Shannon L. Winski

NAD(P)H:quinone oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms

NAD(P)H:quinone oxidoreductase 1 (NQO1) is an obligate two-electron reductase that is involved in chemoprotection and can also bioactivate certain antitumor quinones. This review focuses on detoxification reactions catalyzed by NQO1 and its role in antioxidant defense via the generation of antioxidant forms of ubiquinone and vitamin E. Bioactivation reactions catalyzed by NQO1 are also summarized and the development of new antitumor agents for the therapy of solid tumors with marked NQO1 content is reviewed. NQO1 gene regulation and the role of the antioxidant response element and the xenobiotic response element in transcriptional regulation is summarized. An overview of genetic polymorphisms in NQO1 is presented and biological significance for chemoprotection, cancer susceptibility and antitumor drug action is discussed.

Genotype-phenotype relationships in studies of a polymorphism in NAD(P)H:quinone oxidoreductase 1.

The NAD(P)H:quinone oxidoreductase 1 (NQO1) genotype-phenotype relationship was examined in individuals with a polymorphism in NQO1. The polymorphism comprises a C to T base change at position 609 of the human NQO1 cDNA (C609T) and codes for a proline to serine substitution in the amino acid structure of the NQO1 protein. Genotyping was performed by polymerase chain reaction-restriction fragment length polymorphism analysis of genomic DNA. Phenotyping was performed using enzyme activity assays and/or immunoblotting of human tumor cell lines and of saliva and bone marrow samples from healthy donors. Phenotyping of uninvolved lung and lung tumors from archived biopsy material was performed by immunohistochemistry. NQO1 activity and protein could be detected in wild-type (C/C) human tumor cells (HT-29) under conditions where NQO1 protein could not be detected in cells (BE) homozygous for the C609T change (T/T). Trace levels of NQO1 protein could be detected in BE cells; however, when immunoblots were subjected to chemiluminescence detection for prolonged periods. In saliva samples from 11 individuals carrying the homozygous C609T change (T/T), no NQO1 protein could be detected even after prolonged chemiluminescence detection. The amount of NQO1 protein present in saliva was quantified and found to be significantly less in heterozygous individuals (C/T) than in wild-type individuals (C/C). In bone marrow stromal cultures, both NQO1 activity and protein could be detected in heterozygotes (C/T) and in wild-type (C/C) samples. In a bone marrow stromal culture from an individual genotyped as T/T at position 609, no NQO1 protein or activity could be detected. NQO1 is elevated in non-small cell lung cancers and could be readily observed as intense immunostaining throughout lung adenocarcinomas genotyped as C/C but no immunostaining could be detected in adenocarcinomas genotyped as T/T at position 609. NQO1 is expressed in normal human lung but is localized to respiratory epithelium and to vascular endothelium. In normal lung tissue from individuals genotyped as T/T, no or faint immunostaining for NQO1 could be detected in either respiratory epithelium or vascular endothelium. These results demonstrate that tissues from individuals homozygous for the C609T change have no detectable or, at best, only trace amounts of NQO1 protein and are devoid of NQO1 activity.

Relationship between NAD(P)H:quinone oxidoreductase 1 (NQO1) levels in a series of stably transfected cell lines and susceptibility to antitumor quinones

To investigate the importance of NAD(P)H:quinone oxidoreductase 1 (or DT-diaphorase; NQO1) in the bioactivation of antitumor quinones, we established a series of stably transfected cell lines derived from BE human colon adenocarcinoma cells. BE cells have no NQO1 activity due to a genetic polymorphism. The new cell lines, BE-NQ, stably express wild-type NQO1. BE-NQ7 cells expressed the highest level of NQO1 and were more susceptible [determined by the thiazolyl blue (MTT) assay] to known antitumor quinones and newer clinical candidates. Inhibition of NQO1 by pretreatment with an irreversible inhibitor, ES936 [5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione], protected BE-NQ7 cells from toxicity induced by streptonigrin, ES921 [5-(aziridin-1-yl)-3-(hydroxymethyl)-1,2-dimethylindole-4,7-dione], and RH1 [2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone]. RH1 was evaluated further by clonogenic assay for cytotoxic response and was more cytotoxic to BE-NQ7 cells than to BE cells. Cytotoxicity was abrogated by inhibition of NQO1 with ES936 pretreatment. Using a comet assay to evaluate DNA cross-linking, BE-NQ7 cells demonstrated significantly higher DNA cross-links than did BE cells in response to RH1 treatment. DNA cross-linking in BE-NQ7 cells was observed at very low concentrations of RH1 (5 nM), confirming that NQO1 activates RH1 to a potent cross-linking species. Further studies using streptonigrin, ES921, and RH1 were undertaken to analyze the relationship between NQO1 activity and quinone toxicity. Toxicity of these compounds was measured in a panel of BE-NQ cells expressing a range of NQO1 activity (23-433 nmol/min/mg). Data obtained suggest a threshold for NQO1-induced toxicity above 23 nmol/min/mg and a sharp dose-response curve between the no effect level of NQO1 (23 nmol/min/mg) and the maximal effect level (>77 nmol/min/mg). These data provide evidence that NQO1 can bioactivate antitumor quinones in this system and suggest that a threshold level of NQO1 activity is required to initiate toxic events.

Structure-based development of anticancer drugs: complexes of NAD(P)H:quinone oxidoreductase 1 with chemotherapeutic quinones.

BACKGROUND NAD(P)H:quinone acceptor oxidoreductase (QR1) protects animal cells from the deleterious and carcinogenic effects of quinones and other electrophiles. Remarkably, the same enzyme activates cancer prodrugs that become cytotoxic only after two-electron reduction. QR1s ability to bioactivate quinones and its elevated expression in many human solid tumors makes this protein an excellent target for enzyme-directed drug development. Until now, structural analysis of the mode of binding of chemotherapeutic compounds to QR1 was based on model building using the structures of complexes with simple substrates; no structure of complexes of QR1 with chemotherapeutic prodrugs had been reported. RESULTS Here we report the high-resolution crystal structures of complexes of QR1 with three chemotherapeutic prodrugs: RH1, a water-soluble homolog of dimethylaziridinylbenzoquinone; EO9, an aziridinylindolequinone; and ARH019, another aziridinylindolequinone. The structures, determined to resolutions of 2.0 A, 2.5 A, and 1.86 A, respectively, were refined to R values below 21% with excellent geometry. CONCLUSIONS The structures show that compounds can bind to QR1 in more than one orientation. Surprisingly, the two aziridinylindolequinones bind to the enzyme in different orientations. The results presented here reveal two new factors that must be taken into account in the design of prodrugs targeted for activation by QR1: the enzyme binding site is highly plastic and changes to accommodate binding of different substrates, and homologous drugs with different substituents may bind to QR1 in different orientations. These structural insights provide important clues for the optimization of chemotherapeutic compounds that utilize this reductive bioactivation pathway.

OSI-930: A Novel Selective Inhibitor of Kit and Kinase Insert Domain Receptor Tyrosine Kinases with Antitumor Activity in Mouse Xenograft Models

OSI-930 is a novel inhibitor of the receptor tyrosine kinases Kit and kinase insert domain receptor (KDR), which is currently being evaluated in clinical studies. OSI-930 selectively inhibits Kit and KDR with similar potency in intact cells and also inhibits these targets in vivo following oral dosing. We have investigated the relationships between the potency observed in cell-based assays in vitro, the plasma exposure levels achieved following oral dosing, the time course of target inhibition in vivo, and antitumor activity of OSI-930 in tumor xenograft models. In the mutant Kit-expressing HMC-1 xenograft model, prolonged inhibition of Kit was achieved at oral doses between 10 and 50 mg/kg and this dose range was associated with antitumor activity. Similarly, prolonged inhibition of wild-type Kit in the NCI-H526 xenograft model was observed at oral doses of 100 to 200 mg/kg, which was the dose level associated with significant antitumor activity in this model as well as in the majority of other xenograft models tested. The data suggest that antitumor activity of OSI-930 in mouse xenograft models is observed at dose levels that maintain a significant level of inhibition of the molecular targets of OSI-930 for a prolonged period. Furthermore, pharmacokinetic evaluation of the plasma exposure levels of OSI-930 at these effective dose levels provides an estimate of the target plasma concentrations that may be required to achieve prolonged inhibition of Kit and KDR in humans and which would therefore be expected to yield a therapeutic benefit in future clinical evaluations of OSI-930.

Development of a New Isogenic Cell-Xenograft System for Evaluation of NAD(P)H:Quinone Oxidoreductase-Directed Antitumor Quinones Evaluation of the Activity of RH1

Purpose: The purpose of our study was to develop and validate an isogenic cell line pair that differs only in the expression of NAD(P)H:quinone oxidoreductase (NQO1) that can be used to examine the in vitro and in vivo role of NQO1 in the bioactivation of the antitumor quinone RH1 (2,5-diaziridinyl-3-(hydroxymethyl)-6-methyl-1,4-benzoquinone), a compound currently in Phase I clinical trials. Experimental Design: MDA-MB-468 (MDA468) human breast adenocarcinoma cells, homozygous for a polymorphism in NQO1 (NQO1*2/*2) and with low levels of NQO1 activity, were stably transfected with human NQO1 to generate a clone (NQ16) expressing very high NQO1 activity. We examined levels of other reductases and looked at biochemical systems that might influence response to antitumor quinones to validate that the isogenic cell line pair differed only in the expression of NQO1. The 3-(4,5-dimethylthiazol-2,5-diphenyl)tetrazolium (MTT) assay was used to determine the differential toxicity of various quinones, including the most recent NQO1-directed antitumor quinone, RH1, between the two cell lines. Human tumor xenografts were established from both MDA468 and NQ16 cells, and the antitumor activity of RH1 was evaluated. Results: Levels of cytochrome P450 reductase, cytochrome b5 reductase, soluble thiols, and superoxide dismutase in the NQ16 line were unchanged from the parental line. The functional significance of wild-type NQO1 expression was confirmed by measurement of the differential toxicity of compounds activated or deactivated by NQO1 in the two cell lines. The toxicity of the NQO1-directed antitumor quinones RH1 and streptonigrin were markedly greater and the toxicity of menadione, which is detoxified by NQO1, was ameliorated in the NQ16 line. High levels of NQO1 expression were observed throughout xenograft tumors established from the NQ16 cell line. RH1 treatment was effective at statistically reducing tumor volume in NQ16 xenografts at all of the doses tested [0.1, 0.2, 0.4 mg/kg every day for 5 days), whereas only the highest dose of RH1 resulted in a significant reduction in tumor volume in MDA468 xenografts. Conclusions: The MDA468/NQ16 isogenic cell line pair is a useful model system for evaluating the role of NQO1 in the bioactivation of antitumor quinones in both cell lines and xenografts. In addition, our data demonstrate that the novel antitumor quinone RH1, is effectively activated by NQO1 both in vitro and in vivo.

A Phase I Study of Oral ARRY-614, a p38 MAPK/Tie2 Dual Inhibitor, in Patients with Low or Intermediate-1 Risk Myelodysplastic Syndromes

Purpose: Data suggest that activity of p38 MAPK and Tie2 kinases is dysregulated in myelodysplastic syndromes (MDS) and may be targets for novel therapies. A phase I study of ARRY-614, an oral dual inhibitor of p38 MAPK and Tie2, was conducted in patients with low or intermediate-1 International Prognostic Scoring System risk MDS to evaluate safety, pharmacokinetics (PK), pharmacodynamics (PD), and preliminary responses by International Working Group 2006 criteria. Experimental Design: Forty-five patients received ARRY-614 either once daily or twice daily in dose escalation (400, 600, 900, or 1,200 mg once daily; 200 or 300 mg twice daily) or expansion cohorts. Results: The 300 mg twice daily schedule was not tolerated, and an MTD was not reached for once daily dosing. Treatment-related adverse events were primarily grade 1–2, with the most common being rash, diarrhea, dry skin, fatigue and anorexia. Interpatient PK variability was high, although exposure was sufficient to achieve reduction in p38 MAPK activation in bone marrow and in the levels of circulating biomarkers. Disease responses were observed in 14 of 44 (32%) evaluable patients, 13 (93%) of whom had previously been treated with a hypomethylating agent. Responses were observed in all lineages, with 5 patients experiencing bilineage responses. Three of 25 red blood cell transfusion-dependent (TD) patients achieved transfusion independence (TI) and 5 of 7 platelet TD patients achieved TI. Conclusions: ARRY-614 was well tolerated and has sufficient activity to warrant further evaluation in this patient population. We recommend 1,200 mg once daily as the optimal dose for further study. Clin Cancer Res; 21(5); 985–94. ©2014 AACR.

Indolequinone antitumor agents: relationship between quinone structure and rate of metabolism by recombinant human NQO1.

A series of indolequinones bearing various functional groups has been synthesized, and the effects of substituents on the metabolism of the quinones by recombinant human NAD(P)H:quinone oxidoreductase (NQO1), and on the toxicity toward nonsmall cell lung cancer cells with either high NQO1 activity (H460) or with no detectable activity (H596) were studied.

Abstract 2515: Preclinical Development of ARRY-162, A Potent and Selective MEK 1/2 Inhibitor

Activation of the Ras/Raf/MEK/MAP kinase pathway is implicated in uncontrolled cell proliferation and tumor growth. Inappropriate activation of the RAS pathway can occur through several distinct mechanisms, including activating mutations in Ras and B-raf, or activated growth factor-signaling, cytokines and stress responses. Mutated, oncogenic forms of Ras are found in 50% of colon, 90% of pancreatic, and 30% of lung cancers. Also, B-Raf mutations have been identified in more than 60% of malignant melanomas and from 40-70% of papillary thyroid cancers. MEK, a dual specific kinase, is a key player in this pathway; it is downstream of both Ras and Raf and activates ERK1/2 through phosphorylation of key tyrosine and threonine residues. These data suggest that targeting MEK can inhibit cancer cell signaling mediated by a wide variety of signals, making MEK an attractive target for the treatment of cancer. We have discovered ARRY-162, a novel ATP-uncompetitive inhibitor of MEK 1/2, which is un-competitive with respect to ATP. ARRY-162 has nanomolar activity against purified MEK enzyme (IC50 = 12 nM) and is highly selective. It has been evaluated against 220 serine/threonine and tyrosine kinases with no inhibitory activity observed up to 20 μM. ARRY-162 inhibits both basal and induced levels of ERK phosphorylation in numerous cancer cell lines with IC50s as low as 5 nM. ARRY-162 is especially potent at inhibiting the cell proliferation of mutant B-Raf and Ras cell lines such as HT29, Malme-3M, SK-MEL-2, COLO 205, SK-MEL-28 and A375 (IC50s from 30-250 nM). In vivo, ARRY-162 has demonstrated efficacy in several xenograft tumor models in mice, including HT29, BxPC3, MIA PaCa2, A549, LoVo, Calu6, DU145 and COLO 205. In the HT29 and in the COLO 205 colon carcinoma models, dose-dependent inhibition of tumor growth (up to 75% TGI) was observed at doses ranging from 3 to 30 mg/kg, QD, PO for 21 days. In the Colo-205 colon carcinoma model, significant tumor regressions were observed with 50% partial responses and 13% complete responses at 30 mg/kg, PO, QD. In the BxPC3 pancreatic carcinoma model (which does not harbor either Ras or Raf mutations), tumor growth inhibition (∼70% TGI) and 13% partial responses were seen at doses of 30 mg/kg, QD, PO for 21 days. Consistent with ARRY-1629s mechanism of action, tumor growth inhibition correlates with decreased phospho-ERK levels in tumor xenografts. In addition to its potency against MEK, this compound demonstrates other desirable attributes for development including good physical chemical characteristics, low clearance, medium-to-high Caco-2 permeability and minimal predicted drug-drug interactions. With preclinical efficacy and safety studies on ARRY-162 completed, this compound has entered clinical development for treatment of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2515.

Abstract 852: ARRY-380, a potent, small molecule inhibitor of ErbB2, increases survival in intracranial ErbB2+ xenograft models in mice

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL ARRY-380 is an orally active, potent small molecule targeting ErbB2 inhibitor currently in clinical development in patients with ErbB2+ metastatic breast cancer (MBC). This compound has shown excellent activity in numerous SC mouse tumor models including breast (BT-474, MDA-MB-453), ovarian (SK-OV-3) and gastric (N87) carcinoma models. In breast cancer patients, brain metastases are a serious unmet medical need. Patients with ErbB2+ breast cancer have a significantly increased incidence of brain metastases following trastuzumab therapy. Here we demonstrate significant single agent activity of ARRY-380 in two ErbB2+ intracranial mouse xenograft models. For these studies, female nude mice received intracranial implantations of tumor cells (either NCI-N87 or BT-474) by direct injection into the brain parenchyma (via the sagittal suture). In pilot studies, we demonstrated that the blood brain barrier was not disrupted by mechanical injections and that increasing tumor burden correlates negatively with neurologic outcome, body weight and survival. In the N87 studies, animals received treatments beginning on Day 2 post-implantation and continuing for up to 6 weeks. Dose groups (n=12/group) were vehicle, ARRY-380 at MTD (75 mg/kg, PO, BID) or lapatinib at MTD (50 mg/kg, PO, BID). All animals in the vehicle- or lapatinib-treated groups did not survive beyond Day 22. In the ARRY-380-treated-group, 75% of the animals were alive on Day 43. Brain PK/PD was also evaluated in the N87 model. ARRY-380 and its active metabolite caused a significant reduction in brain pErbB2 (80%). In the BT-474 model, animals received treatments beginning on Day 2 post-implantation and continuing for up to 8 weeks. Dose groups (n=13/group) were vehicle, ARRY-380 at MTD (75 mg/kg, PO, BID), lapatinib at MTD (50 mg/kg, PO, BID) or neratinib at MTD (40 mg/kg, PO, QD). On Day 56, survival in the ARRY-380 group was 69% while survival rates in the vehicle, lapatinib or neratinib-treated groups were 23%, 8% and 23%, respectively. Thus ARRY-380 treatment significantly enhances survival in two ErbB2 driven intracranial tumor xenograft models, with superior activity compared to other ErbB2 agents in these studies. Additionally, ARRY-380 has demonstrated durable clinical activity in heavily pre-treated patients with ErbB2+ MBC. These preclinical and clinical data suggest that ARRY-380 may provide benefit to patients with ErbB2+ MBC with brain metastases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 852. doi:1538-7445.AM2012-852