Tryptophan fuels MYC-dependent liver tumorigenesis through indole 3-pyruvate synthesis.

Cancer cells exhibit distinct metabolic activities and nutritional dependencies compared to normal cells. Thus, characterization of nutrient demands by individual tumor types may identify specific vulnerabilities that can be manipulated to target the destruction of cancer cells. We find that MYC-driven liver tumors rely on augmented tryptophan (Trp) uptake, yet Trp utilization to generate metabolites in the kynurenine (Kyn) pathway is reduced. Depriving MYC-driven tumors of Trp through a No-Trp diet not only prevents tumor growth but also restores the transcriptional profile of normal liver cells. Despite Trp starvation, protein synthesis remains unhindered in liver cancer cells. We define a crucial role for the Trp-derived metabolite indole 3-pyruvate (I3P) in liver tumor growth. I3P supplementation effectively restores the growth of liver cancer cells starved of Trp. These findings suggest that I3P is a potential therapeutic target in MYC-driven cancers. Developing methods to target this metabolite represents a potential avenue for liver cancer treatment.

Targeting of neuroblastoma cells through Kynurenine-AHR pathway inhibition.

Neuroblastoma poses significant challenges in clinical management. Despite its relatively low incidence, this malignancy contributes disproportionately to cancer-related childhood mortality. Tailoring treatments based on risk stratification, including MYCN oncogene amplification, remains crucial, yet high-risk cases often confront therapeutic resistance and relapse. Here, we explore the aryl hydrocarbon receptor (AHR), a versatile transcription factor implicated in diverse physiological functions such as xenobiotic response, immune modulation, and cell growth. Despite its varying roles in malignancies, AHR's involvement in neuroblastoma remains elusive. Our study investigates the interplay between AHR and its ligand kynurenine (Kyn) in neuroblastoma cells. Kyn is generated from tryptophan (Trp) by the activity of the enzymes indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO2). We found that neuroblastoma cells displayed sensitivity to the TDO2 inhibitor 680C91, exposing potential vulnerabilities. Furthermore, combining TDO2 inhibition with retinoic acid or irinotecan (two chemotherapeutic agents used to treat neuroblastoma patients) revealed synergistic effects in select cell lines. Importantly, clinical correlation analysis using patient data established a link between elevated expression of Kyn-AHR pathway genes and adverse prognosis, particularly in older children. These findings underscore the significance of the Kyn-AHR pathway in neuroblastoma progression, emphasizing its potential role as a therapeutic target.

Augmented Concentration of Isopentyl-Deoxynyboquinone in Tumors Selectively Kills NAD(P)H Quinone Oxidoreductase 1-Positive Cancer Cells through Programmed Necrotic and Apoptotic Mechanisms.

Lung and breast cancers rank as two of the most common and lethal tumors, accounting for a substantial number of cancer-related deaths worldwide. While the past two decades have witnessed promising progress in tumor therapy, developing targeted tumor therapies continues to pose a significant challenge. NAD(P)H quinone oxidoreductase 1 (NQO1), a two-electron reductase, has been reported as a promising therapeutic target across various solid tumors. ß-Lapachone (ß-Lap) and deoxynyboquinone (DNQ) are two NQO1 bioactivatable drugs that have demonstrated potent antitumor effects. However, their curative efficacy has been constrained by adverse effects and moderate lethality. To enhance the curative potential of NQO1 bioactivatable drugs, we developed a novel DNQ derivative termed isopentyl-deoxynyboquinone (IP-DNQ). Our study revealed that IP-DNQ treatment significantly increased reactive oxygen species generation, leading to double-strand break (DSB) formation, PARP1 hyperactivation, and catastrophic energy loss. Notably, we discovered that this novel drug induced both apoptosis and programmed necrosis events, which makes it entirely distinct from other NQO1 bioactivatable drugs. Furthermore, IP-DNQ monotherapy demonstrated significant antitumor efficacy and extended mice survival in A549 orthotopic xenograft models. Lastly, we identified that in mice IP-DNQ levels were significantly elevated in the plasma and tumor compared with IB-DNQ levels. This study provides novel preclinical evidence supporting IP-DNQ efficacy in NQO1+ NSCLC and breast cancer cells.

Imaging Brain Injury in Former National Football League Players.

Importance: Pilot studies that involved early imaging of the 18 kDa translocator protein (TSPO) using positron emission tomography (PET) indicated high levels of TSPO in the brains of active or former National Football League (NFL) players. If validated further in larger studies, those findings may have implications for athletes involved in collision sport. Objective: To test for higher TSPO that marks brain injury and repair in a relatively large, unique cohort of former NFL players compared with former elite, noncollision sport athletes. Design, Setting, and Participants: This cross-sectional study used carbon 11-labeled N,N-diethyl-2-(4-methoxyphenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidine-3-acetamide positron emission tomography ([11C]DPA-713 PET) data from former NFL players within 12 years of last participation in the NFL and elite noncollision sport athletes from across the US. Participants were enrolled between April 2018 and February 2023. Main outcomes and measures: Regional [11C]DPA-713 total distribution volume from [11C]DPA-713 PET that is a measure of regional brain TSPO; regional brain volumes on magnetic resonance imaging; neuropsychological performance, including attention, executive function, and memory domains. Results: This study included 27 former NFL players and 27 former elite, noncollision sport athletes. Regional TSPO levels were higher in former NFL players compared with former elite, noncollision sport athletes (unstandardized ß coefficient, 1.08; SE, 0.22; 95% CI, 0.65 to 1.52; P < .001). The magnitude of the group difference depended on region, with largest group differences in TSPO in cingulate and frontal cortices as well as hippocampus. Compared with noncollision sport athletes, former NFL players performed worse in learning (mean difference [MD], -0.70; 95% CI, -1.14 to -0.25; P = .003) and memory (MD, -0.77; 95% CI, -1.24 to -0.30; P = .002), with no correlation between total gray matter TSPO and these cognitive domains. Conclusions and relevance: In this cross-sectional study using [11C]DPA-713 PET, higher brain TSPO was found in former NFL players compared with noncollision sport athletes. This finding is consistent with neuroimmune activation even after cessation of NFL play. Future longitudinal [11C]DPA-713 PET and neuropsychological testing promises to inform whether neuroimmune-modulating therapy may be warranted.

Orthogonal Hydroxyl Functionalization of cGAMP Confers Metabolic Stability and Enables Antibody Conjugation.

cGAMP is a signaling molecule produced by the cGAS-DNA complex to establish antimicrobial and antitumor immunity through STING. Whereas STING activation holds potential as a new strategy to treat cancer, cGAMP is generally considered unsuitable for in vivo use because of the rapid cleavage of its phosphodiester linkages and the limited cellular uptake under physiological conditions. Consequently, phosphorothioation and fluorination are commonly used to improve the metabolic stability and permeability of cGAMP and its synthetic analogues. We now show that methylation of the 3'-hydroxyl group of cGAMP also confers metabolic stability and that acylation of the 2'-hydroxyl group can be achieved directly and selectively to enable receptor-mediated intracellular delivery. Unlike phosphorothioation and fluorination, these modifications do not create a new stereogenic center and do not require laborious building block synthesis. As such, orthogonal hydroxyl functionalization is a simple solution to issues associated with the in vivo use of cGAMP.

KP372-1-Induced AKT Hyperactivation Blocks DNA Repair to Synergize With PARP Inhibitor Rucaparib via Inhibiting FOXO3a/GADD45α Pathway.

Poly (ADP-ribose) polymerase (PARP) inhibitors (PARPi) have exhibited great promise in the treatment of tumors with homologous recombination (HR) deficiency, however, PARPi resistance, which ultimately recovers DNA repair and cell progress, has become an enormous clinical challenge. Recently, KP372-1 was identified as a novel potential anticancer agent that targeted the redox enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), to induce extensive reactive oxygen species (ROS) generation that amplified DNA damage, leading to cancer cell death. To overcome PARPi resistance and expand its therapeutic utility, we investigated whether a combination therapy of a sublethal dose of KP372-1 with a nontoxic dose of PARPi rucaparib would synergize and enhance lethality in NQO1 over-expressing cancers. We reported that the combination treatment of KP372-1 and rucaparib induced a transient and dramatic AKT hyperactivation that inhibited DNA repair by regulating FOXO3a/GADD45α pathway, which enhanced PARPi lethality and overcame PARPi resistance. We further found that PARP inhibition blocked KP372-1-induced PARP1 hyperactivation to reverse NAD+/ATP loss that promoted Ca2+-dependent autophagy and apoptosis. Moreover, pretreatment of cells with BAPTA-AM, a cytosolic Ca2+ chelator, dramatically rescued KP372-1- or combination treatment-induced lethality and significantly suppressed PAR formation and γH2AX activation. Finally, we demonstrated that this combination therapy enhanced accumulation of both agents in mouse tumor tissues and synergistically suppressed tumor growth in orthotopic pancreatic and non-small-cell lung cancer xenograft models. Together, our study provides novel preclinical evidence for new combination therapy in NQO1+ solid tumors that may broaden the clinical utility of PARPi.

Development of a biomarker to monitor target engagement after treatment with dihydroorotate dehydrogenase inhibitors.

Dihydroorotate dehydrogenase (DHODH) catalyzes a key step in pyrimidine biosynthesis and has recently been validated as a therapeutic target for malaria through clinical studies on the triazolopyrimidine-based Plasmodium DHODH inhibitor DSM265. Selective toxicity towards Plasmodium species could be achieved because malaria parasites lack pyrimidine salvage pathways, and DSM265 selectively inhibits Plasmodium DHODH over the human enzyme. However, while DSM265 does not inhibit human DHODH, it inhibits DHODH from several preclinical species, including mice, suggesting that toxicity could result from on-target DHODH inhibition in those species. We describe here the use of dihydroorotate (DHO) as a biomarker of DHODH inhibition. Treatment of mammalian cells with DSM265 or the mammalian DHODH inhibitor teriflunomide led to increases in DHO where the extent of biomarker buildup correlated with both dose and inhibitor potency on DHODH. Treatment of mice with leflunomide (teriflunomide prodrug) caused a large dose-dependent buildup of DHO in blood (up to 16-fold) and urine (up to 5,400-fold) that was not observed for mice treated with DSM265. Unbound plasma teriflunomide levels reached 20-85-fold above the mouse DHODH IC50, while free DSM265 levels were only 1.6-4.2-fold above, barely achieving âˆ¼ IC90 concentrations, suggesting that unbound DSM265 plasma levels are not sufficient to block the pathway in vivo. Thus, any toxicity associated with DSM265 treatment in mice is likely caused by off-target mechanisms. The identification of a robust biomarker for mammalian DHODH inhibition represents an important advance to generally monitor for on-target effects in preclinical and clinical applications of DHODH inhibitors used to treat human disease.

Regional amyloid correlates of cognitive performance in ageing and mild cognitive impairment.

Beta-amyloid deposition is one of the earliest pathological markers associated with Alzheimer's disease. Mild cognitive impairment in the setting of beta-amyloid deposition is considered to represent a preclinical manifestation of Alzheimer's disease. In vivo imaging studies are unique in their potential to advance our understanding of the role of beta-amyloid deposition in cognitive deficits in Alzheimer's disease and in mild cognitive impairment. Previous work has shown an association between global cortical measures of beta-amyloid deposition ('amyloid positivity') in mild cognitive impairment with greater cognitive deficits and greater risk of progression to Alzheimer's disease. The focus of the present study was to examine the relationship between the regional distribution of beta-amyloid deposition and specific cognitive deficits in people with mild cognitive impairment and cognitively normal elderly individuals. Forty-seven participants with multi-domain, amnestic mild cognitive impairment (43% female, aged 57-82 years) and 37 healthy, cognitively normal comparison subjects (42% female, aged 55-82 years) underwent clinical and neuropsychological assessments and high-resolution positron emission tomography with the radiotracer 11C-labelled Pittsburgh compound B to measure beta-amyloid deposition. Brain-behaviour partial least-squares analysis was conducted to identify spatial patterns of beta-amyloid deposition that correlated with the performance on neuropsychological assessments. Partial least-squares analysis identified a single significant (P < 0.001) latent variable which accounted for 80% of the covariance between demographic and cognitive measures and beta-amyloid deposition. Performance in immediate verbal recall (R = -0.46 ± 0.07, P < 0.001), delayed verbal recall (R = -0.39 ± 0.09, P < 0.001), immediate visual-spatial recall (R = -0.39 ± 0.08, P < 0.001), delayed visual-spatial recall (R = -0.45 ± 0.08, P < 0.001) and semantic fluency (R = -0.33 ± 0.11, P = 0.002) but not phonemic fluency (R = -0.05 ± 0.12, P < 0.705) negatively covaried with beta-amyloid deposition in the identified regions. Partial least-squares analysis of the same cognitive measures with grey matter volumes showed similar associations in overlapping brain regions. These findings suggest that the regional distribution of beta-amyloid deposition and grey matter volumetric decreases is associated with deficits in executive function and memory in mild cognitive impairment. Longitudinal analysis of these relationships may advance our understanding of the role of beta-amyloid deposition in relation to grey matter volumetric decreases in cognitive decline.

Serine ADP-ribosylation marks nucleosomes for ALC1-dependent chromatin remodeling.

Serine ADP-ribosylation (ADPr) is a DNA damage-induced post-translational modification catalyzed by the PARP1/2:HPF1 complex. As the list of PARP1/2:HPF1 substrates continues to expand, there is a need for technologies to prepare mono- and poly-ADP-ribosylated proteins for biochemical interrogation. Here, we investigate the unique peptide ADPr activities catalyzed by PARP1 in the absence and presence of HPF1. We then exploit these activities to develop a method that facilitates installation of ADP-ribose polymers onto peptides with precise control over chain length and modification site. Importantly, the enzymatically mono- and poly-ADP-ribosylated peptides are fully compatible with protein ligation technologies. This chemoenzymatic protein synthesis strategy was employed to assemble a series of full-length, ADP-ribosylated histones and show that ADPr at histone H2B serine 6 or histone H3 serine 10 converts nucleosomes into robust substrates for the chromatin remodeler ALC1. We found ALC1 preferentially remodels 'activated' substrates within heterogeneous mononucleosome populations and asymmetrically ADP-ribosylated dinucleosome substrates, and that nucleosome serine ADPr is sufficient to stimulate ALC1 activity in nuclear extracts. Our study identifies a biochemical function for nucleosome serine ADPr and describes a new, highly modular approach to explore the impact that site-specific serine mono- and poly-ADPr have on protein function.

Reducing acetylated tau is neuroprotective in brain injury.

Traumatic brain injury (TBI) is the largest non-genetic, non-aging related risk factor for Alzheimer's disease (AD). We report here that TBI induces tau acetylation (ac-tau) at sites acetylated also in human AD brain. This is mediated by S-nitrosylated-GAPDH, which simultaneously inactivates Sirtuin1 deacetylase and activates p300/CBP acetyltransferase, increasing neuronal ac-tau. Subsequent tau mislocalization causes neurodegeneration and neurobehavioral impairment, and ac-tau accumulates in the blood. Blocking GAPDH S-nitrosylation, inhibiting p300/CBP, or stimulating Sirtuin1 all protect mice from neurodegeneration, neurobehavioral impairment, and blood and brain accumulation of ac-tau after TBI. Ac-tau is thus a therapeutic target and potential blood biomarker of TBI that may represent pathologic convergence between TBI and AD. Increased ac-tau in human AD brain is further augmented in AD patients with history of TBI, and patients receiving the p300/CBP inhibitors salsalate or diflunisal exhibit decreased incidence of AD and clinically diagnosed TBI.

Polymer Microparticles Prolong Delivery of the 15-PGDH Inhibitor SW033291.

As the prevalence of age-related fibrotic diseases continues to increase, novel antifibrotic therapies are emerging to address clinical needs. However, many novel therapeutics for managing chronic fibrosis are small-molecule drugs that require frequent dosing to attain effective concentrations. Although bolus parenteral administrations have become standard clinical practice, an extended delivery platform would achieve steady-state concentrations over a longer time period with fewer administrations. This study lays the foundation for the development of a sustained release platform for the delivery of (+)SW033291, a potent, small-molecule inhibitor of the 15-hydroxyprostaglandin dehydrogenase (15-PGDH) enzyme, which has previously demonstrated efficacy in a murine model of pulmonary fibrosis. Herein, we leverage fine-tuned cyclodextrin microparticles-specifically, ß-CD microparticles (ß-CD MPs)-to extend the delivery of the 15-PGDH inhibitor, (+)SW033291, to over one week.

Inhibition of the de novo pyrimidine biosynthesis pathway limits ribosomal RNA transcription causing nucleolar stress in glioblastoma cells.

Glioblastoma is the most common and aggressive type of cancer in the brain; its poor prognosis is often marked by reoccurrence due to resistance to the chemotherapeutic agent temozolomide, which is triggered by an increase in the expression of DNA repair enzymes such as MGMT. The poor prognosis and limited therapeutic options led to studies targeted at understanding specific vulnerabilities of glioblastoma cells. Metabolic adaptations leading to increased synthesis of nucleotides by de novo biosynthesis pathways are emerging as key alterations driving glioblastoma growth. In this study, we show that enzymes necessary for the de novo biosynthesis of pyrimidines, DHODH and UMPS, are elevated in high grade gliomas and in glioblastoma cell lines. We demonstrate that DHODH's activity is necessary to maintain ribosomal DNA transcription (rDNA). Pharmacological inhibition of DHODH with the specific inhibitors brequinar or ML390 effectively depleted the pool of pyrimidines in glioblastoma cells grown in vitro and in vivo and impaired rDNA transcription, leading to nucleolar stress. Nucleolar stress was visualized by the aberrant redistribution of the transcription factor UBF and the nucleolar organizer nucleophosmin 1 (NPM1), as well as the stabilization of the transcription factor p53. Moreover, DHODH inhibition decreased the proliferation of glioblastoma cells, including temozolomide-resistant cells. Importantly, the addition of exogenous uridine, which reconstitutes the cellular pool of pyrimidine by the salvage pathway, to the culture media recovered the impaired rDNA transcription, nucleolar morphology, p53 levels, and proliferation of glioblastoma cells caused by the DHODH inhibitors. Our in vivo data indicate that while inhibition of DHODH caused a dramatic reduction in pyrimidines in tumor cells, it did not affect the overall pyrimidine levels in normal brain and liver tissues, suggesting that pyrimidine production by the salvage pathway may play an important role in maintaining these nucleotides in normal cells. Our study demonstrates that glioblastoma cells heavily rely on the de novo pyrimidine biosynthesis pathway to generate ribosomal RNA (rRNA) and thus, we identified an approach to inhibit ribosome production and consequently the proliferation of glioblastoma cells through the specific inhibition of the de novo pyrimidine biosynthesis pathway.

MYC promotes tryptophan uptake and metabolism by the kynurenine pathway in colon cancer.

Tumors display increased uptake and processing of nutrients to fulfill the demands of rapidly proliferating cancer cells. Seminal studies have shown that the proto-oncogene MYC promotes metabolic reprogramming by altering glutamine uptake and metabolism in cancer cells. How MYC regulates the metabolism of other amino acids in cancer is not fully understood. Using high-performance liquid chromatography (HPLC)-tandem mass spectrometry (LC-MS/MS), we found that MYC increased intracellular levels of tryptophan and tryptophan metabolites in the kynurenine pathway. MYC induced the expression of the tryptophan transporters SLC7A5 and SLC1A5 and the enzyme arylformamidase (AFMID), involved in the conversion of tryptophan into kynurenine. SLC7A5, SLC1A5, and AFMID were elevated in colon cancer cells and tissues, and kynurenine was significantly greater in tumor samples than in the respective adjacent normal tissue from patients with colon cancer. Compared with normal human colonic epithelial cells, colon cancer cells were more sensitive to the depletion of tryptophan. Blocking enzymes in the kynurenine pathway caused preferential death of established colon cancer cells and transformed colonic organoids. We found that only kynurenine and no other tryptophan metabolite promotes the nuclear translocation of the transcription factor aryl hydrocarbon receptor (AHR). Blocking the interaction between AHR and kynurenine with CH223191 reduced the proliferation of colon cancer cells. Therefore, we propose that limiting cellular kynurenine or its downstream targets could present a new strategy to reduce the proliferation of MYC-dependent cancer cells.

NQO1-dependent, Tumor-selective Radiosensitization of Non-small Cell Lung Cancers.

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 ß-lapachone (ß-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 ß-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: ß-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 ß-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 + ß-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 ß-lap and IR is a viable approach to selectively treat NQO1-overexpressing NSCLC and warrant a clinical trial using low-dose IR + ß-lap against patients with NQO1+ NSCLCs.

Leveraging an NQO1 Bioactivatable Drug for Tumor-Selective Use of Poly(ADP-ribose) Polymerase Inhibitors.

Therapeutic drugs that block DNA repair, including poly(ADP-ribose) polymerase (PARP) inhibitors, fail due to lack of tumor-selectivity. When PARP inhibitors and ß-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 ß-lapachone monotherapy to synergistic tumor-selective, caspase-dependent apoptosis with PARP inhibitors and ß-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.

Esterase-activatable ß-lapachone prodrug micelles for NQO1-targeted lung cancer therapy.

Lung cancer is one of the most lethal forms of cancer and current chemotherapeutic strategies lack broad specificity and efficacy. Recently, ß-lapachone (ß-lap) was shown to be highly efficacious in killing non-small cell lung cancer (NSCLC) cells regardless of their p53, cell cycle and caspase status. Pre-clinical and clinical use of ß-lap (clinical form, ARQ501 or 761) is hampered by poor pharmacokinetics and toxicity due to hemolytic anemia. Here, we report the development and preclinical evaluation of ß-lap prodrug nanotherapeutics consisting of diester derivatives of ß-lap encapsulated in biocompatible and biodegradable poly(ethylene glycol)-b-poly(D,L-lactic acid) (PEG-b-PLA) micelles. Compared to the parent drug, diester derivatives of ß-lap showed higher drug loading densities inside PEG-b-PLA micelles. After esterase treatment, micelle-delivered ß-lap-dC3 and -dC6 prodrugs were converted to ß-lap. Cytotoxicity assays using A549 and H596 lung cancer cells showed that both micelle formulations maintained NAD(P)H: quinone oxidoreductase 1 (NQO1)-dependent cytotoxicity. However, antitumor efficacy study of ß-lap-dC3 micelles against orthotopic A549 NSCLC xenograft-bearing mice showed significantly greater long-term survival over ß-lap-dC6 micelles or ß-lap-HPßCD complexes. Improved therapeutic efficacy of ß-lap-dC3 micelles correlated with higher area under the concentration-time curves of ß-lap in tumors, and enhanced pharmacodynamic endpoints (e.g., PARP1 hyperactivation, γH2AX, and ATP depletion). ß-Lap-dC3 prodrug micelles provide a promising strategy for NQO1-targeted therapy of lung cancer with improved safety and antitumor efficacy.

Identification of DNMT1 selective antagonists using a novel scintillation proximity assay.

A novel scintillation proximity high throughput assay (SPA) to identify inhibitors of DNA methyltransferases was developed and used to screen over 180,000 compounds. The majority of the validated hits shared a quinone core and several were found to generate the reactive oxygen species, H2O2. Inhibition of the production of H2O2 by the addition of catalase blocked the ability of this group of compounds to inhibit DNA methyltransferase (DNMT) activity. However, a related compound, SW155246, was identified that existed in an already reduced form of the quinone. This compound did not generate H2O2, and catalase did not block its ability to inhibit DNA methyltransferase. SW155246 showed a 30-fold preference for inhibition of human DNMT1 versus human or murine DNMT3A or -3B, inhibited global methylation in HeLa cells, and reactivated expression of the tumor suppressor gene RASSF1A in A549 cells. To our knowledge, this work represents the first description of selective chemical inhibitors of the DNMT1 enzyme.

An NQO1 substrate with potent antitumor activity that selectively kills by PARP1-induced programmed necrosis.

Agents, such as ß-lapachone, that target the redox enzyme, NAD(P)H:quinone oxidoreductase 1 (NQO1), to induce programmed necrosis in solid tumors have shown great promise, but more potent tumor-selective compounds are needed. Here, we report that deoxynyboquinone kills a wide spectrum of cancer cells in an NQO1-dependent manner with greater potency than ß-lapachone. Deoxynyboquinone lethality relies on NQO1-dependent futile redox cycling that consumes oxygen and generates extensive reactive oxygen species (ROS). Elevated ROS levels cause extensive DNA lesions, PARP1 hyperactivation, and severe NAD+ /ATP depletion that stimulate Ca2+ -dependent programmed necrosis, unique to this new class of NQO1 "bioactivated" drugs. Short-term exposure of NQO1+ cells to deoxynyboquinone was sufficient to trigger cell death, although genetically matched NQO1- cells were unaffected. Moreover, siRNA-mediated NQO1 or PARP1 knockdown spared NQO1+ cells from short-term lethality. Pretreatment of cells with BAPTA-AM (a cytosolic Ca2+ chelator) or catalase (enzymatic H2O2 scavenger) was sufficient to rescue deoxynyboquinone-induced lethality, as noted with ß-lapachone. Investigations in vivo showed equivalent antitumor efficacy of deoxynyboquinone to ß-lapachone, but at a 6-fold greater potency. PARP1 hyperactivation and dramatic ATP loss were noted in the tumor, but not in the associated normal lung tissue. Our findings offer preclinical proof-of-concept for deoxynyboquinone as a potent chemotherapeutic agent for treatment of a wide spectrum of therapeutically challenging solid tumors, such as pancreatic and lung cancers.

Specific inhibition of mitochondrial oxidative stress suppresses inflammation and improves cardiac function in a rat pneumonia-related sepsis model.

Using a mitochondria-targeted vitamin E (Mito-Vit-E) in a rat pneumonia-related sepsis model, we examined the role of mitochondrial reactive oxygen species in sepsis-mediated myocardial inflammation and subsequent cardiac contractile dysfunction. Sepsis was produced in adult male Sprague-Dawley rats via intratracheal injection of S. pneumonia (4 × 10(6) colony formation units per rat). A single dose of Mito-Vit-E, vitamin E, or control vehicle, at 21.5 µmol/kg, was administered 30 min postinoculation. Blood was collected, and heart tissue was harvested at various time points. Mito-Vit-E in vivo distribution was confirmed by mass spectrometry. In cardiac mitochondria, Mito-Vit-E improved total antioxidant capacity and suppressed H(2)O(2) generation, whereas vitamin E offered little effect. In cytosol, both antioxidants decreased H(2)O(2) levels, but only vitamin E strengthened antioxidant capacity. Mito-Vit-E protected mitochondrial structure and function in the heart during sepsis, demonstrated by reduction in lipid and protein oxidation, preservation of mitochondrial membrane integrity, and recovery of respiratory function. While both Mito-Vit-E and vitamin E suppressed sepsis-induced peripheral and myocardial production of proinflammatory cytokines (tumor necrosis factor-α, interleukin-1ß, and interleukin-6), Mito-Vit-E exhibited significantly higher efficacy (P < 0.05). Stronger anti-inflammatory action of Mito-Vit-E was further shown by its near-complete inhibition of sepsis-induced myeloperoxidase accumulation in myocardium, suggesting its effect on neutrophil infiltration. Echocardiography analysis indicated that Mito-Vit-E ameliorated cardiac contractility of sepsis animals, shown by improved fractional shortening and ejection fraction. Together, our data suggest that targeted scavenging of mitochondrial reactive oxygen species protects mitochondrial function, attenuates tissue-level inflammation, and improves whole organ activities in the heart during sepsis.

Regulation of VEGF-induced endothelial cell migration by mitochondrial reactive oxygen species.

Endothelial migration is a crucial aspect of a variety of physiologic and pathologic conditions including atherosclerosis and vascular repair. Reactive oxygen species (ROS) function as second messengers during endothelial migration. Multiple intracellular sources of ROS are regulated by cellular context, external stimulus, and the microenvironment. However, the predominant source of ROS during endothelial cell (EC) migration and the mechanisms by which ROS regulate cell migration are incompletely understood. In this study, we tested the hypothesis that mitochondria-derived ROS (mtROS) regulate EC migration. In cultured human umbilical vein endothelial cells, VEGF increased mitochondrial metabolism, promoted mtROS production, and induced cell migration. Either the targeted mitochondrial delivery of the antioxidant, vitamin E (Mito-Vit-E), or the depletion of mitochondrial DNA abrogated VEGF-mediated mtROS production. Overexpression of mitochondrial catalase also inhibited VEGF-induced mitochondrial metabolism, Rac activation, and cell migration. Furthermore, these interventions suppressed VEGF-stimulated EC migration and blocked Rac1 activation in endothelial cells. Constitutively active Rac1 reversed Mito-Vit-E-induced inhibition of EC migration. Mito-Vit-E also attenuated carotid artery reendothelialization in vivo. These results provide strong evidence that mtROS regulate EC migration through Rac-1.