Summary Therapeutic drugs that block DNA repair, including poly(ADP-ribose) polymerase (PARP) inhibitors, fail due to lack of tumor-selectivity. When PARP inhibitors and β-lapachone are combined, synergistic antitumor activity results from sustained NAD(P)H levels that refuel NQO1-dependent futile redox drug recycling. Significant oxygen consumption rate/reactive oxygen species cause dramatic DNA lesion increases that are not repaired due to PARP inhibition. In NQO1+ cancers, such as non-small cell lung, pancreatic and breast, cell death mechanism switches from PARP1 hyperactivation-mediated programmed necrosis with β-lapachone monotherapy to synergistic tumor-selective, caspase-dependent apoptosis with PARP inhibitors and β-lapachone. Synergistic antitumor efficacy and prolonged survival were noted in human orthotopic pancreatic and non-small cell lung xenograft models, expanding use and efficacy of PARP inhibitors for human cancer therapy.

BackgroundPancreatic ductal adenocarcinomas (PDA) activate a glutamine-dependent pathway of cytosolic nicotinamide adenine dinucleotide phosphate (NADPH) production to maintain redox homeostasis and support proliferation. Enzymes involved in this pathway (GLS1 (mitochondrial glutaminase 1), GOT1 (cytoplasmic glutamate oxaloacetate transaminase 1), and GOT2 (mitochondrial glutamate oxaloacetate transaminase 2)) are highly upregulated in PDA, and among these, inhibitors of GLS1 were recently deployed in clinical trials to target anabolic glutamine metabolism. However, single-agent inhibition of this pathway is cytostatic and unlikely to provide durable benefit in controlling advanced disease.ResultsHere, we report that reducing NADPH pools by genetically or pharmacologically (bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES) or CB-839) inhibiting glutamine metabolism in mutant Kirsten rat sarcoma viral oncogene homolog (KRAS) PDA sensitizes cell lines and tumors to ß-lapachone (ß-lap, clinical form ARQ761). ß-Lap is an NADPH:quinone oxidoreductase (NQO1)-bioactivatable drug that leads to NADPH depletion through high levels of reactive oxygen species (ROS) from the futile redox cycling of the drug and subsequently nicotinamide adenine dinucleotide (NAD)+ depletion through poly(ADP ribose) polymerase (PARP) hyperactivation. NQO1 expression is highly activated by mutant KRAS signaling. As such, ß-lap treatment concurrent with inhibition of glutamine metabolism in mutant KRAS, NQO1 overexpressing PDA leads to massive redox imbalance, extensive DNA damage, rapid PARP-mediated NAD+ consumption, and PDA cell death—features not observed in NQO1-low, wild-type KRAS expressing cells.ConclusionsThis treatment strategy illustrates proof of principle that simultaneously decreasing glutamine metabolism-dependent tumor anti-oxidant defenses and inducing supra-physiological ROS formation are tumoricidal and that this rationally designed combination strategy lowers the required doses of both agents in vitro and in vivo. The non-overlapping specificities of GLS1 inhibitors and ß-lap for PDA tumors afford high tumor selectivity, while sparing normal tissue.Electronic supplementary materialThe online version of this article (doi:10.1186/s40170-015-0137-1) contains supplementary material, which is available to authorized users.

Nicotinamide phosphoribosyltransferase (NAMPT) inhibitors (e.g., FK866) target the most active pathway of NAD+ synthesis in tumor cells, but lack tumor-selectivity for use as a single agent. Reducing NAD+ pools by inhibiting NAMPT primed pancreatic ductal adenocarcinoma (PDA) cells for poly(ADP ribose) polymerase (PARP1)-dependent cell death induced by the targeted cancer therapeutic, β-lapachone (β-lap, ARQ761), independent of poly(ADP ribose) (PAR) accumulation. β-Lap is bioactivated by NADPH:quinone oxidoreductase 1 (NQO1) in a futile redox cycle that consumes oxygen and generates high levels of reactive oxygen species (ROS) that cause extensive DNA damage and rapid PARP1-mediated NAD+ consumption. Synergy with FK866+β-lap was tumor-selective, only occurring in NQO1-overexpressing cancer cells, which is noted in a majority (∼85%) of PDA cases. This treatment strategy simultaneously decreases NAD+ synthesis while increasing NAD+ consumption, reducing required doses and treatment times for both drugs and increasing potency. These complementary mechanisms caused profound NAD(P)+ depletion and inhibited glycolysis, driving down adenosine triphosphate levels and preventing recovery normally observed with either agent alone. Cancer cells died through an ROS-induced, μ-calpain-mediated programmed cell death process that kills independent of caspase activation and is not driven by PAR accumulation, which we call NAD+-Keresis. Non-overlapping specificities of FK866 for PDA tumors that rely heavily on NAMPT-catalyzed NAD+ synthesis and β-lap for cancer cells with elevated NQO1 levels affords high tumor-selectivity. The concept of reducing NAD+ pools in cancer cells to sensitize them to ROS-mediated cell death by β-lap is a novel strategy with potential application for pancreatic and other types of NQO1+ solid tumors.

Base excision repair (BER) is an essential pathway for pancreatic ductal adenocarcinoma (PDA) survival. Attempts to target this repair pathway have failed due to lack of tumor-selectivity and very limited efficacy. The NAD(P)H:Quinone Oxidoreductase 1 (NQO1) bioactivatable drug, ß-lapachone (ARQ761 in clinical form), can provide tumor-selective and enhanced synergy with BER inhibition. ß-Lapachone undergoes NQO1-dependent futile redox cycling, generating massive intracellular hydrogen peroxide levels and oxidative DNA lesions that stimulate poly(ADP-ribose) polymerase 1 (PARP1) hyperactivation. Rapid NAD+/ATP depletion and programmed necrosis results. To identify BER modulators essential for repair of ß-lapachone-induced DNA base damage, a focused synthetic lethal RNAi screen demonstrated that silencing the BER scaffolding protein, XRCC1, sensitized PDA cells. In contrast, depleting OGG1 N-glycosylase spared cells from ß-lap-induced lethality and blunted PARP1 hyperactivation. Combining ß-lapachone with XRCC1 knockdown or methoxyamine (MeOX), an apyrimidinic/apurinic (AP)-modifying agent, led to NQO1-dependent synergistic killing in PDA, NSCLC, breast and head and neck cancers. OGG1 knockdown, dicoumarol-treatment or NQO1- cancer cells were spared. MeOX + ß-lapachone exposure resulted in elevated DNA double-strand breaks, PARP1 hyperactivation and TUNEL+ programmed necrosis. Combination treatment caused dramatic antitumor activity, enhanced PARP1-hyperactivation in tumor tissue, and improved survival of mice bearing MiaPaca2-derived xenografts, with 33% apparent cures. Significance: Targeting base excision repair (BER) alone has limited therapeutic potential for pancreatic or other cancers due to a general lack of tumor-selectivity. Here, we present a treatment strategy that makes BER inhibition tumor-selective and NQO1-dependent for therapy of most solid neoplasms, particularly for pancreatic cancer.

Ionizing radiation (IR) is a key therapeutic regimen for many head and neck cancers (HNCs). However, the 5-year overall survival rate for locally-advanced HNCs is ∼50% and better therapeutic efficacy is needed. NAD(P)H:quinone oxidoreductase 1 (NQO1) is over-expressed in many cancers, and β-lapachone (β-lap), an unique NQO1 bioactivatable drug, exploits this enzyme to release massive reactive oxygen species (ROS) levels that synergizes with IR to kill by programmed necrosis. β-Lap represents a novel therapeutic opportunity in HNC leading to tumor-selective lethality that will enhance the efficacy of ionizing radiation. Immunohistochemical staining and western blot assays were used to assess the expression levels of NQO1 in HNC cells and tumors. Forty-five percent of endogenous HNCs express elevated NQO1 levels. In addition, multiple HNC cell lines and tumors demonstrated elevated levels of NQO1 expression and activity and were tested for anticancer lethality and radiosensitization by β-lap using long-term survival assays. The combination of nontoxic β-lap doses and IR significantly enhanced NQO1-dependenttumor cell lethality, increased ROS, TUNEL positive cells, DNA damage, NAD+ and adenosine triphosphate (ATP) consumption, and resulted in significantantitumor efficacy and prolonged survival in two xenograft murine HNC models, demonstrating β-Lap radiosensitization of HNCs through a NQO1-dependent mechanism. This translational study offers a potential biomarker-driven strategy using NQO1 expression to select tumors susceptible to β-lap-induced radiosensitization.