Abstract
Development of tumor-specific therapies for the treatment of recalcitrant non–small cell lung cancers (NSCLC) is urgently needed. Here, we investigated the ability of β-lapachone (β-lap, ARQ761 in clinical form) to selectively potentiate the effects of ionizing radiation (IR, 1–3 Gy) in NSCLCs that overexpress NAD(P)H:Quinone Oxidoreductase 1 (NQO1).
The mechanism of lethality of low-dose IR in combination with sublethal doses of β-lap was evaluated in NSCLC lines in vitro and validated in subcutaneous and orthotopic xenograft models in vivo. Pharmacokinetics and pharmacodynamics (PK/PD) studies comparing single versus cotreatments were performed to validate therapeutic efficacy and mechanism of action.
β-Lap administration after IR treatment hyperactivated PARP, greatly lowered NAD+/ATP levels, and increased double-strand break (DSB) lesions over time in vitro. Radiosensitization of orthotopic, as well as subcutaneous, NSCLCs occurred with high apparent cures (>70%), even though 1/8 β-lap doses reach subcutaneous versus orthotopic tumors. No methemoglobinemia or long-term toxicities were noted in any normal tissues, including mouse liver that expresses the highest level of NQO1 (∼12 units) of any normal tissue. PK/PD responses confirm that IR + β-lap treatments hyperactivate PARP activity, greatly lower NAD+/ATP levels, and dramatically inhibit DSB repair in exposed NQO1+ cancer tissue, whereas low NQO1 levels and high levels of catalase in associated normal tissue were protective.
Our data suggest that combination of sublethal doses of β-lap and IR is a viable approach to selectively treat NQO1-overexpressing NSCLC and warrant a clinical trial using low-dose IR + β-lap against patients with NQO1+ NSCLCs.
Lung cancer remains the leading cause of cancer-related death in men and women in North America. Patients diagnosed with non–small-cell lung cancer (NSCLC) have few effective treatment options, which cause undesirable side effects due to lack of tumor selectivity when used at higher doses. There is an urgent need for personalized strategies to selectively target cancer while sparing normal tissue. This study delineates the mechanistic basis for tumor-selective potentiation of ionizing radiation (IR) in combination with β-lapachone (β-lap, ARQ761 in clinical form), an NQO1-bioactivatable agent that induces lethal DNA damage in recalcitrant NSCLCs (∼90% overexpress NQO1 as a biomarker). Nontoxic doses of β-lap can be combined after treatment with IR to selectively and synergistically kill NSCLCs with no apparent normal tissue toxicity in vivo. Our findings warrant a clinical trial to combine sublethal doses of IR + β-lap against patients with NQO1+ NSCLCs for precision-guided medicine.
Introduction
We previously demonstrated that β-lapachone (β-lap) was a unique substrate for NAD(P)H:Quinone Oxidoreductase 1 (NQO1; E.C.1.6.5.2; refs. 1–10), wherein the enzyme performs a futile two-electron oxidoreduction of the compound using NAD(P)H to generate an unstable hydroquinone form of β-lap (7, 10, 11). Unique to this quinone, its hydroquinone form is highly unstable and spontaneously undergoes two oxidoreductive back-reactions to its original drug form (1, 3–7, 10–15). NQO1 thereby constantly undergoes a futile redox cycle using NAD(P)H and creating two moles of superoxide in the two-step back-reaction (6, 7). A tremendous pool of NAD(P)+ forms (4, 6–9, 12, 16, 17), along with very high levels of superoxide, which are rapidly converted to hydrogen peroxide (H2O2). High doses of H2O2 eventually damage the DNA of NQO1+ cancer cells (15, 18, 19). The reaction can also cause a strong lethal effect in adjacent NQO1Low cancer cells in the same tumor via an H2O2-dependent bystander effect mediated by low catalase levels in these cancers (15). AP (apurinic, apyrimidinic, or abasic) sites, DNA single-strand breaks (SSB), and DNA double-strand breaks (DSB) form in NQO1+ cancer cells exposed to β-lap via a strong PARP1 hyperactivation (6, 7, 12, 16), which then degrades the heightened NAD+ levels resulting from NQO1-dependent redox cycling of this unique quinone (3, 4, 7, 10, 15, 20). This process is mediated by H2O2-induced release of calcium from endoplasmic reticulum stores (12, 16). Delineating the mechanism of action of β-lap has revealed exciting synergistic combinations, for example, using glutamate synthase inhibitors (BEPTES, CB089; ref. 18), DNA base excision repair inhibitors (methoxyamine and XRCC1 depletion; ref. 19), as well as PARP inhibitors (10). Analyses of numerous NQO1-expressing breast, prostate, lung, pancreatic, and head and neck cancer cells show that β-lap (a prototypic NQO1-bioactivatable drug) kills cancer cells by an NQO1-dependent ischemia–reperfusion mechanism independent of oncogenic driver or passenger statuses, regardless of p53 or cell-cycle status, and independent of overexpressed antiapoptotic mechanisms (e.g., Bcl2, Bax loss) that may drive resistance (10).
We previously exploited β-lap as a radiosensitizer of cancer cells in vitro, initially without knowing the mechanism by which synergistic killing with IR occurred (21, 22). After determining the mechanism of action of β-lap, we then used NQO1 overexpressing human prostate or head and neck cancer xenograft mouse models to show how the compound radiosensitized human cancers in vivo (11, 23). Our data showed that synergy could be accomplished using IR + β-lap via creation of normal, rapidly repaired DNA lesions that simultaneously hyperactivate PARP1, where IR or drug alone did not separately meet the DNA damage required. Here, we demonstrate that β-lap suppresses IR-induced DSB repair and we illustrate the efficacy of IR + β-lap treatments against non–small cell lung cancers (NSCLC) that commonly have significantly elevated levels of NQO1, but concomitantly low catalase levels (10). We also tested the hypotheses that massive PARP1 hyperactivation reactions after IR + β-lap led to dramatic accompanying NAD+/ATP losses that ultimately inhibit DSB repair processes (initiated by IR exposure) and may lead to NQO1-dependent NAD+-Keresis cell death (14) responses specific for NQO1+ NSCLC tumors. No methemoglobinemia or toxicities to normal tissues were found in IR + β-lap–exposed mice. Data presented in this study warrant a clinical trial using low-dose radiotherapy with the clinically available NQO1-bioactivatable drug (ARQ761) against NSCLCs containing wild-type NQO1 as a biomarker for personalized medicine.
Materials and Methods
Reagents and chemicals
β-lap was synthesized and purified by us (6). Dicoumarol (DIC), hydrogen peroxide (H2O2), Hoechst 33342, BSA, and cytochrome c were purchased from Sigma-Aldrich. HPβCD (>98% purity) was purchased from Cyclodextrin Technologies Development, Inc. and β-lap-HPβCD was prepared as described previously (24).
NQO1 enzyme assays
NQO1 enzyme activities from cancer cells or tumor or normal tissues were measured as DIC-inhibited units either with or without cytochrome c (1, 25).
Cell lines and tissue culture
Cell culture irradiations and colony-forming ability assays
All cell culture irradiations were performed using a Mark 1 irradiator with a 137Cesium source delivering a dose rate of 3.49 Gy/minute (JL Shepherd & Associates). Varying doses of β-lap dissolved in DMSO (± DIC) were added immediately after irradiation and incubated with the drug for 2 hours in a humidified incubator (37°C, 5% CO2). After 2-hour treatment, media were replaced with fresh complete media and allowed to grow for several doubling periods. Colony-forming ability assays were performed using 500–15,000 cells per 100-mm plates as described previously (21, 22). Colonies of >100 healthy normal-appearing cells were counted and normalized to vehicle-treated control cells.
Antibodies
Antibodies used for immunofluorescence and Western blotting included: NQO1 (A180), PARP1 (SC-8007, Santa Cruz Biotechnology), β-actin (C4, Santa Cruz Biotechnology), PAR (Trevigen), 53BP1 (Santa Cruz Biotechnology), γ-H2AX (JBW301, Millipore), phosphorylated ATM (p-ATM-s1981), total-ATM (t-ATM), and α-tubulin (Santa Cruz Biotechnology).
Western blot analysis
Western blots were performed using ECL chemiluminescent detection and density analyses using NIH ImageJ with intensity normalization (6, 7, 10, 25).
DSB detection and repair
Cells were imaged by immunofluorescence using Leica DM5500 fluorescent microscopy for a marker of DSB formation. The number of 53BP1 foci in a given cell nucleus was quantified and graphed as percent (%) nuclei with ≥10 53BP1 foci/nucleus (6, 7, 25). Alternatively, for assessments in vivo, γH2AX levels were screened and quantified by Western blotting analysis (10).
Tumor irradiations and antitumor activity assays
Luciferase-labeled tumors (LLC-luc or NSCLC-luc) were generated by injecting 5 × 106 tumor cells into the subcutaneous space on the backs, or 1 × 106 into tail veins to generate orthotopic xenografts, of female athymic nude mice or NOD scid mice weighing 18–20 grams obtained from the UT Southwestern Institutional Breeding Core. In general, survival and tumor volume data were graphed from two separate studies, each with 10 combined mice/group. Bioluminescence imaging (BLI), antitumor activity, and survival studies using subcutaneous (300 mm3) or orthotopic NSCLC (A549) xenograft-bearing athymic nude or NSG mice were performed. Subcutaneous LLC tumors (200 mm3) were raised in a same manner. Four weeks after implantation, mice were treated with or without various doses of IR (Gy), using specific collimators for delivery only to the lung or subcutaneous tumor flank areas every other day for 5 treatments. Immediately following IR treatments, mice were then exposed intravenously with or without HPβCD or sublethal doses of β-lap-HPβCD by tail vein injection (9). Animals were irradiated using an X-RAD 320 Small-Animal Irradiator (Precision X-Ray). For combination IR + β-lap-HPβCD treatments, β-lap-HPβCD was administered within 30 minutes post-IR. Tumor volumes were either directly measured using calipers (for subcutaneous models) and/or using BLI-based tumor volume estimates for orthotopic models (7, 10). Long-term survival and target validation assays were performed with log-rank tests for survival (7, 9, 10). All animal studies were carried out under a UT Southwestern Medical Center Institutional Animal Care and Use Committee (IACUC)-approved protocol and in accordance with the guidelines for ethical conduct in the care and use of animals in research.
Biomarker and pharmacokinetic analyses
Pharmacokinetics of β-lap levels in blood, tumor, and normal tissues were assessed by LC/MS-MS analyses following extraction of plasma, tumor, or normal tissue homogenates with acetonitrile (7, 9, 10, 27). An unpaired t test (GraphPad QuickCalcs) was used to evaluate significant differences in β-lap concentrations after different treatments. Pharmacodynamic parameters of PAR formation and γH2AX in normal tissue and tumors were simultaneously performed using pooled samples from three animals per time point (7, 10, 27). Please refer to the Supplementary Information for a more detailed description of the ATP, NAD+/NADH quantification in tumor lysates at indicated treatment conditions.
Statistical analysis
Data (means, ± SDs) were graphed and ANOVA used to compare groups. Two-tailed Student t tests for independent measures with Holm–Sidak correction for multiple comparisons, if >1 comparisons were performed. Minimum replicate size for any experiment was n = 3. Alpha was set to 0.05. Curve fitting and calculation of LD50 values, ANOVA, and two-tailed Student t tests for statistical significance were performed in GraphPad Prism 6.0. Images were representative of experiments or stainings repeated three times (*, P < 0.05; **, P < 0.01; ***, P < 0.001; and ****, P < 0.0001).
Synergy calculations
Synergy interactions between two drugs were evaluated as described previously (10) using the following two methods: (i) direct comparisons made between the effect of combined treatments and the effect of individual drugs in each experiment; and (ii) formal synergy effects evaluations used a strict method proposed by Chou and Talalay (28) and Lee and colleagues, (29) where pooled, multiple dose responses for each of the treatments were required.
Results
β-lap exerts NQO1-dependent radiosensitization of NSCLC cells
A549 NSCLC cells express high levels of NQO1 typical of most NSCLC tumors, with relatively low expression of catalase, so they are a good representative cell line model to use (10). A549 cells are hypersensitive to β-lap, where inhibiting NQO1 activity with DIC greatly suppressed lethality (Fig. 1A). A striking dose response with NQO1+ cells is noted, where 3 μmol/L β-lap (2 hours) results in cell stress with minimal lethality (∼80% survival) and 4 μmol/L (2 hours) causes significant cell death (>LD50) as previously demonstrated to be through programmed necrosis (10). Irradiation of A549 cells with lower doses of IR (1–3 Gy) and then exposing cells immediately thereafter with sublethal doses of β-lap (1–3 μmol/L, Fig. 1A) resulted in significant dose-response increases in lethality (Fig. 1B). Radiosensitization was also noted when cells were exposed to higher IR doses (>3 Gy) in combination with lower concentrations (<LD50) of β-lap (1–3 μmol/L; Fig. 1B). Administration of DIC, a well-utilized NQO1 inhibitor (10), prevented lethality of irradiated (2–7 Gy) A549 cells cotreated with β-lap (3 μmol/L). Similarly, H1650 NSCLC cells exposed to IR (2 Gy) and then treated immediately with various concentrations of β-lap (1–5 μmol/L) for 2 hours postirradiation, exhibited significant synergistic responses compared with 2 Gy IR dose alone, or to various low doses of β-lap alone (Fig. 1C). Radiosensitization of IR + β-lap was prevented by inhibiting NQO1 via coadministration of 50 μmol/L DIC immediately following IR (Fig. 1C), suggesting NQO1-dependent lethal effects. H1650 cells express approximately 100 units of NQO1, which is sufficient to induce a synergistic effect. As reported previously (21, 22), adding low doses of β-lap before IR treatment could lead to no discernable synergistic responses in A549 or H1650 NSCLC cells.
IR-induced DSB repair is inhibited by β-lap
Exposure of NQO1+ A549 cells with β-lap alone causes specific DNA lesions, including AP sites and DNA SSBs (18, 19) that result in the hyperactivation of PARP activity, noted by a significant accumulation of poly(ADP-ribose)–PARP (PAR–PARP) posttranslational protein modification (Fig. 2A); note that PAR–PARP may represent an inactive form of the enzyme (6, 10). PARP hyperactivation is strongly induced within 5–10 minutes of β-lap exposure. Thereafter, PAR–PARP1 levels were dramatically decreased because of loss of NAD+ levels (PARP substrate to produce PAR; refs. 6, 7, 10) within 20–30 minutes during exposure and simultaneous removal of PAR moieties from the PARP1 protein by Poly(ADP-ribosyl) glycohydrolase (PARG; Fig. 2A). Interestingly, the activation of ATM (formation of pS1981-ATM) and formation of γH2AX were significantly delayed in cells exposed to β-lap and were not significantly apparent until after PARP hyperactivation was exhausted. These data suggest that PARP activity binds and protects AP sites and SSBs, but once PARP activity is exhausted cells attempt to either replicate over the damage, or the damage becomes hypersensitive to the stress caused by high levels of β-lap–induced H2O2 (7, 10), and DSBs are formed. ATM is then activated, which phosphorylates H2AX (γH2AX; Fig. 2A). Although major reductions of NAD+/ATP levels are observed in these cells, there appears to be significant levels of nuclear dATP to drive ATM activation and γH2AX formation over the 2-hour time period (ref. 17; Fig. 2A and B).
We hypothesized that exposure of NQO1+ NSCLC cells to IR + β-lap resulted in the inhibition of DSB repair, due to significant losses of NAD+/ATP resulting from PARP hyperactivation and conversion of DNA single-strand lesions caused by IR + β-lap to elevated DSBs. A549 cells were exposed to IR (2 Gy) and then treated with a sublethal dose of β-lap, shown to result in significant radiosensitization (Fig. 1B), to monitor DSB repair over time using 53BP1 foci formation/nucleus assessments (Fig. 2C and D). Data were then graphed showing DSB formation (% nucleus with ≥10 53BP1 foci formation/nucleus) over time (Fig. 2D, Supplementary Fig. S1). β-lap treatment of IR-exposed A549 cells resulted in substantial formation of DSBs per cell nucleus within the 2-hour treatment (Fig. 2C and D) followed by a significant inhibition of DSB repair compared with IR-treated or β-lap–exposed cells alone 4 hours posttreatment (Supplementary Fig. S1A and S1B). Approximately, less than 10% of cells repaired any significant level of DSBs over the 120-minute postirradiation time when β-lap was administered (Fig. 2C and D). In contrast, the significant formation of DSBs noted after IR alone were rapidly repaired within 120 minutes (Fig. 2C and D). Exposure of A549 cells to 3 μmol/L β-lap only caused a low level of delayed DSB formation at 120 minutes, which does not lead to a lethal effect with this dose of drug (Fig. 1B). The effects of β-lap on IR-induced DSB repair were reversed by NQO1 inhibition by DIC.
Radiosensitization of subcutaneous NSCLC by β-lap
We then established subcutaneous xenografts (∼400 mm3) using A549 cells in female athymic nude mice and treated these mice every other day for 5 injections with: (i) HPβCD vehicle alone (intravenously); (ii) IR (2 Gy) alone, only around the xenograft with collimator shielding; (iii) β-lap (20 mg/kg, i.v.) alone; or (iv) IR (2 Gy) + 20 mg/kg HPβCD-β-lap, administered intravenously immediately after IR treatment. Treatment of subcutaneous A549 NSCLC xenografts with β-lap (20 mg/kg, i.v.) alone is a very ineffective therapeutic regimen, where some growth suppression occurs, but regrowth occurs in a rapid manner (ref. 7; Fig. 3A–C). Likewise, IR (2 Gy) alone, given every other day for 5 doses had no significant antitumor or long-term survival effects compared with vehicle (HPβCD) alone (Fig. 3A–C). In contrast, treatment with IR (2 Gy) + β-lap (20 mg/kg, i.v.) exhibited synergistic antitumor effects, with tumor regression from days 5 to 16 compared with vehicle or IR alone and a significant overall apparent cure rate of approximately 80% of exposed mice bearing xenografts. No methemoglobinemia nor normal tissue toxicities were noted at 90 days posttreatment and apparently cured mice lived to >300 days posttreatment. At euthanasia at 350 days, no notable tumor was found within the legs of these IR + β-lap–treated mice. Pharmacokinetic analyses of mice bearing subcutaneous versus orthotopic A549 xenografts grown at the same time in female NOD scid mice revealed that subcutaneous xenografts accumulated 8-fold less drug (β-lap) than in orthotopic A549 xenografts (Fig. 3D, Supplementary Fig. S2). Levels of β-lap in A549 tumor, plasma, lung, and other tissue over time (minutes) are represented in Supplementary Fig. S3, where significant levels of β-lap were found in lung tissue and relatively lower levels found in subcutaneous A549 tumor tissue. The higher levels of the drug in lung tissue, suggested that we examine orthotopic NSCLC antitumor and survival effects. Moreover, A549 NSCLC subcutaneous tumors (200–400 mm3) show significant synergistic responses to 8 Gy (Supplementary Fig. S4A) or 10 Gy (Supplementary Fig. S4B) + β-lap (30 mg/kg, i.v.) given every other day for 5 injections, with no significant methemoglobinemia or weight loss (Supplementary Fig. S5).
Radiosensitization of subcutaneous LLC xenografts that express lower levels of NQO1
LLC cells express significantly less NQO1 activity (∼50 units/mg tissue) compared with A549 NSCLC cells, which express >350 units/mg tissue (10). LLC levels of NQO1 are still approximately 5- to 100-fold above normal tissue expression of NQO1, with liver tissue NQO1 levels being the highest in these mice at approximately 10.3 ± 0.2 units/mg tissue. We examined the ability of β-lap to radiosensitize low NQO1-expressing LLC xenografts (Fig. 4), exposing 300 mm3 xenografts with varying doses of IR (0, 2, 4, 8 Gy) alone, varying doses of β-lap (10 or 20 mg/kg) alone or with 2 Gy IR + 10 or 20 mg/kg β-lap given every other day for 5 treatments (Fig. 4). Minimal antitumor effects were noted with 20 mg/kg β-lap or IR (2 Gy) alone, or with combination IR (2 Gy) + 10 mg/kg β-lap. However, exposure of LLC xenograft–bearing athymic mice to IR (2 Gy) + 20 mg/kg β-lap i.v. immediately after IR treatment caused a significant increase in survival, with approximately 50% apparent cures at 40 days (Fig. 4). These mice exhibited no weight loss or methemolobinemia responses (trouble breathing, lethargy) and remained alive with no apparent LLC tumor levels out to 200 days. In addition, necropsy at 200 days showed no toxicities to normal tissue nor tumor tissues.
β-lap radiosensitizes orthotopic NSCLC tumors with low drug levels
We then examined the ability of β-lap to radiosensitize orthotopic A549 NSCLCs. A549-luc–derived cells were used to develop orthotopic A549 NSCLC xenografts in athymic nude mice (Fig. 5A), where animal CT scanning of orthotopic tumors was visualized and tumor tissue confirmed by IHC analyses (Fig. 5A). Mice bearing significant A549 tumor volumes (Fig. 5A) were treated every other day, for five total doses, with IR (2 Gy) using collimator-guided irradiations with or without 10, 20, or 30 mg/kg HPβCD-β-lap, i.v.. Mice were also treated with β-lap alone at the same doses and frequency. Antitumor effects were noted with 20 and 30 mg/kg β-lap, as well as IR (2 Gy X 5) alone, but significant antitumor effects were noted at 40 days posttreatment with IR + 10, 20, or 30 mg/kg β-lap (Fig. 5B). β-lap alone caused significant increases in survival in a dose-dependent manner, with 10 mg/kg affording little antitumor (Fig. 5B) or overall survival (OS) advantage. In contrast, 20 and 30 mg/kg β-lap alone caused dramatic increases in antitumor effects (Fig. 5B) and survival advantages, with 35- and 50-day increases in survival at LD50 levels (Fig. 5C). IR alone caused significant increases in antitumor effects (Fig. 5B) and survival (Fig. 5C), but no overall apparent cures in A549-bearing mice. In contrast, a significant enhancement of antitumor effects (Fig. 5B) and OS (Fig. 5C), where 50% (for 10 mg/kg β-lap) and 60% (for 20 and 30 mg/kg β-lap) apparent cures were noted at 300 days; synergy in terms of OS between IR + β-lap versus IR or β-lap alone was noted as per Chou and Talalay calculations.(28) No overall weight loss or methemoglobinemia was noted with any IR alone, β-lap alone, or IR + β-lap treatments (Fig. 5D).
Biomarker analyses reveal DSB repair inhibition from PARP hyperactivation in vivo in A549 orthotopic xenografts
As treated above, athymic nude mice bearing A549 orthotopic xenografts were exposed to IR (2 Gy) alone, β-lap (20 mg/kg, i.v.) alone, or to 2 Gy + β-lap (20 mg/kg, i.v.). Mice (3/group) were then euthanized over time for assessment of PAR–PARP levels (Fig. 6A), DSBs via γH2AX (Fig. 6B), as well as NAD+ and ATP levels (Fig. 6C and D). Although β-lap alone caused significant increases in PAR–PARP levels (Fig. 6A), with late-forming DSBs (Fig. 6B) and decreases in NAD+ and ATP (Fig. 6C and D), changes in these same levels in A549 xenografts after IR + β-lap were dramatically greater (Fig. 6A–D). We noted that after IR + β-lap, PAR–PARP formation occurred early within 5–30 minutes then decreased by 120 minutes, whereas DSB formation increased late between 30–120 minutes. These data are consistent with effects of IR + β-lap noted in vitro in A549 culture cells (Fig. 2A and B). In contrast, exposure of tumors with IR (2 Gy) alone resulted in no increases in PAR–PARP formation, DSBs [i.e., γ-H2AX (at 30 or 60 minutes)], or in NAD+ or ATP level losses (Fig. 6A–D). The kinetics of PAR–PARP formation and NAD+ and ATP losses are shown in Supplementary Fig. S6A–S6C.
Discussion
NQO1-bioactivatable drugs, such as β-lap, are competent tumor-selective agents for selective use against cancers that express elevated levels of NQO1, such as found in NSCLCs (ref. 10). Here, we demonstrated that the drug is an efficacious radiosensitizing agent if added immediately after IR treatment in NQO1+ NSCLC. Interestingly, the drug does not work if pretreated prior to IR exposure. It is possible that radiotherapy first transiently primes the tumors to facilitate the accumulation and delivery of β-lap-HPβCD by modifying the local tumor microenvironment (i.e., vascular bursting) as previously reported with other chemical agents (30–32). It is also conceivable that IR treatment could induce NQO1 protein expression, and thus increased enzymatic activity, to generate more reactive oxygen species with the addition of β-lap (33). However, we have previously reported that the lethal effect of β-lap (LD50 values) remains relatively constant at ≥100 units of NQO1 activity in multiple cell lines and cancer types (23). These possibilities require further optimization and investigation. Mechanistically, we envision that the generation of potentially clustered ROS-induced nucleobase damage, DNA SSBs and extensive AP sites by both agents at low doses lead to the hyperactivation of PARP, with dramatic losses of NAD+ and ATP. The contribution of DSBs by IR treatment, with minimally significant levels induced by β-lap (Fig. 2C and D; Supplementary Fig. S1; Fig. 6B), immediately causes lethal DNA lesions with the combination treatments that then may not be repaired efficiently due to the dramatic losses of NAD+ and ATP, which are the energetic currency utilized by various DNA damage response and repair factors for efficient function (Supplementary Fig. S7). Increased PAR–PARP formation with concomitantly lowered levels of NAD+ and ATP are consistent with PARP hyperactivation. We monitored increased DSB formation through assessment of 53BP1 (Fig. 2C and D; Supplementary Fig. S1) and γH2AX (Fig. 6B) when IR + β-lap treatments were given in vitro and in vivo, respectively. All of the radiosensitizing effects of β-lap in NSCLC (Fig. 1C), prostate, (11), or head and neck (23) cancers were prevented by DIC, and were not seen in NQO1− cancer cells. The net effect of IR + β-lap is efficacious radiosensitization of NQO1+ cancer cells, with minimal effects on methemoglobinemia or normal tissue toxicities. The efficacy of IR + β-lap in preclinical models using NSCLCs (Figs. 3–6) against both subcutaneous as well as orthotopic xenografts warrants a phase I clinical trial. ARQ761 is currently in two phase I clinical trials as a monotherapy against solid cancers, as well as used in combination with gemcitabine and abraxane against pancreatic cancers. Therefore, the phase I MTD is already known and will be reported soon.
A major impact of the IR + β-lap combination therapy is the specificity of the combination against specific NQO1 overexpressing cancers. Recently, we reported high levels of NQO1 expression in a majority of NSCLC (10), pancreatic (18, 19), prostate (11), breast (15), and head and neck (23) cancers. Interestingly, we also reported that most NSCLC and pancreatic cancers that overexpressed NQO1 concomitantly underexpress catalase (10) that neutralizes H2O2, thus contributing to the efficacy of IR + β-lap by elevating and prolonging H2O2 half-lives and increasing tumor-selective DNA damage needed for PARP hyperactivation and NAD+ degradation. The exhaustion of PARP activity through hyperactivation most likely further elevates DSB break formation over time. Thus, we anticipate that IR + β-lap treatments would be an extremely efficacious tumor-selective therapy against a wide range of solid cancers. The therapy would allow significant lowering of IR and β-lap doses compared with using either agent alone. The data presented in this study reveal a number of novel biomarkers that could be used to examine the efficacy of β-lap (ARQ761 in clinical form) when specifically combined with IR therapies against NSCLC. Collectively, our in vitro and in vivo data may provide the impetus needed to (i) translate our findings in the clinic; (ii) examine other types of tumors that overexpress NQO1 as a biomarker for selective and synergistic lethality using our combination therapy; and (iii) investigate other novel NQO1-bioactivatable drugs in conjunction with ionizing radiation.
Disclosure of Potential Conflicts of Interest
D. E. Gerber reports receiving commercial research grants from ArQule, Inc. No potential conflict of interests was disclosed by other authors.
Authors' Contributions
Conception and design: E.A. Motea, X. Huang, D.E. Gerber, E.A. Bey, D.A. Boothman
Development of methodology: E.A. Motea, X. Huang, N. Singh, E.A. Bey, D.A. Boothman
Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): E.A. Motea, X. Huang, N. Singh, J.A. Kilgore, N.S. Williams, D.A. Boothman, E.A. Bay
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): E.A. Motea, X. Huang, N. Singh, J.A. Kilgore, N.S. Williams, X.-J. Xie, D.E. Gerber, E.A. Bey, D.A. Boothman
Writing, review, and/or revision of the manuscript: E.A. Motea, X. Huang, N. Singh, D.E. Gerber, M.S. Beg, E.A. Bey, D.A. Boothman
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): X.-J. Xie, D.A. Boothman
Study supervision: E.A. Bey, D.A. Boothman
Others (assisted with acquisition, analysis, and interpretation of pharmacokinetic data): N.S. Williams
Acknowledgments
We are grateful to the UT Southwestern Simmons Comprehensive Cancer Center support grant (5P30CA142543) for use of cores (biostatistics, bioinformatics, preclinical pharmacology) and Jaideep Chaudhary for technical assistance. This work was supported by NIH/NCI (R01CA224493-01, to D.A. Boothman).
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.