Sodium Salicylate Has a Priming Effect When Combined with Azacitidine in Pre-Clinical Models of AML

Introduction: Current therapy for acute myeloid leukemia (AML) is inadequate. Treatment of older patients considered unfit for standard induction therapy is particularly challenging (Juliusson et al. Blood 2009). Hypomethylating agents (HMA) are commonly used alternatives for these patients (Fenaux et al. JCO 2010). In the case of azacitidine, complete and partial response rates range between 15-30% (Maurillo et al. Cancer 2012) and the addition of other drugs (e.g. lenalidomide and vorinostat) has been limited by toxicity. In this study, we sought to improve the activity of azacitidine by adding the non-steroidal anti-inflammatory drug (NSAID), sodium salicylate (NSal). NSAIDs exert anti-cancer effects through impaired signal transduction (inhibition of NF-kB and Wnt/B catenin pathways) (Kop et al. Science 1994; Reya et al. Nature 2003), epigenetic modulation and disruption of cellular metabolism (activation of AMPK) (Wang et al. CMLS 2013). In addition, previous reports have demonstrated that NSal combines synergistically with established anti-leukemic agents (daunorubicin) (Klampfer et al. Blood 1999), through down-regulation of the anti-apoptotic protein Mcl-1. Importantly, NSal has no effect on platelet function and a small pilot trial (N=11) confirmed the clinical feasibility of this agent in patients with refractory myeloid neoplasms (Klimek et al. Leukemia Research 2012). In this study, therapeutic plasma concentrations of NSal were safely achieved without excessive NSAID class effects (GI and renal toxicity, bleeding). Here, we report the in vitro effects of NSal combined with azacitidine and suggest a rationale for exploring this combination further in the clinic.

Methods: AML cell lines (HL-60, KG-1a and THP-1) were treated with increasing concentrations of NSal (0.2–20mM), azacitidine (0.02-10µM) and both agents combined. Consequent effects on cell viability were measured by trypan blue exclusion and cell proliferation assay (MTT based). Apoptosis was measured by FACS analysis for propidium iodide (PI) and annexin V binding. Synergistic lethality was calculated using the Chou-Talalay method (Calcusyn software). Critical mediators underlying mechanism were measured by immunoblotting (Mcl-1).

Results: The degree of apoptosis in AML cells treated with sub-lethal concentrations of NSal (0.2-20mM) and azacitidine (0.02-10µM) was compared to the degree of apoptosis induced by the combination. Little effect on the viability of HL-60, KG-1a and THP-1 cell lines was observed after 48 hours exposure to either agent alone, however, in the presence of 2 µM azacitidine and 2mM NSal, 39.2% of THP-1 cells were dead at 48 hours (annexin V binding). The combination of 0.2 µM azacitidine and 2mM NSal had significantly inhibited cell proliferation at 72 hours. Cell proliferation decreased by 47.4%, 25%, and 66.1% in HL-60, KG-1a, and THP-1, cell lines, respectively. Combination index (CI) values (Calcusyn software) were less than 1 indicating synergistic activity. The expression of Mcl-1 decreased in a dose- and time-dependent manner in salicylate treated cells which might account for its ability to enhance cell killing by azacitidine.

Conclusions: Sodium salicylate has a priming effect when combined with azacitidine in pre-clinical models of AML. Despite the high pre-clinical concentrations of NSal evaluated, previous studies have confirmed the feasibility of achieving therapeutically active plasma concentrations in patients (Klimek et al. Leukemia Research 2012). This study forms a rationale for a clinical investigation of this approach. Additional in vitro mechanistic studies elucidating the combined effects of NSal and azacitidine will be presented.