Lung cancer is the number one cause of cancer-related deaths in the world. Patients treated with current chemotherapies for non-smallcell lung cancers (NSCLCs) have a survival rate of Ϸ15% after 5 years. Novel approaches are needed to treat this disease. We show elevated NAD(P)H:quinone oxidoreductase-1 (NQO1) levels in tumors from NSCLC patients. ␤-Lapachone, an effective chemotherapeutic and radiosensitizing agent, selectively killed NSCLC cells that expressed high levels of NQO1. Isogenic H596 NSCLC cells that lacked or expressed NQO1 along with A549 NSCLC cells treated with or without dicoumarol, were used to elucidate the mechanism of action and optimal therapeutic window of ␤-lapachone. NSCLC cells were killed in an NQO1-dependent manner by ␤-lapachone (LD 50, Ϸ4 M) with a minimum 2-h exposure. Kinetically, ␤-lapachone-induced cell death was characterized by the following: (i) dramatic reactive oxygen species (ROS) formation, eliciting extensive DNA damage; (ii) hyperactivation of poly(ADP-ribose)polymerase-1 (PARP-1); (iii) depletion of NAD ؉ /ATP levels; and (iv) proteolytic cleavage of p53/PARP-1, indicating -calpain activation and apoptosis. ␤-Lapachone-induced PARP-1 hyperactivation, nucleotide depletion, and apoptosis were blocked by 3-aminobenzamide, a PARP-1 inhibitor, and 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid acetoxymethyl ester (BAPTA-AM), a Ca 2؉ chelator. NQO1 ؊ cells (H596, IMR-90) or dicoumarol-exposed NQO1 ؉ A549 cells were resistant (LD50, >40 M) to ROS formation and all cytotoxic effects of ␤-lapachone. Our data indicate that the most efficacious strategy using ␤-lapachone in chemotherapy was to deliver the drug in short pulses, greatly reducing cytotoxicity to NQO1 ؊ ''normal'' cells. ␤-Lapachone killed cells in a tumorselective manner and is indicated for use against NQO1 ؉ NSCLC cancers.DNA repair inhibitor ͉ non-small-cell lung cancer ͉ NQO1 ͉ apoptosis ͉ -calpain cell death

Alterations in the initiation and regulation of caspase-mediated apoptosis are associated with an array of pathological disease states, including chemotherapy resistance in cancer (1). Therefore, elucidating mechanisms that initiate non-caspasemediated cell death are crucial for the development and use of novel anticancer agents.A growing number of chemotherapeutic approaches focus on targeting specific DNA repair enzymes. In particular, inhibitors of poly(ADP-ribose) polymerase-1 (PARP-1) 2 that sensitize cells to DNA-damaging agents are under extensive investigation (2). PARP-1 functions as a DNA damage sensor that responds to both single-and/or double-strand DNA breaks (SSBs, DSBs), facilitating DNA repair and cell survival. After binding to DNA breaks, PARP-1 converts ␤-NAD ϩ (NAD ϩ ) into polymers of branched or linear poly(ADP-ribose) units (PAR) and attaches them to various nuclear acceptor proteins, including XRCC1, histones, and PARP-1 for its autoregulation (3). However, in response to extensive DNA damage, PARP-1 can be hyperactivated, eliciting rapid cellular NAD ϩ and ATP pool depletion. PARP-1-mediated NAD ϩ and ATP losses have affects on mitochondrial function by decreasing the levels of pyruvate and NADH. Loss of mitochondrial membrane potential (MMP) ensues, causing caspase-independent cell death by as yet unknown mechanisms (3). PARP-1 hyperactivation was documented in the cellular response to trauma, such as ischemia-reperfusion, myocardial infarction, and reactive oxygen species (ROS)-induced injury (3). In each case, inhibition of PARP-1 was necessary for the long-term survival of damaged cells (4).␤-lapachone (␤-lap) elicits a unique cell death process in various human breast, lung, and prostate cancers that have elevated levels of the two-electron oxidoreductase, NAD(P)H: quinone oxidoreductase 1 (NQO1) (EC 1.6.99.2) (5). ␤-lap induces an NQO1-dependent form of cell death wherein PARP-1 and p53 proteolytic cleavage fragments were noted (6), concomitant with -calpain activation (7). ␤-lap-induced lethality and proteolysis were abrogated by dicoumarol (an NQO1 inhibitor), and were muted in cells deficient in NQO1

Improving patient outcome by personalized therapy involves a thorough understanding of an agent’s mechanism of action. β-Lapachone (clinical forms, Arq501/Arq761) has been developed to exploit dramatic cancer-specific elevations in the phase II detoxifying enzyme, NAD(P)H:quinone oxidoreductase (NQO1). NQO1 is dramatically elevated in solid cancers, including primary and metastatic (e.g., triple-negative (ER-, PR-, Her2/Neu-)) breast cancers. To define cellular factors that influence the efficacy of β-lapachone using knowledge of its mechanism of action, we confirmed that NQO1 was required for lethality and mediated a futile redox cycle where ~120 moles of superoxide were formed per mole of β-lapachone in 5 min. β-Lapachone induced reactive oxygen species (ROS), stimulated DNA single strand break-dependent PARP1 hyperactivation, caused dramatic loss of essential nucleotides (NAD+/ATP) and elicited programmed necrosis in breast cancer cells. While PARP1 hyperactivation and NQO1 expression were major determinants of β-lapachone-induced lethality, alterations in catalase expression, including treatment with exogenous enzyme, caused marked cytoprotection. Thus, catalase is an important resistance factor, and highlights H2O2 as an obligate ROS for cell death from this agent. Exogenous superoxide dismutase (SOD) enhanced catalase-induced cytoprotection. β-Lapachone-induced cell death included AIF translocation from mitochondria to nuclei, TUNEL+ staining, atypical PARP1 cleavage, and GAPDH S-nitrosylation, which were abrogated by catalase. We predict that the ratio of NQO1:catalase activities in breast cancer versus associated normal tissue are likely to be the major determinants affecting the therapeutic window of β-lapachone and other NQO1 bioactivatable drugs.