Synergistic Activity of Adenosine A2A and Beta-2 Adrenergic Receptor Agonists in Myeloma Cells in the Context of Tumor-Stromal Interactions
Authors: McMillin Douglas W. , Rickles Richard , Negri Joseph , Delmore Jake , Ooi Melissa G. , Farwell Melissa , Crowe David , Chen Mei , Avery William , Kansra Vikram , Anderson Kenneth C. , Lee Margaret S. , Mitsiades Constantine S. 
Published: April 2020

Abstract
Context: The bone marrow microenvironment, including bone marrow stroma cells (BMSCs) attenuates response of multiple myeloma (MM) cells to various conventional and experimental agents. Development of novel agents for the treatment of MM depends on attenuating the protective interactions that occur between the tumor cells and its microenvironment. Utilizing a combination high throughput screening platform (cHTS™) and the compartment-specific bioluminescence imaging (CS-BLI) co-culture system we have identified 2 novel classes of agents, adenosine A2A receptor agonists and β2-adrenergic receptor (bAR) agonists, with increased activity in the context of the microenvironment.
Methods/Results: We tested 10 human MM cell lines in both the cHTS and CS-BLI systems in the presence and absence of IL-6 and discovered that multiple compounds that agonize A2A and bAR receptors had potent anti-MM activity in MM.1S cells. To further explore this activity, we evaluated CRx-501, a potent selective A2A agonist and salmeterol, a prototypical bAR agonist, which were found to have IC50 values in MM.1S cells of 15 nM and 0.2 nM, respectively. Individual single agent activities of CRx-501 and salmeterol were enhanced in the presence of exogenous IL-6 (10ng/mL). We also observed enhancement of CRx-501 activity in the presence of HS-5 human BMSCs. CRx-501 and salmeterol were also observed to synergize with bortezomib, dexamethasone, lenalidomide, and doxorubicin both in the presence and absence of IL-6 or stromal cells. These interactions were highly synergistic, as determined by the Loewe additivity model, demonstrating an increase in efficacy with combination indices ranging from 0.17 to 0.74. To further explore the anti-tumor activity of A2A and bAR agonists, CRx-501 and salmeterol were evaluated in an MM.1S tumor xenograft model. SCID CB17 mice received subcutaneous inoculation of MM.1S cells and, upon tumors becoming palpable, were treated with vehicle, dexamethasone (1 mg/kg s.c.), salmeterol (10 mg/kg s.c), CRx-501 (3 mg/kg s.c.) as single agents or pair-wise combinations of salmeterol or CRx-501 with dexamethasone. After 34 days of treatment with dexamethasone, salmeterol or CRx-501, we observe 34%, 42% and 72% reduction in tumor volume compared to vehicle control, respectively. Interestingly, in vivo activity of dexamethasone was enhanced to 86% and 89% with the combination of CRx-501 or salmeterol, respectively, with minimal toxicity (e.g. 7% fluctuation in animal weight during the course of the study).
Conclusion: The development of active agents and drug combinations for MM depends on the evaluation and modeling of microenvironmental factors that play a role in the pathophysiology of the disease. Utilizing a novel combination screening method and co-culture screening system we identified and evaluated 2 novel mechanisms that are active in the context of the microenvironment and synergize with many standard therapies. These studies provide the framework for further preclinical evaluation and possible trials for these combinations for the treatment of MM.