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Alterations in the NRF2/KEAP1 pathway result in the constitutive activation of NRF2, leading to the aberrant induction of antioxidant and detoxification enzymes, including NQO1. The NQO1 bioactivatable agent β-lapachone can target cells with high NQO1 expression but relies in the generation of reactive oxygen species (ROS), which are actively scavenged in cells with NRF2/KEAP1 mutations. However, whether NRF2/KEAP1 mutations influence the response to β-lapachone treatment remains unknown. To address this question, we assessed the cytotoxicity of β-lapachone in a panel of NSCLC cell lines bearing either wild-type or mutant KEAP1. We found that, despite overexpression of NQO1, KEAP1 mutant cells were resistant to β-lapachone due to enhanced detoxification of ROS, which prevented DNA damage and cell death. To evaluate whether specific inhibition of the NRF2-regulated antioxidant enzymes could abrogate resistance to β-lapachone, we systematically inhibited the four major antioxidant cellular systems using genetic and/or pharmacologic approaches. We demonstrated that inhibition of the thioredoxin-dependent system or copper-zinc superoxide dismutase (SOD1) could abrogate NRF2-mediated resistance to β-lapachone, while depletion of catalase or glutathione was ineffective. Interestingly, inhibition of SOD1 selectively sensitized KEAP1 mutant cells to β-lapachone exposure. Our results suggest that NRF2/KEAP1 mutational status might serve as a predictive biomarker for response to NQO1-bioactivatable quinones in patients. Further, our results suggest SOD1 inhibition may have potential utility in combination with other ROS inducers in patients with KEAP1/NRF2 mutations., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: 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)., Experimental Design: 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., Results: 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., Conclusions: 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., (©2019 American Association for Cancer Research.)

Tremendous progress has been made in the development of delivery carriers for small RNA therapeutics. However, most achievements have focused on the treatment of liver-associated diseases because conventional lipid and lipidoid nanoparticles (LNPs) readily accumulate in the liver after intravenous (i.v.) administration. Delivering RNAs to other organs and tumor tissues remains an ongoing challenge. Here, we utilized a 540-member combinatorial functional polyester library to discover nanoparticles (NPs) that enable efficacious siRNA delivery to A549 lung cancer cells in vitro and in vivo. PE4K-A13-0.33C6 and PE4K-A13-0.33C10 NPs were efficiently internalized into A549-Luc cells within 4 h. The addition of PEG 2000 DMG lipid or Pluronic F-127 onto the surface of the polyplexes reduced the surface charge of NPs, resulting in an increase of serum stability. We then explored aerosol delivery of stabilized PE4K-A13-0.33C6 and PE4K-A13-0.33C10 NPs to implanted orthotopic lung tumors. We found that by altering the administration route from i.v. to aerosol, the NPs could avoid liver accumulation and instead be specifically localized only in the lungs. This resulted in significant gene silencing in the A549 orthotopic lung tumors. Due to the ability to deliver siRNA to non-liver targets, this approach provides a privileged route for gene silencing in the lungs., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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 cancers, 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., (Published by Elsevier Inc.)

Purpose: Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related deaths worldwide due to the limited availability of effective therapeutic options. For instance, there are no effective strategies for NSCLCs that harbor mutant KRAS, the most commonly mutated oncogene in NSCLC. Thus, our purpose was to make progress toward the generation of a novel therapeutic strategy for NSCLC., Experimental Design: We characterized the effects of suppressing focal adhesion kinase (FAK) by RNA interference (RNAi), CRISPR/CAS9 gene editing or pharmacologic approaches in NSCLC cells and in tumor xenografts. In addition, we tested the effects of suppressing FAK in association with ionizing radiation (IR), a standard-of-care treatment modality., Results: FAK is a critical requirement of mutant KRAS NSCLC cells. With functional experiments, we also found that, in mutant KRAS NSCLC cells, FAK inhibition resulted in persistent DNA damage and susceptibility to exposure to IR. Accordingly, administration of IR to FAK-null tumor xenografts causes a profound antitumor effect in vivo CONCLUSIONS: FAK is a novel regulator of DNA damage repair in mutant KRAS NSCLC and its pharmacologic inhibition leads to radiosensitizing effects that could be beneficial in cancer therapy. Our results provide a framework for the rationale clinical testing of FAK inhibitors in NSCLC patients. Clin Cancer Res; 22(23); 5851-63. ©2016 AACR., Competing Interests: No potential conflicts of interest were disclosed DISCLOSURE OF POTENTIAL CONFLICT OF INTEREST The other authors disclosed no potential conflicts of interest., (©2016 American Association for Cancer Research.)

Unlabelled: The fundamental role that NAD(P)H/quinone oxidoreductase 1 (NQO1) plays, in normal cells, as a cytoprotective enzyme guarding against stress induced by reactive oxygen species (ROS) is well documented. However, what is not known is whether the observed overexpression of NQO1 in neoplastic cells contributes to their survival. The current study discovered that depleting NQO1 expression in A549 and H292 lung adenocarcinoma cells caused an increase in ROS formation, inhibited anchorage-independent growth, increased anoikis sensitization, and decreased three-dimensional tumor spheroid invasion. These in vivo data further implicate tumor-NQO1 expression in a protumor survival role, because its depletion suppressed cell proliferation and decreased lung tumor xenograft growth. Finally, these data reveal an exploitable link between tumor-NQO1 expression and the survival of lung tumors because NQO1 depletion significantly decreased the percentage of ALDH((high)) cancer cells within the tumor population., Implications: Loss of tumor-NQO1 expression inhibits tumor growth and suggests that novel therapeutics directed at tumor-NQO1 may have clinical benefit., (©2015 American Association for Cancer Research.)

Purpose: Preclinical studies demonstrated anti-tumor efficacy of the combination of the histone deacetylase (HDAC) inhibitor romidepsin plus erlotinib in non-small cell lung cancer (NSCLC) models that were insensitive to erlotinib monotherapy. We therefore studied this combination in a phase 1 clinical trial in previously treated advanced NSCLC., Methods: Romidepsin (8 or 10mg/m(2)) was administered intravenously on days 1, 8, and 15 every 28 days in combination with erlotinib (150 mg orally daily), with romidepsin monotherapy lead-in during Cycle 1. Correlative studies included peripheral blood mononuclear cell HDAC activity and histone acetylation status, and EGFR pathway activation status in skin biopsies., Results: A total of 17 patients were enrolled. Median number of prior lines of therapy was 3 (range 1-5). No cases had a sensitizing EGFR mutation. The most common related adverse events were nausea, vomiting, and fatigue (each 82%), diarrhea (65%), anorexia (53%), and rash (41%). Dose-limiting nausea and vomiting occurred at the romidepsin 10 mg/m(2) level despite aggressive antiemetic prophylaxis and treatment. Among 10 evaluable patients, the best response was stable disease (n=7) and progressive disease (n=3). Median progression-free survival (PFS) was 3.3 months (range 1.4-16.5 months). Prolonged PFS (>6 months) was noted in a KRAS mutant adenocarcinoma and a squamous cell cancer previously progressed on erlotinib monotherapy. Romidepsin monotherapy inhibited HDAC activity, increased histone acetylation status, and inhibited EGFR phosphorylation., Conclusions: Romidepsin 8 mg/m(2) plus erlotinib appears well tolerated, has evidence of disease control, and exhibits effects on relevant molecular targets in an unselected advanced NSCLC population., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Lung cancer is the leading cause of cancer-related deaths with current chemotherapies lacking adequate specificity and efficacy. Beta-lapachone (beta-lap) is a novel anticancer drug that is bioactivated by NAD(P)H:quinone oxidoreductase 1, an enzyme found specifically overexpressed in non-small cell lung cancer (NSCLC). Herein, we report a nanotherapeutic strategy that targets NSCLC tumors in two ways: (a) pharmacodynamically through the use of a bioactivatable agent, beta-lap, and (b) pharmacokinetically by using a biocompatible nanocarrier, polymeric micelles, to achieve drug stability, bioavailability, and targeted delivery. Beta-lap micelles produced by a film sonication technique were small ( approximately 30 nm), displayed core-shell architecture, and possessed favorable release kinetics. Pharmacokinetic analyses in mice bearing subcutaneous A549 lung tumors showed prolonged blood circulation (t(1/2), approximately 28 h) and increased accumulation in tumors. Antitumor efficacy analyses in mice bearing subcutaneous A549 lung tumors and orthotopic Lewis lung carcinoma models showed significant tumor growth delay and increased survival. In summary, we have established a clinically viable beta-lap nanomedicine platform with enhanced safety, pharmacokinetics, and antitumor efficacy for the specific treatment of NSCLC tumors., ((c)2010 AACR.)

Non-small cell lung cancer (NSCLC) is a leading cause of cancer-related death worldwide. NSCLC often harbors oncogenic K-RAS mutations that lead to the aberrant activation of several intracellular networks including the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) signaling pathway. Oncogenic K-RAS predicts poor prognosis and resistance to treatment with ionizing radiation (IR). Oncogenic K-Ras expression in the respiratory epithelium is sufficient to initiate NSCLC tumorigenesis, which requires the catalytic subunit of PI3K. Thus, effective inhibition of the PI3K signaling should lead to significant antitumor effects. However, therapy with rapamycin analogues has yielded disappointing results due in part to compensatory up-regulation of AKT. We hypothesized that dual PI3K/mTOR blockade would overcome these limitations. We tested this hypothesis with BEZ235, a novel dual PI3K/mTOR inhibitor that has recently entered clinical development. We found that BEZ235 induces a striking antiproliferative effect both in transgenic mice with oncogenic K-RAS-induced NSCLC and in NSCLC cell lines expressing oncogenic K-RAS. We determined that treatment with BEZ235 was not sufficient to induce apoptosis. However, we found that dual PI3K/mTOR blockade effectively sensitizes NSCLC expressing oncogenic K-RAS to the proapoptotic effects of IR both in vitro and in vivo. We conclude that dual PI3K/mTOR blockade in combination with IR may benefit patients with NSCLC expressing oncogenic K-RAS. These findings may have general applicability in cancer therapy, because aberrant activation of PI3K occurs frequently in human cancer.

Magnetic resonance imaging is a powerful clinical imaging technique that allows for noninvasive tomographic visualization of anatomic structures with high spatial resolution and soft tissue contrast. However, its application in molecular imaging of cancer has been limited by the lack of sensitivity and detection accuracy in depicting the biochemical expression of these diseases. Here, we combine an ultrasensitive design of superparamagnetic polymeric micelles (SPPM) and an off-resonance saturation (ORS) method to enhance the imaging efficacy of tumor biomarkers in vivo. SPPM nanoparticles encoded with cyclic(RGDfK) were able to target the alpha(v)beta(3)-expressing microvasculature in A549 non-small cell lung tumor xenografts in mice. ORS greatly improved tumor detection accuracy over the conventional T(2)*-weighted method by its ability to turn "ON" the contrast of SPPM. This combination of ORS imaging with a tumor vasculature-targeted, ultrasensitive SPPM design offers new opportunities in molecular imaging of cancer.

Lung cancer is the number one cause of cancer-related deaths in the world. Patients treated with current chemotherapies for non-small-cell lung cancers (NSCLCs) have a survival rate of approximately 15% 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. beta-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 beta-lapachone. NSCLC cells were killed in an NQO1-dependent manner by beta-lapachone (LD50, approximately 4 microM) with a minimum 2-h exposure. Kinetically, beta-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 mu-calpain activation and apoptosis. Beta-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 Ca2+ chelator. NQO1- cells (H596, IMR-90) or dicoumarol-exposed NQO1+ A549 cells were resistant (LD50, >40 microM) to ROS formation and all cytotoxic effects of beta-lapachone. Our data indicate that the most efficacious strategy using beta-lapachone in chemotherapy was to deliver the drug in short pulses, greatly reducing cytotoxicity to NQO1- "normal" cells. beta-Lapachone killed cells in a tumorselective manner and is indicated for use against NQO1+ NSCLC cancers.

In order to investigate the biochemical events involved in potentially lethal DNA damage repair (PLDR), we have identified a pleiotropic protein expression response that is activated upon X-irradiation of confluence-arrested human malignant melanoma (U1-Mel) cells. Plateau-phase U1-Mel cells were selected because of their extraordinary capacity for PLDR. Eight major X-ray-induced polypeptides (XIPs) of Mr 126,000-275,000 (i.e., XIP126 through XIP275) were detected by resolving L-[35S]methionine-labeled whole cell extracts using two-dimensional gel electrophoresis. XIPs were found in unirradiated, proliferating U1-Mel cells, shut off under plateau-phase conditions and resynthesized in response to X-irradiation. The expression of three classes of proteins was affected by X-irradiation. Class I proteins, XIP145 and XIP269, were induced linearly with increasing X-ray doses. The rate of synthesis of class II proteins, XIP126, XIP135, XIP138, XIP141, XIP147, and XIP275, increased linearly with low X-irradiation doses, but plateaued at doses of 150-250 cGy. In contrast, the expression of class III proteins, 47,000 and 254,000 Mr proteins, decreased with increasing X-ray doses. Tumor, cancer-prone, and normal human cells, which represent a wide range of cells with varied repair capacities, were investigated to better understand the role of XIPs in DNA damage responses. X-irradiated normal and tumor cells induced the synthesis of XIP145 and XIP269. A strong correlation between the induction of XIP269 and PLDR capacity, as measured by delayed plating of plateau-phase cells, was noted. XIP269 was present in six of seven normal and tumor cells types, but was completely absent in cells from patients with Bloom's syndrome and ataxia telangiectasia. X-irradiated Fanconi's anemia and xeroderma pigmentosum cells synthesized low levels of XIP269. The majority of XIPs synthesized by X-irradiated cells from cancer-prone patients were of low molecular weights. A number of XIP expression characteristics suggest their role in either gross chromosomal PLDR and/or in X-ray adaptivity responses: (a) XIP expression was inhibited by 1 microgram/ml cycloheximide, a dose which decreased survival 6-fold during PLDR holding and resulted in greater than 80% inhibition of protein synthesis; (b) XIP expression was specific for ionizing radiation damage, since heat shock, hypoxia, and alkylating agents failed to induce their synthesis; (c) the time course of expression was long, with the first appearance of XIPs at 3 h and maximal expression at 4 h.

The metabolic products formed and incorporated into the nucleic acids (RNA and DNA) of mice bearing Lewis lung carcinoma (LLC) following optimal doses of 5-fluorouracil (FUra), 5-fluoro-2'-deoxyuridine (FdUrd), and 5-fluoro-2'-deoxycytidine (FdCyd) coadministered with tetrahydrouridine (H4Urd), a potent inhibitor of cytidine deaminase, were examined. Treatment with FdCyd plus H4Urd resulted in a tumor-selective incorporation and formation of antimetabolites compared to either FUra or FdUrd treatments. Between 45- and greater than 5400-fold higher levels of the potent thymidylate synthetase inhibitor, 5-fluoro-2'-deoxyuridylate (FdUMP), were formed in tumor than in any of the normal tissues analyzed. RNA-level antimetabolites (FUra, 5-fluorouridine, and 5-fluorouridylate) were also between 3 and greater than 990-fold higher in tumor compared to normal tissue following FdCyd plus H4Urd administration. DNA-level antimetabolites (FdCyd, 5-fluorodeoxycytidylate, FdUrd, and FdUMP) were from 2- to 6-fold higher in tumor compared to normal tissue. FUra and FdUrd treatments resulted in between 3 and greater than 1300-fold higher RNA-level antimetabolites and from 4 to greater than 1020-fold higher FdUMP pools in normal tissues than FdCyd plus H4Urd treatment. DNA-level antimetabolites were also from 4- to 32-fold higher in normal tissues following optimal doses of FUra or FdUrd. In tumor tissue, optimal doses of FUra or FdUrd resulted in lower (a) FdUMP levels (5- to 2-fold), (b) RNA-level antimetabolites (6- to 3-fold), and (c) DNA-level antimetabolites (10- to 4-fold) compared to an optimal dosage of FdCyd plus H4Urd. In serum, the administration of H4Urd resulted in the protection of FdCyd from systemic catabolism, unlike that found with FUra or FdUrd. Substantial levels of FdUMP, FUrd, and FUMP were noted in serum following FUra or FdUrd treatment. The formation of di- and triphosphate antimetabolite pools and the incorporation of antimetabolites into the RNA and DNA of normal and tumor tissues demonstrated trends similar to those mentioned above with nucleoside, mononucleotide, and free base pools. H4Urd treatment of 25 mg/kg did not affect the elevated levels of deoxycytidine kinase or deoxycytidylate deaminase in LLC tumor tissue or the low levels found in normal tissue. A critical feature of this chemotherapeutic strategy using FdCyd plus H4Urd was that the elevated level of cytidine deaminase in LLC tumor tissue was inhibited less than 10% by the administration of 25 mg/kg H4Urd, whereas deoxycytidine deaminase activities in normal tissues (including bone marrow and intestine) were inhibited greater than 93%.(ABSTRACT TRUNCATED AT 400 WORDS)