratios over a wide concentration range. (C) To assess synergy, each observed point is compared with the dose-additive model (expectation for a drug crossed with itself) that is calculated at every test point in the matrix using the single-agent responses at the edges (see “Proliferation assays” for more information). (D) Activity in excess of the dose-additivity model is calculated (subtraction of dose-additive values from the observed data) and assigned a synergy score (see “Proliferation assays” for more information). (E) Dose-matrix analysis, dose-additivity excess, and isobologram analysis for Chloro-IB-MECA combined with papaverine in the MM.1S cell line and (F) the MM.1R cell line. The isobologram error bars are the result of comparison of drug-treated cells versus untreated controls.