A2A or Beta-2 Adrenergic Receptor Agonist Synergies Induce Distinct Patterns of Gene Expression Relevant to Multiple Myeloma Cell Survival.

Abstract 3831

Poster Board III-767

Using a high-throughput combination screening strategy, we have demonstrated that adenosine A₂A receptors (A₂AR) or beta-2 adrenergic receptors (bAR) agonists, in low nano-molar range, exhibit highly synergistic anti-proliferative activity when combined with Multiple Myeloma drugs, such as dexamethasone, lenalidomide, bortezomib, melphalan and doxorubicin at clinically relevant concentrations. Synergy and selectivity have been observed in multiple myeloma (MM) cell lines and ex vivo using patient tumor cells. To understand this synergistic mechanism, we employed the Affymetrix U133 plus 2.0 cDNA microarray to investigate the differentially expressed genes in a multiple myeloma MM1.S cell line treated with A₂AR agonists, bAR agonists, dexamethasone, or in combinations (A₂AR/ bAR agonists and dex) for six hours. Using conventional hierarchical clustering, unsupervised analyses clearly separate the A₂AR/ bAR agonists or the dexamethosone groups from the combination groups, suggesting a unique mechanism underlying the combination treatments. With SAM (Significance Analysis of Microarrays), we found 314 and 309 genes that showed statistically significant up-regulation or down-regulation respectively in the combination groups compared to the untreated control, with a false discovery rate=<1%. Interestingly, only a few genes were statistically different between A₂AR agonists with dex and bAR agonists with dex, indicating that they may function with a similar mechanism. To further investigate the genes that are coordinately expressed, we performed gene set enrichment analyses (GESA) with the published or curated gene sets available from Board Institute (Cambridge, MA) or GeneGo pathway analysis software (Encinitas, CA). We found that the cells treated with drug combinations were enriched in genes involved in multiple cancer relevant pathways including TGF-beta receptor signaling, WNT signaling, MAPK/Erk signaling, PKA signaling, IGF-RI signaling, the stress response pathway and the myc pathway. Bim1 is one of the genes upregulated by single agent and combination drug treatment and is important for cell killing as siRNA targeting Bim1 reduces single agent and combination effects. Down-regulation of IRF4 by combination treatments is particularly interesting. High levels of IRF-4 expression have been suggested as an independent risk factor for poor survival in multiple myeloma patients (Heintel D et. al., Leukemia 2008). Moreover, IRF4 has recently been shown to be critical for proliferation and/or survival in a number of multiple myleoma cell lines (Shaffer et al., Nature 2008). Consistent with these observations, flow cytometric analysis of annexinV/PI stained MM1.S cells transfected with siRNA against IRF4 confirms that IRF-4 is important for MM.1S survival. Furthermore, direct or indirect downstream targets of IRF4 (e.g. MYC, HK2, PDK1, and SUB1) are coordinately down-regulated in MM1.S cells when IRF4 expression is reduced by A₂AR / bAR agonists with dex. These data suggest that A2A and bAR agonist combinations coordinately down-regulate both myc and IRF4, which form a positive autoregulatory loop important for MM proliferation/survival and further underscore the utility of systematic combination screening in the identification of pathway and chemical interactions relevant to disease.