Adenosine A2A Receptor Agonism and PDE Inhibition: A Synergistic Multi-Target Mechanism Discovered through Systematic Combination Screening in Multiple Myeloma
Authors: Rickles Richard J , Pierce Laura , Giordano Thomas , Tam Winnie F. , McMillin Douglas W. , Anderson Kenneth C. , Mitsiades Constantine , Lee Margaret S 
Published: October 2019

Abstract
Using a combination high throughput screening technology, we have discovered an unexpected synergistic interaction between adenosine A2A receptor (A2A) agonism and phosphodiesterase (PDE) inhibition that displays substantial activity in preclinical Multiple Myeloma (MM) models. High throughput combination screening allows the systematic testing of combinations of approved drugs and other biologically active molecules in cell based assays of tumor cell proliferation and viability. In this approach we generate a dose matrix for each chemical combination, capturing the combined activity of two compounds over a broad range of single agent concentrations. Quantitative scoring of dose response matrices using models of drug interactions allows insight into the biological mechanism of action of drug combinations and the discovery of novel therapeutic applications. Using a panel of 4 MM cell lines (H929, MM.1S, MM.1R and RPMI-8226) we evaluated a defined set of combinations of approved drugs and molecular probes. A total of 2841 unique combinations were evaluated, 648 of which were assayed in all 4 cell lines. Multiple combinations were identified that exceeded the Loewe additivity model including enhancers of known anti-cancer drugs and combinations of targets not previously know in multiple myeloma. Multiple classes of drugs and targeted agents were identified that synergize with dexamethasone. Backcrosses of these active agents revealed multiple dexamethasone enhancers that also synergized with each other. Two of these classes, A2A agonists and PDE inhibitors demonstrated high levels of synergy and good breadth of activity across several MM cell lines. Interestingly, while the majority of the combinations screened had little or no synergistic effect in the glucocorticoid resistant MM.1R cell line, combinations of PDE inhibitors and A2A agonists demonstrated substantial efficacy (>90% inhibition of proliferation) and striking synergy (combination indices<0.3) in this cell line. We have used panels of selective adenosine receptor agonists, antagonists and PDE inhibitors as well as siRNAs targeting specific molecular isoforms of these proteins to dissect the molecular mechanism of this synergy. All adenosine receptor agonists tested synergize with the multi-PDE isoform inhibitor trequinsin. Synergy between the A2A agonist Chloro-IB-MECA and trequinsin is suppressed by the adenosine A2A antagonist SCH58261 at 78 nM but not by the A1, A2B or A3 antagonists DPCPX, MRS1754 and MRS1523 respectively, demonstrating that A2A agonism is the necessary component of the synergistic multi-target mechanism. Furthermore, siRNA knockdown of adenosine A2A but not A1, A2B or A3 receptors blocks the activity of all adenosine receptor agonists examined. Similarly, using 22 subtype specific PDE inhibitors, we find that the specific subset of PDE2, 3, 4 and 7, are able to synergize with A2A agonists. By combining subtype inhibitors and/or siRNAs targeting specific PDE isoforms we find that maximal activity is observed when more than one PDE is targeted. Inhibition of both PDE3 and 4 results in the greatest synergy, with further enhancement from the addition of PDE2 and/or PDE7 inhibition. During treatment with A2A agonists and PDE inhibitors there is a compensatory 13-fold up-regulation of PDE4B, as measured by qPCR, suggesting that inhibition of this isoform is particularly important for maximal antiproliferative activity. We find that neither A2A agonism nor PDE inhibition alone induces cAMP accumulation in MM cell lines. However, the combination of A2A activation and PDE inhibition leads to elevated intracellular cAMP and cell death. Notably, the activity of A2A agonists is enhanced in the presence of 10 ng/mL interleukin-6 and HS-5 human bone marrow stromal cells and the synergy between A2A agonists and PDE inhibitors is preserved under these conditions. In summary, we describe the use of cHTS screening to discover new synergistic multi-target mechanisms and prioritize synergistic anti-proliferative combinations for preclinical evaluation. This approach has resulted in the discovery of adenosine A2A receptor agonism and PDE inhibition as a highly selective and synergistic multi-target mechanism with therapeutic potential in MM.