Abstract C25: Targeting cancer stem cells using ALDH-dependent 5-nitrofuran pro-drugs

Abstract

Abstract We hypothesise that cancer stem cells with high aldehyde dehydrogenase (ALDHhigh) activity present a new therapeutic target and will be selectively sensitive to 5-nitrofuran pro-drugs. Cancers are heterogeneous and contain subpopulations of ALDHhigh cells with tumour initiating potential. ALDH enzymes metabolize toxic aldehydes, and are highly expressed in somatic and cancer stem cells (CSCs), although their function in stem cells is not fully understood. In a small molecule screen coupled with target ID, we recently discovered that clinically active 5-nitrofurans (5-NFNs) are substrates of ALDH2 (Zhou et al., 2012). 5-NFNs are a class of pro-drug widely used to treat bacterial and parasitic infections where their relative specificity is driven by nitroreductases, but little is known about the enzymes that bio-activate 5-NFNs in humans. Recent clinical cancer research has found that the 5-NFN nifurtimox has anti-cancer properties and it is currently in Phase 2 clinical trials for neuroblastoma and medulloblastoma (ClinicalTrials.gov Identifier: NCT00601003), however the mechanism underlying this anti-cancer activity is unknown. In melanoma and other cancers, ALDH1A1 and ALDH1A3 are highly expressed in CSCs. We find that cancer cell lines are highly sensitive to 5-NFNs in cell viability assays, where we use a logarithmic drug dose range and assess cell viability by PrestoBlue™ (e.g. A375 melanoma cells EC50 = 86nM). To test if ALDH1 isoforms are substrates of 5-NFNs, we preformed in vitroactivity assays by monitoring NADH production (λ = 340nm). We find that the clinically active 5-NFNs nifuroxazide and nifurtimox, in addition to our own newly synthesised 5-NFNs, are competitive substrates for human ALDH1A3 activity in vitro (p<0.05). Notably, nifuroxazide was not a substrate for ALDH2, suggesting that nifuroxazide may show selectivity toward ALDH1 isoforms. Consistent with our enzymatic activity assays, we find that 5-NFNs are competitive substrates for ALDH activity in melanoma cells by Aldefluor™ in vivo, with 5-NFNs displaying a prolonged competitive inhibition compared with the known inhibitor, DEAB. Importantly, no-nitro control compounds show no activity toward ALDH enzymes in vitro or in vivo. Computational docking studies reveal that 5-NFNs have the potential to fit within the interior of the ALDH enzymatic cavity and interact with the catalytic cysteine, thereby offering a potential mechanism for 5-NFN bio-activation. Kinetic living-cell imaging (IncuCyte ZOOM®) reveals that ALDH1A3 siRNA transfected A375 cells are protected from 5-NFN toxicity (p>0.05) and apoptosis (DRAQ7™: p<0.0001), demonstrating a functional role for ALDH1A3 in mediating 5-NFN activity in cancer cells. Our work demonstrates a novel and biologically relevant 5-NFN-ALDH1 interaction in cancer cells. We propose 5-NFNs have the potential to target ALDHhigh CSCs within a tumour and advance the repurposing of clinical 5-NFN pro-drug antibiotics as anti-cancer therapeutics. Citation Format: Richard Crispin, Nathalie Spockeli, Val Brunton, Neil Carragher, Charlie Gourley, Douglas Houston, Asier Unciti-Broceta, E. Elizabeth Patton. Targeting cancer stem cells using ALDH-dependent 5-nitrofuran pro-drugs. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C25.