Abstract
Using a high throughput combination screening strategy, we have discovered that agonism of either adenosine A2A receptors (A2A) or beta-2 adrenergic receptors (bAR) demonstrate significant, synergistic, anti-proliferative effects in preclinical Multiple Myeloma (MM) models. Using quantitative synergy analysis, we observe that A2A and bAR agonists have significant anti-proliferative effects in a broad panel of 10 MM cell lines when combined with each other or with standard MM agents. Individual A2A agonists CGS-21680 and HE-NECA inhibited proliferation 25–80% with EC50s ranging from 2–20 nM. Individual bAR agonists salmeterol and formoterol inhibited proliferation 35–75% with EC50s ranging from 10–30 pM. Potent, highly synergistic, inhibition of proliferation, up to 95%, was demonstrated with combinations of A2A or bAR agonists and multiple agents including dexamethasone, lenalidomide, bortezomib, melphalan, doxorubicin, HDAC inhibitors and HSP90 inhibitors at clinically relevant concentrations. These combinations exceeded Loewe additivity, and demonstrated both substantial increases in efficacy over maximal single agent levels as well as significant potency shifting with many combination indices (CIs) in the range of 0.1 to 0.3. Synergistic anti-proliferative effects were observed broadly across several MM cell lines and when using cell lines unresponsive to standard MM drugs, e.g. A2A agonists CGS-21680 and HE-NECA in combination with dexamethasone inhibited 75–85% of the proliferation of EJM, and MOLP-8 dexamethasone-insensitive cell lines as compared to 35–60% for the single agent A2A agonists. The selective A2A antagonist SCH58261 but not A1, A2B and A3 selective antagonists DPCPX, MRS1754 and MRS1523 blocked the synergy and antiproliferative activity of HE-NECA, demonstrating that the effect is mediated via the A2A receptor. siRNA directed against adenosine and adrenergic receptor isoforms, caused a concomitant reduction in the antiproliferative effects of HE-NECA and salmeterol. Synergy (CI<0.4) observed between A2A and bAR agonists suggested that while both these targets signal through Gs coupled signaling pathways, the two targets contribute to the antiproliferative effect via distinct molecular mechanisms. Anti-proliferative effects occurred through a synergistic induction of apoptosis. Combinations of either agonist with dexamethasone demonstrate 50–75% Annexin V positive MM.1S cells after 24 hours treatment whereas single agents show less than 10%. The activity, synergy and selectivity of A2A and bAR combinations were observed in xenograft models of MM. SCID CB17 mice received subcutaneous inoculation of RPMI-8226 cells and once tumors were palpable, mice were treated with vehicle, bortezomib (0.5 mg/kg IV Q3D), salmeterol (10 mg/kg s.c QD) or the combination of both agents. After 19 days of treatment, the combination showed significantly greater reduction in tumor volume than either of the single agents alone (70% vs. 34%; p<0.05, ANOVA). High throughput combination screening facilitated the discovery of two novel and related classes of drug targets with highly synergistic and selective anti-tumor activity in MM. These preclinical data provide a strong rationale for the investigation of A2A and bAR agonists in the treatment of MM.
Adenosine A2A and Beta-2 Adrenergic Receptor Agonists: Novel Selective and Synergistic Multiple Myeloma Targets Discovered through Systematic Combination Screening
