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Background: Cigarette smoking induces cytochrome P450 1A2 (CYP1A2) expression and activity, while smoking cessation normalizes the levels of this enzyme. The aim of this publication is to summarize the data on CYP1A2 gene expression and activity in preclinical and clinical studies on the Tobacco Heating System (THS), currently marketed as IQOS® with HEETs® , and to summarize the potential effects on CYP1A2 to be expected upon switching to reduced-risk products (RRPs)., Methods: We summarized PMI's preclinical and clinical data on the effects of switching from cigarette smoking to THS., Results: Data from four preclinical mouse and rat studies showed that, upon either cessation of cigarette smoke exposure or switching to THS exposure, the upregulation of CYP1A2 observed with exposure to cigarette smoke reverted close to fresh-air levels. Data from four clinical studies yielded similar results on CYP1A2 activity within a time frame of five days. Furthermore, the effects of switching to THS were similar to those seen after smoking cessation., Conclusions: Because smoking cessation and switching to either electronic cigarettes or THS seem to have similar effects on CYP1A2 activity, the same measures taken for patients treated with narrow therapeutic index drugs that are metabolized by CYP1A2 and who quit smoking should be recommended for those switching to RRPs., Competing Interests: The authors declare no conflict of interest., (© 2020 The Author(s).)

Cigarette smoking causes major preventable diseases, morbidity, and mortality worldwide. Smoking cessation and prevention of smoking initiation are the preferred means for reducing these risks. Less harmful tobacco products, termed modified-risk tobacco products (MRTP), are being developed as a potential alternative for current adult smokers who would otherwise continue smoking. According to a regulatory framework issued by the US Food and Drug Administration, a manufacturer must provide comprehensive scientific evidence that the product significantly reduces harm and the risk of tobacco-related diseases, in order to obtain marketing authorization for a new MRTP. For new tobacco products similar to an already approved predicate product, the FDA has foreseen a simplified procedure for assessing "substantial equivalence". In this article, we present a use case that bridges the nonclinical evidence from previous studies demonstrating the relatively reduced harm potential of two heat-not-burn products based on different tobacco heating principles. The nonclinical evidence was collected along a "causal chain of events leading to disease" (CELSD) to systematically follow the consequences of reduced exposure to toxicants (relative to cigarette smoke) through increasing levels of biological complexity up to disease manifestation in animal models of human disease. This approach leverages the principles of systems biology and toxicology as a basis for further extrapolation to human studies. The experimental results demonstrate a similarly reduced impact of both products on apical and molecular endpoints, no novel effects not seen with cigarette smoke exposure, and an effect of switching from cigarettes to either MRTP that is comparable to that of complete smoking cessation. Ideally, a subset of representative assays from the presented sequence along the CELSD could be sufficient for predicting similarity or substantial equivalence in the nonclinical impact of novel products; this would require further validation, for which the present use case could serve as a starting point., Competing Interests: The work reported in this publication involved candidate/potential modified risk tobacco products developed by Philip Morris International. Philip Morris International is the sole source of funding and sponsor of this research. All authors are employees of PMI R&D or had worked for PMI R&D under contractual agreements., (© 2020 Published by Elsevier B.V.)

Background: Inflammatory bowel disease (IBD) is the collective term for chronic immune-mediated diseases of unknown, multifactorial etiology, arising from the interplay between genetic and environmental factors and including two main disease manifestations: ulcerative colitis (UC) and Crohn's disease. In the last few decades, naturally occurring alkaloids have gained interest because of their substantial anti-inflammatory effects in several animal models of disease. Studies on mouse models of IBD have demonstrated the anti-inflammatory action of the main tobacco alkaloid, nicotine. In addition, anatabine, a minor tobacco alkaloid also present in peppers, tomato, and eggplant presents anti-inflammatory properties in vivo and in vitro. In this study, we aimed to evaluate the anti-inflammatory properties of nicotine and anatabine in a dextran sulfate sodium (DSS) mouse model of UC., Results: Oral administration of anatabine, but not nicotine, reduced the clinical symptoms of DSS-induced colitis. The result of gene expression analysis suggested that anatabine had a restorative effect on global DSS-induced gene expression profiles, while nicotine only had limited effects. Accordingly, MAP findings revealed that anatabine reduced the colonic abundance of DSS-associated cytokines and increased IL-10 abundance., Conclusions: Our results support the amelioration of inflammatory effects by anatabine in the DSS mouse model of UC, and suggest that anatabine constitutes a promising therapeutic agent for IBD treatment., Competing Interests: Competing interestsAll authors are employees of Philip Morris International., (© The Author(s) 2020.)

The use of flavoring substances is an important element in the development of reduced-risk products for adult smokers to increase product acceptance and encourage switching from cigarettes. In a first step towards characterizing the sub-chronic inhalation toxicity of neat flavoring substances, a study was conducted using a mixture of the substances in a base solution of e-liquid, where the standard toxicological endpoints of the nebulized aerosols were supplemented with transcriptomics analysis. The flavor mixture was produced by grouping 178 flavors into 26 distinct chemical groups based on structural similarities and potential metabolic and biological effects. Flavoring substances predicted to show the highest toxicological effect from each group were selected as the flavor group representatives (FGR). Following Organization for Economic Cooperation and Development Testing Guideline 413, rats were exposed to three concentrations of the FGR mixture in an e-liquid composed of nicotine (23 µg/L), propylene glycol (1520 µg/L), and vegetable glycerin (1890 µg/L), while non-flavored and no-nicotine mixtures were included as references to identify potential additive or synergistic effects between nicotine and the flavoring substances. The results indicated that the inhalation of an e-liquid containing the mixture of FGRs caused very minimal local and systemic toxic effects. In particular, there were no remarkable clinical (in-life) observations in flavored e-liquid-exposed rats. The biological effects related to exposure to the mixture of neat FGRs were limited and mainly nicotine-mediated, including changes in hematological and blood chemistry parameters and organ weight. These results indicate no significant additive biological changes following inhalation exposure to the nebulized FGR mixture above the nicotine effects measured in this sub-chronic inhalation study. In a subsequent study, e-liquids with FGR mixtures will be aerosolized by thermal treatment and assessed for toxicity.

Cigarette smoke (CS) exposure is one of the leading risk factors for human health. Nicotine-containing inhalable products, such as e-cigarettes, can effectively support tobacco harm reduction approaches. However, there are limited comparative data on the effects of the aerosols generated from electronic vapor products (e-vapor) and CS on bone. Here, we report the effects of e-vapor aerosols and CS on bone morphology, structure, and strength in a 6-month inhalation study. Eight-week-old ApoE -/- mice were exposed to aerosols from three different e-vapor formulations-CARRIER (propylene glycol and vegetable glycerol), BASE (CARRIER and nicotine), TEST (BASE and flavor)-to CS from 3R4F reference cigarettes at matched nicotine concentrations (35 µg/L) or to fresh air (Sham) (N = 10 per group). Tibiae were analyzed for bone morphology by µCT imaging, biomechanics by three-point bending, and by histological analysis. CS inhalation caused a significant decrease in cortical and total bone volume fraction and bone density relative to e-vapor aerosols. Additionally, CS exposure caused a decrease in ultimate load and stiffness. In contrast, bone structural and biomechanical parameters were not significantly affected by e-vapor aerosol or Sham exposure. At the dissection time point, there was no significant difference in body weight or tibia bone weight or length among the groups. Histological findings revealed microcracks in cortical bone areas among all exposed groups compared to Sham control. In conclusion, because of the bone-preserving effect of e-vapor aerosols relative to CS exposure, e-vapor products could potentially constitute less harmful alternatives to cigarettes in situations in which bone health is of importance.

Cigarette smoke (CS) causes adverse health effects and, for smoker who do not quit, modified risk tobacco products (MRTPs) can be an alternative to reduce the risk of developing smoking-related diseases. Standard toxicological endpoints can lack sensitivity, with systems toxicology approaches yielding broader insights into toxicological mechanisms. In a 6-month systems toxicology study on ApoE -/- mice, we conducted an integrative multi-omics analysis to assess the effects of aerosols from the Carbon Heated Tobacco Product (CHTP) 1.2 and Tobacco Heating System (THS) 2.2-a potential and a candidate MRTP based on the heat-not-burn (HnB) principle-compared with CS at matched nicotine concentrations. Molecular exposure effects in the lungs were measured by mRNA/microRNA transcriptomics, proteomics, metabolomics, and lipidomics. Integrative data analysis included Multi-Omics Factor Analysis and multi-modality functional network interpretation. Across all five data modalities, CS exposure was associated with an increased inflammatory and oxidative stress response, and lipid/surfactant alterations. Upon HnB aerosol exposure these effects were much more limited or absent, with reversal of CS-induced effects upon cessation and switching to CHTP 1.2. Functional network analysis revealed CS-induced complex immunoregulatory interactions across the investigated molecular layers (e.g., itaconate, quinolinate, and miR-146) and highlighted the engagement of the heme-Hmox-bilirubin oxidative stress axis by CS. This work exemplifies how multi-omics approaches can be leveraged within systems toxicology studies and the generated multi-omics data set can facilitate the development of analysis methods and can yield further insights into the effects of toxicological exposures on the lung of mice., (© 2020 The Authors.)

For many diseases, there is an unmet need for new or better biomarkers for improved disease risk assessment and monitoring, as available markers lack sufficient specificity. Lipids are drawing major interest as potential candidates for filling these gaps. This has recently been demonstrated by the identification of selective ceramides for prediction of cardiovascular mortality, enabling improved risk assessment of cardiovascular disease compared with conventional clinical markers. In this review, we discuss current lipid biomarker findings and the possible connection between cardiovascular disease, chronic obstructive pulmonary disease, and aging. Moreover, we discuss how to overcome the current roadblocks facing lipid biomarker research. We stress the need for improved quantification, standardization of methodologies, and establishment of initial reference values to allow for an efficient transfer path of research findings into the clinical landscape, and, ultimately, to put newly identified biomarkers into practical use., Competing Interests: Declaration of Competing Interest KE is the founder and CEO of Lipidomics Consulting Ltd. OL, BT, PC, JH, and NVI are employees of Philip Morris International., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Purpose: There is a continuous search for imaging techniques with high sensitivity and specificity for brain tumors. Positron emission tomography (PET) imaging has shown promise, though many PET agents either have a low tumor specificity or impractical physical half-lives. [ 124 I]CLR1404 is a small molecule alkylphosphocholine analogue that is thought to bind to plasma membrane lipid rafts and has shown high tumor-to-background ratios (TBR) in a previous pilot study in brain tumor patients. This study attempts to define the clinical value of [ 124 I]CLR1404 PET/CT (aka CLR124)., Procedures: Adult patients with new or suspected recurrence of high-grade primary or metastatic brain tumors (N = 27) were injected with [ 124 I]CLR1404 followed by PET/CT at 6, 24, and 48 h. Standard uptake values (SUV) and TBR values were calculated for all time points. Uptake of [ 124 I]CLR1404 was qualitatively assessed, compared with magnetic resonance imaging (MRI), and correlated with clinical outcome. Final diagnosis (N = 25) was established based on surgically resected tissue or long-term follow-up., Results: Positive uptake with high TBR was detected in all but one patient with a final diagnosis of primary/recurrent brain tumor (12/13) and in less than half of patients with treatment-related changes (5/12). Concordance between [ 124 I]CLR1404 uptake and contrast enhancement on MRI was seen in < 40 %, with no concordance between T 2 -hyperintensities and uptake. No significant difference in overall outcome was found between patients with and without [ 124 I]CLR1404 uptake., Conclusions: The uptake pattern in these patients suggests a very high sensitivity of [ 124 I]CLR1404 PET/CT for diagnosing tumor tissue; however, tumor specificity needs to be further defined. Relative lack of concordance with standard MRI characteristics suggests that [ 124 I]CLR1404 PET/CT provides additional information about brain tumors compared to MRI alone, potentially improving clinical decision-making.

Exposure to cigarette smoke (CS) causes detrimental health effects, increasing the risk of cardiovascular, pulmonary diseases and carcinogenesis in exposed individuals. The impact of CS on Inflammatory Bowel Disease (IBD) has been established by a number of epidemiological and clinical studies. In fact, CS is associated with a higher risk of developing Crohn's disease (CD) while inversely correlates with the development, disease risks, and relapse rate of ulcerative colitis (UC). To investigate the effect of CS exposure on experimental colitis, we performed a comprehensive and integrated comparative analysis of colon transcriptome and microbiome in mice exposed to dextran sodium sulfate (DSS) and CS. Colon transcriptome analysis revealed that CS downregulated specific pathways in a concentration-dependent manner, affecting both the inflammatory state and composition of the gut microbiome. Metagenomics analysis demonstrated that CS can modulate DSS-induced dysbiosis of specific bacterial genera, contributing to resolve the inflammation or accelerate recovery. The risks of smoking far outweigh any possible benefit, thus smoking cessation must always be encouraged because of its significant health benefits. However, the inverse association between active smoking and the development of UC cannot be ignored and the present study lays the foundation for investigating potential molecular mechanisms responsible for the attenuation of colitis by certain compounds of tobacco when decoupled from combustion.

Smoking cigarettes is harmful to the cardiovascular system. Considerable attention has been paid to the reduced harm potential of alternative nicotine-containing inhalable products such as e-cigarettes. We investigated the effects of E-vapor aerosols or cigarette smoke (CS) on atherosclerosis progression, cardiovascular function, and molecular changes in the heart and aorta of female apolipoprotein E-deficient (ApoE -/- ) mice. The mice were exposed to aerosols from three different E-vapor formulations: 1 ) carrier (propylene glycol and vegetable glycerol), 2 ) base (carrier and nicotine), or 3 ) test (base and flavor) or to CS from 3R4F reference cigarettes for up to 6 mo. Concentrations of CS and base or test aerosols were matched at 35 µg nicotine/L. Exposure to CS, compared with sham-exposed fresh air controls, accelerated atherosclerotic plaque formation, whereas no such effect was seen for any of the three E-vapor aerosols. Molecular changes indicated disease mechanisms related to oxidative stress and inflammation in general, plus changes in calcium regulation, and altered cytoskeletal organization and microtubule dynamics in the left ventricle. While ejection fraction, fractional shortening, cardiac output, and isovolumic contraction time remained unchanged following E-vapor aerosols exposure, the nicotine-containing base and test aerosols caused an increase in isovolumic relaxation time similar to CS. A nicotine-related increase in pulse wave velocity and arterial stiffness was also observed, but it was significantly lower for base and test aerosols than for CS. These results demonstrate that in comparison with CS, E-vapor aerosols induce substantially lower biological responses associated with smoking-related cardiovascular diseases. NEW & NOTEWORTHY Analysis of key urinary oxidative stress markers and proinflammatory cytokines showed an absence of oxidative stress and inflammation in the animals exposed to E-vapor aerosols. Conversely, animals exposed to conventional cigarette smoke had high urinary levels of these markers. When compared with conventional cigarette smoke, E-vapor aerosols induced smaller atherosclerotic plaque surface area and volume. Systolic and diastolic cardiac function, as well as endothelial function, were further significantly less affected by electronic cigarette aerosols than conventional cigarette smoke. Molecular analysis demonstrated that E-vapor aerosols induce significantly smaller transcriptomic dysregulation in the heart and aorta compared with conventional cigarette smoke.

Aim: To investigate the molecular, structural, and functional impact of aerosols from candidate modified risk tobacco products (cMRTP), the Carbon Heated Tobacco Product (CHTP) 1.2 and Tobacco Heating System (THS) 2.2, compared with that of mainstream cigarette smoke (CS) on the cardiovascular system of ApoE -/- mice., Methods: Female ApoE -/- mice were exposed to aerosols from THS 2.2 and CHTP 1.2 or to CS from the 3R4F reference cigarette for up to 6 months at matching nicotine concentrations. A Cessation and a Switching group (3 months exposure to 3R4F CS followed by filtered air or CHTP 1.2 for 3 months) were included. Cardiovascular effects were investigated by echocardiographic, histopathological, immunohistochemical, and transcriptomics analyses., Results: Continuous exposure to cMRTP aerosols did not affect atherosclerosis progression, heart function, left ventricular (LV) structure, or the cardiovascular transcriptome. Exposure to 3R4F CS triggered atherosclerosis progression, reduced systolic ejection fraction and fractional shortening, caused heart LV hypertrophy, and initiated significant dysregulation in the transcriptomes of the heart ventricle and thoracic aorta. Importantly, the structural, functional, and molecular changes caused by 3R4F CS were improved in the smoking cessation and switching groups., Conclusion: Exposure to cMRTP aerosols lacked most of the CS exposure-related functional, structural, and molecular effects. Smoking cessation or switching to CHTP 1.2 aerosol caused similar recovery from the 3R4F CS effects in the ApoE -/- model, with no further acceleration of plaque progression beyond the aging-related rate., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Justyna Szostak, Bjoern Titz, Emmanuel Guedj, Alain Sewer, Blaine Phillips, Patrice Leroy, Neau Laurent, Keyur Trivedi, Florian Martin, Nikolai V. Ivanov, Patrick Vanscheeuwijck, Manuel C. Peitsch, Julia Hoeng are employee of Philip Morris Products S.A., Neuchâtel, Switzerland. The work reported was paid by PMP S.A. Dr. Walter Schlage and Dr. Ansgar Buettner are consultants paid by Philip Morris Products S.A., Neuchâtel, Switzerland. Philip Morris Products S.A. is the sole source of funding and sponsor of this project., (Copyright © 2019. Published by Elsevier B.V.)

We previously proposed a systems toxicology framework for in vitro assessment of e-liquids. The framework starts with the first layer aimed at screening the potential toxicity of e-liquids, followed by the second layer aimed at investigating the toxicity-related mechanism of e-liquids, and finally, the third layer aimed at evaluating the toxicity-related mechanism of the corresponding aerosols. In this work, we applied this framework to assess the impact of the e-liquid MESH Classic Tobacco and its aerosol compared with that of cigarette smoke (CS) from the 3R4F reference cigarette. In the first layer, we evaluated the cytotoxicity profile of the MESH Classic Tobacco e-liquid (containing humectants, nicotine, and flavors) and its Base e-liquid (containing humectant and nicotine only) in comparison with total particulate matter (TPM) of 3R4F CS using primary bronchial epithelial cell cultures. In the second layer, the same culture model was used to explore changes in specific markers using high-content screening assays to identify potential toxicity-related mechanisms induced by the MESH Classic Tobacco and Base e-liquids beyond cell viability in comparison with the 3R4F CS TPM-induced effects. Finally, in the third layer, we compared the impact of exposure to the MESH Classic Tobacco or Base aerosols with 3R4F CS using human organotypic air-liquid interface buccal and small airway epithelial cultures. The results showed that the cytotoxicity of the MESH Classic Tobacco liquid was similar to the Base liquid but lower than 3R4F CS TPM at comparable nicotine concentrations. Relative to 3R4F CS exposure, MESH Classic Tobacco aerosol exposure did not cause tissue damage and elicited lower changes in the mRNA, microRNA, and protein markers. In the context of tobacco harm reduction strategy, the framework is suitable to assess the potential-reduced impact of electronic cigarette aerosol relative to CS.

Smoking is one of the major modifiable risk factors in the development and progression of chronic obstructive pulmonary disease (COPD) and cardiovascular disease (CVD). Modified-risk tobacco products (MRTP) are being developed to provide substitute products for smokers who are unable or unwilling to quit, to lessen the smoking-related health risks. In this study, the ApoE -/- mouse model was used to investigate the impact of cigarette smoke (CS) from the reference cigarette 3R4F, or aerosol from two potential MRTPs based on the heat-not-burn principle, carbon heated tobacco product 1.2 (CHTP1.2) and tobacco heating system 2.2 (THS 2.2), on the cardiorespiratory system over a 6-month period. In addition, cessation or switching to CHTP1.2 after 3 months of CS exposure was assessed. A systems toxicology approach combining physiology, histology and molecular measurements was used to evaluate the impact of MRTP aerosols in comparison to CS. CHTP1.2 and THS2.2 aerosols, compared with CS, demonstrated lower impact on the cardiorespiratory system, including low to absent lung inflammation and emphysematous changes, and reduced atherosclerotic plaque formation. Molecular analyses confirmed the lower engagement of pathological mechanisms by MRTP aerosols than CS. Both cessation and switching to CHTP1.2 reduced the observed CS effects to almost sham exposure levels., (Copyright © 2019. Published by Elsevier Ltd.)

Swedish snus is a smokeless tobacco product that contains reduced levels of harmful compounds compared with cigarette smoke. In Sweden, where snus use exceeds smoking among men, relatively low rates of major smoking-related diseases have been recorded. To better understand how snus use could align with current tobacco harm reduction strategies, its potential mechanisms of toxicity must be investigated. This study aimed to determine, via a systems toxicology approach, the biological impact of repeated 72-hour exposure of human gingival epithelial organotypic cultures to extracts from both a commercial and a reference snus and the total particulate matter (TPM) from cigarette smoke. At concentrations relevant for human use, cultures treated with snus extracts induced mild, generally reversible biological changes, while TPM treatment induced substantial morphological and inflammatory alterations. Network enrichment analysis and integrative analysis of the global mRNA and miRNA expression profiles indicated a limited and mostly transient impact of the snus extracts, in particular on xenobiotic metabolism, while the effects of TPM were marked and sustained over time. High-confidence miRNAs that might be related to pathological conditions in vivo were identified. This study highlights the limited biological impact of Swedish snus extract on human organotypic gingival cultures., (Copyright © 2018. Published by Elsevier Ltd.)

We compared early biological changes in mice after inhalation exposures to cigarette smoke or e-vapor aerosols (MarkTen ® cartridge with Carrier, Test-1, or Test-2 formulations; 4% nicotine). Female C57BL/6 mice were exposed to 3R4F cigarette smoke or e-vapor aerosols by nose-only inhalation for up to 4 hours/day, 5 days/week, for 3 weeks. The 3R4F and e-vapor exposures were set to match the target nose port aerosol nicotine concentration (∼41 µg/L). Only the 3R4F group showed postexposure clinical signs such as tremors and lethargy. At necropsy, the 3R4F group had significant increases in lung weight and changes in bronchoalveolar lavage parameters, as well as microscopic findings in the respiratory tract. The e-vapor groups had minimal microscopic changes, including squamous metaplasia in laryngeal epiglottis, and histiocytic infiltrates in the lung (Test-2 group only). The 3R4F group had a higher incidence and severity of microscopic findings compared to any e-vapor group. Transcriptomic analysis also showed that the 3R4F group had the highest number of differentially expressed genes compared to Sham Control. Among e-vapor groups, Test-2 group had more differentially expressed genes but the magnitude of gene expression-based network perturbations in all e-vapor groups was ∼94% less than the 3R4F group. On proteome analysis in the lung, differentially regulated proteins were detected in the 3R4F group only. In conclusion, 3-weeks of 3R4F exposure induced molecular and microscopic changes associated with smoking-related diseases in the respiratory tract, while e-vapor exposures showed substantially reduced biological activities.

Inflammatory bowel disease (IBD) represents a group of progressive disorders characterized by recurrent chronic inflammation of the gut. Ulcerative colitis and Crohn's disease are the major manifestations of IBD. While our understanding of IBD has progressed in recent years, its etiology is far from being fully understood, resulting in suboptimal treatment options. Complementing other biological endpoints, bioanalytical "omics" methods that quantify many biomolecules simultaneously have great potential in the dissection of the complex pathogenesis of IBD. In this review, we focus on the rapidly evolving proteomics and lipidomics technologies and their broad applicability to IBD studies; these range from investigations of immune-regulatory mechanisms and biomarker discovery to studies dissecting host⁻microbiome interactions and the role of intestinal epithelial cells. Future studies can leverage recent advances, including improved analytical methodologies, additional relevant sample types, and integrative multi-omics analyses. Proteomics and lipidomics could effectively accelerate the development of novel targeted treatments and the discovery of complementary biomarkers, enabling continuous monitoring of the treatment response of individual patients; this may allow further refinement of treatment and, ultimately, facilitate a personalized medicine approach to IBD.

Within the framework of a systems toxicology approach, the inhalation toxicity of aerosol from a novel tobacco-heating potentially modified risk tobacco product (MRTP), the carbon-heated tobacco product (CHTP) 1.2, was characterized and compared with that of mainstream smoke (CS) from the 3R4F reference cigarette in a 90-day nose-only rat inhalation study in general accordance with OECD TG 413. CHTP1.2 is a heat-not-burn product using a carbon heat source to produce an aerosol that contains nicotine and tobacco flavor. At equal or twice the nicotine concentration in the test atmospheres, inhalation of CHTP1.2 aerosol led to a significantly lower exposure to harmful constituents and induced less respiratory tract irritation, systemic, and pathological effects compared with CS. Nasal epithelial changes were less pronounced in the CHTP1.2- than in the CS-exposed groups and reverted in the nicotine concentration-matched group after a recovery period. Lung inflammation was minimal in the CHTP1.2-treated groups compared with the moderate extent seen in the 3R4F groups. Many other toxicological endpoints evaluated did not show CHTP1.2 aerosol exposure-related effects, and no effects not seen for 3R4F were observed. These observations were consistent with findings from previous studies in which rats were exposed to MRTP aerosols containing similar nicotine concentrations., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cigarette smoke (CS) is affecting considerably the oral mucosa. Heating, instead of burning, tobacco reduces consistently the amount of toxic compounds and may exert a lower impact on oral health than combusted cigarettes. The carbon-heated tobacco product 1.2 (CHTP1.2) is a potential modified risk tobacco product (MRTP) based on heat-not-burn technology. Using a systems toxicology assessment framework, we compared the effects of exposure to CHTP1.2 aerosol with those of CS from a reference cigarette (3R4F). Human organotypic cultures derived from buccal and gingival epithelia were exposed acutely (28-min) or repeatedly (28 min/day for 3 days), respectively, to two matching concentrations of CHTP1.2 aerosol or 3R4F CS, and a non-diluted (100%) CHTP1.2 aerosol. The results showed an absence of cytotoxicity, reduction in pathophysiological alterations, toxicological marker proteins, and inflammatory mediators following exposure to CHTP1.2 aerosol compared with 3R4F CS. Changes in mRNA and miRNA expression were linked by an integrative analysis approach, suggesting a regulatory role of miRNAs in several smoke/disease-relevant biological processes induced by 3R4F CS. The identification of mechanisms by which potential MRTPs can reduce the impact of tobacco use on biological systems is of great importance in understanding the molecular basis of the smoking harm reduction paradigm., (Copyright © 2018 PMI R&D, Philip Morris Products, S.A. Published by Elsevier Ltd.. All rights reserved.)

Modified risk tobacco products (MRTPs) have the potential to reduce smoking-related health risks. The Carbon Heated Tobacco Product 1.2 (CHTP1.2) is a potential MRTP that uses a pressed carbon heat source to generate an aerosol by heating tobacco. Here, we report the results from the systems toxicology arm of a 90-day rat inhalation study (OECD test guideline 413) to assess the effects of CHTP1.2 aerosol compared with cigarette smoke (CS). Transcriptomics, proteomics, and lipidomics analyses complemented the standard endpoints. In the respiratory nasal epithelium, CS induced an adaptive tissue and inflammatory response, which was much weaker after CHTP1.2 aerosol exposure, mostly limited to the highest CHTP1.2 concentration (at twice the 3R4F CS concentration: 50 vs. 23 μg nicotine/L), in female rats. In the lungs, the effects of CS exposure included inflammatory and cellular stress responses, which were absent or much lower after CHTP1.2 aerosol exposure. Outside of the respiratory tract, CS and CHTP1.2 aerosol induced effects that were previously associated with exposure to any nicotine-containing aerosol, e.g., lower lipid concentrations in serum. Overall, this systems toxicology analysis complements and confirms the results from classical toxicological endpoints and further suggests potentially reduced respiratory health risks of CHTP1.2., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The biological impact of an aerosol of a potential modified-risk tobacco product, carbon heated tobacco product 1.2 (CHTP1.2), was comprehensively assessed for the first time in vitro using human small airway and nasal epithelial models following a systems toxicology approach. The potentially reduced effects of CHTP1.2 aerosol exposure were benchmarked against those of 3R4F cigarette smoke at similar nicotine concentrations. Experimental repetitions were conducted for which new batches of small airway and nasal cultures were exposed to CHTP1.2 aerosol or 3R4F smoke for 28 minutes. The biological impacts were determined based on a collection of endpoints including morphology, cytotoxicity, proinflammatory mediator profiles, cytochrome P450 1A1/1B1 activity, global mRNA and microRNA changes and proteome profiles. Alterations in mRNA expression were detected in cultures exposed to CHTP1.2 aerosol, without noticeable morphological changes and cytotoxicity, and minimal impact on proinflammatory mediator and proteome profiles. The changes linked to CHTP1.2 aerosol exposure, when observed, were transient. However, the impact of 3R4F smoke exposure persisted long post-exposure and greater than CHTP1.2 aerosol. Morphological changes were observed only in cultures exposed to 3R4F smoke. The lower biological effects of CHTP1.2 aerosol than 3R4F smoke exposure were observed similarly in both small airway and nasal epithelial cultures., (Copyright © 2018 PMI R&D, Philip Morris Products S.A. Published by Elsevier Ltd.. All rights reserved.)

Purpose: Auger electrons emitted by radioisotopes such as 125 I have a high linear energy transfer and short mean-free path in tissue (<10 μm), making them suitable for treating micrometastases while sparing normal tissues. The authors developed and subsequently investigated a cancer cell-selective small molecule phospholipid ether analog to deliver 125 I to triple-negative breast cancer (TNBC) cells in vivo., Methods: A Current Good Manufacturing Practice (cGMP) method to radiolabel 125 I-CLR1404 (CLR 125) with >95% radiochemical purity was established. To estimate CLR 125 in vivo dosimetry and identify dose-limiting organs, the biodistribution of the analog compound 124 I-CLR1404 (CLR 124) was investigated using micro-positron emission tomography (PET)/computed tomography (CT) in conjunction with a Monte Carlo dosimetry platform to estimate CLR 125 dosimetry. In vivo antitumor efficacy was tested by injecting nude mice bearing either MDA-MB-231-luc orthotopic xenografts or lung metastases with 74 MBq (3.7 GBq/kg) of CLR 125 or an equivalent mass amount of nonradiolabeled CLR 125. Longitudinal tumor measurements using calipers and bioluminescence imaging were obtained for the xenografts and lung metastases, respectively., Results: Dosimetry analysis estimated that CLR 125 would impart the largest absorbed dose to the tumor per injected activity (0.261 ± 0.023 Gy/MBq) while the bone marrow, which is generally the dose-limiting organ for CLR1404, appears to have the lowest (0.063 ± 0.005 Gy/MBq). At administered activities of up to 74 MBq (3.7 GBq/kg), mice did not experience signs of toxicity. In addition, a single dose of CLR 125 reduced the volume of orthotopic primary TNBC xenografts by ∼60% compared to control vehicle (p < 0.001) and significantly extended survival. In addition, CLR 125 was efficacious against preclinical metastatic TNBC models by inhibiting the progression of micrometastases (p < 0.01)., Conclusions: Targeted radionuclide therapy with CLR 125 displayed significant antitumor efficacy in vivo, suggesting promise for treatment of TNBC micrometastases.

While the toxicity of the main constituents of electronic cigarette (ECIG) liquids, nicotine, propylene glycol (PG), and vegetable glycerin (VG), has been assessed individually in separate studies, limited data on the inhalation toxicity of them is available when in mixtures. In this 90-day subchronic inhalation study, Sprague-Dawley rats were nose-only exposed to filtered air, nebulized vehicle (saline), or three concentrations of PG/VG mixtures, with and without nicotine. Standard toxicological endpoints were complemented by molecular analyses using transcriptomics, proteomics, and lipidomics. Compared with vehicle exposure, the PG/VG aerosols showed only very limited biological effects with no signs of toxicity. Addition of nicotine to the PG/VG aerosols resulted in effects in line with nicotine effects observed in previous studies, including up-regulation of xenobiotic enzymes (Cyp1a1/Fmo3) in the lung and metabolic effects, such as reduced serum lipid concentrations and expression changes of hepatic metabolic enzymes. No toxicologically relevant effects of PG/VG aerosols (up to 1.520  mg PG/L + 1.890 mg VG/L) were observed, and no adverse effects for PG/VG/nicotine were observed up to 438/544/6.6 mg/kg/day. This study demonstrates how complementary systems toxicology analyses can reveal, even in the absence of observable adverse effects, subtoxic and adaptive responses to pharmacologically active compounds such as nicotine., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

CLR1404 is a cancer-selective alkyl phosphocholine (APC) analog that can be radiolabeled with 124 I for PET imaging, 131 I for targeted radiotherapy and/or SPECT imaging, or 125 I for targeted radiotherapy. Studies have demonstrated avid CLR1404 uptake and prolonged retention in a broad spectrum of preclinical tumor models. The purpose of this pilot trial was to demonstrate avidity of 124 I-CLR1404 in human brain tumors and develop a framework to evaluate this uptake for use in larger studies. 12 patients (8 men and 4 women; mean age of 43.9 ± 15.1 y; range 23-66 y) with 13 tumors were enrolled. Eleven patients had suspected tumor recurrence and 1 patient had a new diagnosis of high grade tumor. Patients were injected with 185 MBq ± 10% of 124 I-CLR1404 followed by PET/CT imaging at 6-, 24-, and 48-hour. 124 I-CLR1404 PET uptake was assessed qualitatively and compared with MRI. After PET image segmentation SUV values and tumor to background ratios were calculated. There was no significant uptake of 124 I-CLR1404 in normal brain. In tumors, uptake tended to increase to 48 hours. Positive uptake was detected in 9 of 13 lesions: 5/5 high grade tumors, 1/2 low grade tumors, 1/1 meningioma, and 2/4 patients with treatment related changes. 124 I-CLR1404 uptake was not detected in 1/2 low grade tumors, 2/4 lesions from treatment related changes, and 1/1 indeterminate lesion. For 6 malignant tumors, the average tumor to background ratios (TBR) were 9.32 ± 4.33 (range 3.46 to 15.42) at 24 hours and 10.04 ± 3.15 (range 5.17 to 13.17) at 48 hours. For 2 lesions from treatment related change, the average TBR were 5.05 ± 0.4 (range 4.76 to 5.33) at 24 hours and 4.88 ± 1.19 (range 4.04 to 5.72) at 48 hours. PET uptake had areas of both concordance and discordance compared with MRI. 124 I-CLR1404 PET demonstrated avid tumor uptake in a variety of brain tumors with high tumor-to-background ratios. There were regions of concordance and discordance compared with MRI, which has potential clinical relevance. Expansion of these studies is required to determine the clinical significance of the 124 I-CLR1404 PET findings., Competing Interests: None.

Variations in tumor volume segmentation methods in targeted radionuclide therapy (TRT) may lead to dosimetric uncertainties. This work investigates the impact of PET and MRI threshold-based tumor segmentation on TRT dosimetry in patients with primary and metastatic brain tumors. In this study, PET/CT images of five brain cancer patients were acquired at 6, 24, and 48 h post-injection of 124 I-CLR1404. The tumor volume was segmented using two standardized uptake value (SUV) threshold levels, two tumor-to-background ratio (TBR) threshold levels, and a T1 Gadolinium-enhanced MRI threshold. The dice similarity coefficient (DSC), jaccard similarity coefficient (JSC), and overlap volume (OV) metrics were calculated to compare differences in the MRI and PET contours. The therapeutic 131 I-CLR1404 voxel-level dose distribution was calculated from the 124 I-CLR1404 activity distribution using RAPID, a Geant4 Monte Carlo internal dosimetry platform. The TBR, SUV, and MRI tumor volumes ranged from 2.3-63.9 cc, 0.1-34.7 cc, and 0.4-11.8 cc, respectively. The average  ±  standard deviation (range) was 0.19  ±  0.13 (0.01-0.51), 0.30  ±  0.17 (0.03-0.67), and 0.75  ±  0.29 (0.05-1.00) for the JSC, DSC, and OV, respectively. The DSC and JSC values were small and the OV values were large for both the MRI-SUV and MRI-TBR combinations because the regions of PET uptake were generally larger than the MRI enhancement. Notable differences in the tumor dose volume histograms were observed for each patient. The mean (standard deviation) 131 I-CLR1404 tumor doses ranged from 0.28-1.75 Gy GBq -1 (0.07-0.37 Gy GBq -1 ). The ratio of maximum-to-minimum mean doses for each patient ranged from 1.4-2.0. The tumor volume and the interpretation of the tumor dose is highly sensitive to the imaging modality, PET enhancement metric, and threshold level used for tumor volume segmentation. The large variations in tumor doses clearly demonstrate the need for standard protocols for multimodality tumor segmentation in TRT dosimetry.

Smoking is one of the major lifestyle-related risk factors for periodontal diseases. Modified risk tobacco products (MRTP) offer a promising alternative in the harm reduction strategy for adult smokers unable to quit. Using a systems toxicology approach, we investigated and compared the exposure effects of a reference cigarette (3R4F) and a heat-not-burn technology-based candidate MRTP, the Tobacco Heating System (THS) 2.2. Human gingival epithelial organotypic cultures were repeatedly exposed (3 days) for 28 min at two matching concentrations of cigarette smoke (CS) or THS2.2 aerosol. Results showed only minor histopathological alterations and minimal cytotoxicity upon THS2.2 aerosol exposure compared to CS (1% for THS2.2 aerosol vs. 30% for CS, at the high concentration). Among the 14 proinflammatory mediators analyzed, only 5 exhibited significant alterations with THS2.2 exposure compared with 11 upon CS exposure. Transcriptomic and metabolomic analysis indicated a general reduction of the impact in THS2.2 aerosol-exposed samples with respect to CS (∼79% lower biological impact for the high THS2.2 aerosol concentration compared to CS, and 13 metabolites significantly perturbed for THS2.2 vs. 181 for CS). This study indicates that exposure to THS2.2 aerosol had a lower impact on the pathophysiology of human gingival organotypic cultures than CS., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Modified risk tobacco products (MRTPs) are being developed with the aim of reducing smoking-related health risks. The Tobacco Heating System 2.2 (THS2.2) is a candidate MRTP that uses the heat-not-burn principle. Here, systems toxicology approaches were engaged to assess the respiratory effects of mentholated THS2.2 (THS2.2M) in a 90-day rat inhalation study (OECD test guideline 413). The standard endpoints were complemented by transcriptomics and quantitative proteomics analyses of respiratory nasal epithelium and lung tissue and by lipidomics analysis of lung tissue. The adaptive response of the respiratory nasal epithelium to conventional cigarette smoke (CS) included squamous cell metaplasia and an inflammatory response, with high correspondence between the molecular and histopathological results. In contrast to CS exposure, the adaptive tissue and molecular changes to THS2.2M aerosol exposure were much weaker and were limited mostly to the highest THS2.2M concentration in female rats. In the lung, CS exposure induced an inflammatory response, triggered cellular stress responses, and affected sphingolipid metabolism. These responses were not observed or were much lower after THS2.2M aerosol exposure. Overall, this system toxicology analysis complements and reconfirms the results from classical toxicological endpoints and further suggests potentially reduced health risks of THS2.2M., (Copyright © 2016. Published by Elsevier Inc.)

The toxicity of a mentholated version of the Tobacco Heating System (THS2.2M), a candidate modified risk tobacco product (MRTP), was characterized in a 90-day OECD inhalation study. Differential gene and protein expression analysis of nasal epithelium and lung tissue was also performed to record exposure effects at the molecular level. Rats were exposed to filtered air (sham), to THS2.2M (at 15, 23 and 50 μg nicotine/l), to two mentholated reference cigarettes (MRC) (at 23 μg nicotine/l), or to the 3R4F reference cigarette (at 23 μg nicotine/l). MRCs were designed to meet 3R4F specifications. Test atmosphere analyses demonstrated that aldehydes were reduced by 75%-90% and carbon monoxide by 98% in THS2.2M aerosol compared with MRC smoke; aerosol uptake was confirmed by carboxyhemoglobin and menthol concentrations in blood, and by the quantities of urinary nicotine metabolites. Systemic toxicity and alterations in the respiratory tract were significantly lower in THS2.2M-exposed rats compared with MRC and 3R4F. Pulmonary inflammation and the magnitude of the changes in gene and protein expression were also dramatically lower after THS2.2M exposure compared with MRCs and 3R4F. No menthol-related effects were observed after MRC mainstream smoke-exposure compared with 3R4F., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Smoking is a major risk factor for several diseases including chronic obstructive pulmonary disease (COPD). To better understand the systemic effects of cigarette smoke exposure and mild to moderate COPD-and to support future biomarker development-we profiled the serum lipidomes of healthy smokers, smokers with mild to moderate COPD (GOLD stages 1 and 2), former smokers, and never-smokers ( n = 40 per group) (ClinicalTrials.gov registration: NCT01780298). Serum lipidome profiling was conducted with untargeted and targeted mass spectrometry-based lipidomics. Guided by weighted lipid co-expression network analysis, we identified three main trends comparing smokers, especially those with COPD, with non-smokers: a general increase in glycero(phospho)lipids, including triglycerols; changes in fatty acid desaturation (decrease in ω-3 polyunsaturated fatty acids, and an increase in monounsaturated fatty acids); and an imbalance in eicosanoids (increase in 11,12- and 14,15-DHETs (dihydroxyeicosatrienoic acids), and a decrease in 9- and 13-HODEs (hydroxyoctadecadienoic acids)). The lipidome profiles supported classification of study subjects as smokers or non-smokers, but were not sufficient to distinguish between smokers with and without COPD. Overall, our study yielded further insights into the complex interplay between smoke exposure, lung disease, and systemic alterations in serum lipid profiles., Competing Interests: The authors except for Terhi Vihervaar and Kim Ekroos are employees of Philip Morris International. Terhi Vihervaara and Kim Ekroos are employees of Zora Biosciences. Philip Morris International is the sole source of funding and sponsor of this study.

A prominent mechanism of acquired resistance to BRAF inhibitors in BRAF (V600) -mutant melanoma is associated with the upregulation of receptor tyrosine kinases. Evidences suggested that this resistance mechanism is part of a more complex cellular adaptation process. Using an integrative strategy, we found this mechanism to invoke extensive transcriptomic, (phospho-) proteomic and phenotypic alterations that accompany a cellular transition to a de-differentiated, mesenchymal and invasive state. Even short-term BRAF-inhibitor exposure leads to an early adaptive, differentiation state change-characterized by a slow-cycling, persistent state. The early persistent state is distinct from the late proliferative, resistant state. However, both differentiation states share common signaling alterations including JUN upregulation. Motivated by the similarities, we found that co-targeting of BRAF and JUN is synergistic in killing fully resistant cells; and when used up-front, co-targeting substantially impairs the formation of the persistent subpopulation. We confirmed that JUN upregulation is a common response to BRAF inhibitor treatment in clinically treated patient tumors. Our findings demonstrate that events shared between early- and late-adaptation states provide candidate up-front co-treatment targets.

Unlabelled: Smoking is associated with several serious diseases, such as lung cancer and chronic obstructive pulmonary disease (COPD). Within our systems toxicology framework, we are assessing whether potential modified risk tobacco products (MRTP) can reduce smoking-related health risks compared to conventional cigarettes. In this article, we evaluated to what extent 2D-PAGE/MALDI MS/MS (2D-PAGE) can complement the iTRAQ LC-MS/MS results from a previously reported mouse inhalation study, in which we assessed a prototypic MRTP (pMRTP). Selected differentially expressed proteins identified by both LC-MS/MS and 2D-PAGE approaches were further verified using reverse-phase protein microarrays. LC-MS/MS captured the effects of cigarette smoke (CS) on the lung proteome more comprehensively than 2D-PAGE. However, an integrated analysis of both proteomics data sets showed that 2D-PAGE data complement the LC-MS/MS results by supporting the overall trend of lower effects of pMRTP aerosol than CS on the lung proteome. Biological effects of CS exposure supported by both methods included increases in immune-related, surfactant metabolism, proteasome, and actin cytoskeleton protein clusters. Overall, while 2D-PAGE has its value, especially as a complementary method for the analysis of effects on intact proteins, LC-MS/MS approaches will likely be the method of choice for proteome analysis in systems toxicology investigations., Significance: Quantitative proteomics is anticipated to play a growing role within systems toxicology assessment frameworks in the future. To further understand how different proteomics technologies can contribute to toxicity assessment, we conducted a quantitative proteomics analysis using 2D-PAGE and isobaric tag-based LC-MS/MS approaches and compared the results produced from the 2 approaches. Using a prototypic modified risk tobacco product (pMRTP) as our test item, we show compared with cigarette smoke, how 2D-PAGE results can complement and support LC-MS/MS data, demonstrating the much lower effects of pMRTP aerosol than cigarette smoke on the mouse lung proteome. The combined analysis of 2D-PAGE and LC-MS/MS data identified an effect of cigarette smoke on the proteasome and actin cytoskeleton in the lung., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

We used clinical tissue from lethal metastatic castration-resistant prostate cancer (CRPC) patients obtained at rapid autopsy to evaluate diverse genomic, transcriptomic, and phosphoproteomic datasets for pathway analysis. Using Tied Diffusion through Interacting Events (TieDIE), we integrated differentially expressed master transcriptional regulators, functionally mutated genes, and differentially activated kinases in CRPC tissues to synthesize a robust signaling network consisting of druggable kinase pathways. Using MSigDB hallmark gene sets, six major signaling pathways with phosphorylation of several key residues were significantly enriched in CRPC tumors after incorporation of phosphoproteomic data. Individual autopsy profiles developed using these hallmarks revealed clinically relevant pathway information potentially suitable for patient stratification and targeted therapies in late stage prostate cancer. Here, we describe phosphorylation-based cancer hallmarks using integrated personalized signatures (pCHIPS) that shed light on the diversity of activated signaling pathways in metastatic CRPC while providing an integrative, pathway-based reference for drug prioritization in individual patients., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Approximately 75% of melanomas have known driver oncogenic mutations in BRAF, NRAS, GNA11 or GNAQ, while the mutations providing constitutive oncogenic signaling in the remaining melanomas are not known. We established a melanoma cell line from a tumor with none of the common driver mutations. This cell line demonstrated a signaling profile similar to BRAF-mutants, but lacked sensitivity to the BRAF inhibitor vemurafenib. RNA-seq mutation data implicated CRAF R391W as the alternative driver mutation of this melanoma. CRAF R391W was homozygous and over expressed. These melanoma cells were highly sensitive to CRAF, but not BRAF knockdown. In reconstitution experiments, CRAF R391W, but not CRAF WT, transformed NIH3T3 cells in soft-agar colony formation assays, increased kinase activity in vitro, induced MAP kinase signaling and conferred vemurafenib resistance. MAP kinase inducing activity was dependent on CRAF dimerization. Thus, CRAF is a bona fide alternative oncogene for BRAF/NRAS/GNAQ/GNA11 wild type melanomas.

The liver is one of the most important organs involved in elimination of xenobiotic and potentially toxic substances. Cigarette smoke (CS) contains more than 7000 chemicals, including those that exert biological effects and cause smoking-related diseases. Though CS is not directly hepatotoxic, a growing body of evidence suggests that it may exacerbate pre-existing chronic liver disease. In this study, we integrated toxicological endpoints with molecular measurements and computational analyses to investigate effects of exposures on the livers of Apoe(-/- )mice. Mice were exposed to 3R4F reference CS, to an aerosol from the Tobacco Heating System (THS) 2.2, a candidate modified risk tobacco product (MRTP) or to filtered air (Sham) for up to 8 months. THS2.2 takes advantage of a "heat-not-burn" technology that, by heating tobacco, avoids pyrogenesis and pyrosynthesis. After CS exposure for 2 months, some groups were either switched to the MRTP or filtered air. While no group showed clear signs of hepatotoxicity, integrative analysis of proteomics and transcriptomics data showed a CS-dependent impairment of specific biological networks. These networks included lipid and xenobiotic metabolism and iron homeostasis that likely contributed synergistically to exacerbating oxidative stress. In contrast, most proteomic and transcriptomic changes were lower in mice exposed to THS2.2 and in the cessation and switching groups compared to the CS group. Our findings elucidate the complex biological responses of the liver to CS exposure. Furthermore, they provide evidence that THS2.2 aerosol has reduced biological effects, as compared with CS, on the livers of Apoe(-/- )mice.

The impact of cigarette smoke (CS), a major cause of lung diseases, on the composition and metabolism of lung lipids is incompletely understood. Here, we integrated quantitative lipidomics and proteomics to investigate exposure effects on lung lipid metabolism in a C57BL/6 and an Apolipoprotein E-deficient (Apoe(-/-)) mouse study. In these studies, mice were exposed to high concentrations of 3R4F reference CS, aerosol from potential modified risk tobacco products (MRTPs) or filtered air (Sham) for up to 8 months. The 2 assessed MRTPs, the prototypical MRTP for C57BL/6 mice and the Tobacco Heating System 2.2 for Apoe(-/-) mice, utilize "heat-not-burn" technologies and were each matched in nicotine concentrations to the 3R4F CS. After 2 months of CS exposure, some groups were either switched to the MRTP or underwent cessation. In both mouse strains, CS strongly affected several categories of lung lipids and lipid-related proteins. Candidate surfactant lipids, surfactant proteins, and surfactant metabolizing proteins were increased. Inflammatory eicosanoids, their metabolic enzymes, and several ceramide classes were elevated. Overall, CS induced a coordinated lipid response controlled by transcription regulators such as SREBP proteins and supported by other metabolic adaptations. In contrast, most of these changes were absent in the mice exposed to the potential MRTPs, in the cessation group, and the switching group. Our findings demonstrate the complex biological response of the lungs to CS exposure and support the benefits of cessation or switching to a heat-not-burn product using a design such as those employed in this study., (© The Author 2015. Published by Oxford University Press on behalf of the Society of Toxicology.)

Smoking of combustible cigarettes has a major impact on human health. Using a systems toxicology approach in a model of chronic obstructive pulmonary disease (C57BL/6 mice), we assessed the health consequences in mice of an aerosol derived from a prototype modified risk tobacco product (pMRTP) as compared to conventional cigarettes. We investigated physiological and histological endpoints in parallel with transcriptomics, lipidomics, and proteomics profiles in mice exposed to a reference cigarette (3R4F) smoke or a pMRTP aerosol for up to 7 months. We also included a cessation group and a switching-to-pMRTP group (after 2 months of 3R4F exposure) in addition to the control (fresh air-exposed) group, to understand the potential risk reduction of switching to pMRTP compared with continuous 3R4F exposure and cessation. The present manuscript describes the study design, setup, and implementation, as well as the generation, processing, and quality control analysis of the toxicology and 'omics' datasets that are accessible in public repositories for further analyses.

Neuroblastoma (NB) is the most common extracranial solid tumor in children and is associated with high mortality in advanced stages. Survivors suffer from long-term treatment-related sequelae. Thus, new targeted treatment options are urgently needed. 18-(p-[(127)I] iodophenyl) octadecyl phosphocholine (CLR1404) is a novel, broadly tumor targeted small molecule drug suitable for intravenous injection with highly selective tumor uptake. As a carrier molecule for radioactive iodine, CLR1404 is in clinical trials as cancer imaging agent and radiotherapeutic drug. Chemically, CLR1404 belongs to the anti-tumor alkyl phospholipids, a class of drugs known to have intrinsic cytotoxic effects on cancer cells. Therefore, we hypothesized that CLR1404 could be a tumor-targeted anti-cancer agent for neuroblastoma, and investigated its effect in vitro and in vivo. CLR1404 was taken up by NB cells in a highly tumor-selective manner both in vitro and in vivo, confirmed by flow cytometry and PET/CT imaging of mouse flank xenografts with (124)I-CLR1404, respectively. Using flow cytometry, MTT assay, Western blotting and caspase 3/7 assay, we confirm that in vitro treatment with CLR1404 leads to robust apoptosis and cell death in multiple NB cell lines and is associated with Akt inhibition, while sparing normal cells. Treatment with CLR1404 in doses of 10 or 30 mg/kg administered by intravenous injection once weekly for 7 weeks significantly inhibited the tumor growth rate in a mouse flank xenograft model of NB (P<0.001) when compared to control cohorts, without causing drug-related hematotoxicity or other noticeable adverse effects, which was determined by serial tumor volume measurements, complete blood counts, and monitoring of animal-specific health parameters. We conclude that CLR1404 warrants clinical exploration as a novel, tumor selective anticancer agent in NB and potentially other cancers.

Chronic obstructive pulmonary disease (COPD) is one of the most prevalent lung diseases. Cigarette smoking is the main risk factor for COPD. In this parallel-group clinical study we investigated to what extent the transitions in a chronic-exposure-to-disease model are reflected in the proteome and cellular transcriptome of induced sputum samples. We selected 60 age- and gender-matched individuals for each of the four study groups: current asymptomatic smokers, smokers with early stage COPD, former smokers, and never smokers. The cell-free sputum supernatant was analyzed by quantitative proteomics and the cellular mRNA fraction by gene expression profiling. The sputum proteome of current smokers clearly reflected the common physiological responses to smoke exposure, including alterations in mucin/trefoil proteins and a prominent xenobiotic/oxidative stress response. The latter response also was observed in the transcriptome, which additionally demonstrated an immune-cell polarization change. The former smoker group showed nearly complete attenuation of these biological effects. Thirteen differentially abundant proteins between the COPD and the asymptomatic smoker group were identified including TIMP1, APOA1, C6orf58, and BPIFB1 (LPLUNC1). In summary, our study demonstrates that sputum profiling can capture the complex and reversible physiological response to cigarette smoke exposure, which appears to be only slightly modulated in early-stage COPD., (Copyright © 2015. Published by Elsevier B.V.)

Chemosensitivity and resistance assays (CSRAs) aim to direct therapies based upon ex vivo responses of patient tumor cells to chemotherapeutic drugs. However, successful CSRAs are yet to be developed. Here, we exposed primary CD138(+) multiple myeloma (MM) cells to bortezomib, a clinical proteasome inhibitor, in microfluidic-cis-coculture (MicroC(3)) incorporating the patient's own CD138(-) tumor-companion mononuclear cells to integrate some of the patients' own tumor microenvironment components in the CSRA design. Statistical clustering techniques segregated MicroC(3) responses into two groups which correctly identified all seventeen patients as either clinically responsive or non-responsive to bortezomib-containing therapies. In contrast, when the same patient MM samples were analyzed in the absence of the CD138(-) cells (monoculture), the tumor cell responses did not segregate into clinical response clusters. Thus, MicroC(3) identified bortezomib-therapy MM patient responses making it a viable CSRA candidate toward enabling personalized therapy.

Modified risk tobacco products (MRTP) are designed to reduce smoking-related health risks. A murine model of chronic obstructive pulmonary disease (COPD) was applied to investigate classical toxicology end points plus systems toxicology (transcriptomics and proteomics). C57BL/6 mice were exposed to conventional cigarette smoke (3R4F), fresh air (sham), or a prototypic MRTP (pMRTP) aerosol for up to 7 months, including a cessation group and a switching-to-pMRTP group (2 months of 3R4F exposure followed by fresh air or pMRTP for up to 5 months respectively). 3R4F smoke induced the typical adaptive changes in the airways, as well as inflammation in the lung, associated with emphysematous changes (impaired pulmonary function and alveolar damage). At nicotine-matched exposure concentrations of pMRTP aerosol, no signs of lung inflammation and emphysema were observed. Both the cessation and switching groups showed a similar reversal of inflammatory responses and no progression of initial emphysematous changes. A significant impact on biological processes, including COPD-related inflammation, apoptosis, and proliferation, was identified in 3R4F-exposed, but not in pMRTP-exposed lungs. Smoking cessation or switching reduced these perturbations to near sham-exposed levels. In conclusion, the mouse model indicated retarded disease progression upon cessation or switching to pMRTP which alone had no adverse effects., (Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.)

B cells are selected for an intermediate level of B-cell antigen receptor (BCR) signalling strength: attenuation below minimum (for example, non-functional BCR) or hyperactivation above maximum (for example, self-reactive BCR) thresholds of signalling strength causes negative selection. In ∼25% of cases, acute lymphoblastic leukaemia (ALL) cells carry the oncogenic BCR-ABL1 tyrosine kinase (Philadelphia chromosome positive), which mimics constitutively active pre-BCR signalling. Current therapeutic approaches are largely focused on the development of more potent tyrosine kinase inhibitors to suppress oncogenic signalling below a minimum threshold for survival. We tested the hypothesis that targeted hyperactivation--above a maximum threshold--will engage a deletional checkpoint for removal of self-reactive B cells and selectively kill ALL cells. Here we find, by testing various components of proximal pre-BCR signalling in mouse BCR-ABL1 cells, that an incremental increase of Syk tyrosine kinase activity was required and sufficient to induce cell death. Hyperactive Syk was functionally equivalent to acute activation of a self-reactive BCR on ALL cells. Despite oncogenic transformation, this basic mechanism of negative selection was still functional in ALL cells. Unlike normal pre-B cells, patient-derived ALL cells express the inhibitory receptors PECAM1, CD300A and LAIR1 at high levels. Genetic studies revealed that Pecam1, Cd300a and Lair1 are critical to calibrate oncogenic signalling strength through recruitment of the inhibitory phosphatases Ptpn6 (ref. 7) and Inpp5d (ref. 8). Using a novel small-molecule inhibitor of INPP5D (also known as SHIP1), we demonstrated that pharmacological hyperactivation of SYK and engagement of negative B-cell selection represents a promising new strategy to overcome drug resistance in human ALL.

Constitutively active BCR-ABL kinase fusions are causative mutations in the pathogenesis of hematopoietic neoplasias including chronic myelogenous leukemia (CML). Although these fusions have been successfully targeted with kinase inhibitors, drug-resistance and relapse continue to limit long-term survival, highlighting the need for continued innovative drug discovery. We developed a time-resolved Förster resonance energy transfer (TR-FRET) -based assay to identify compounds that disrupt stimulation of the ABL kinase by blocking its ability to bind the positive regulator RIN1. This assay was used in a high throughput screen (HTS) of two small molecule libraries totaling 444,743 compounds. 708 confirmed hits were counter-screened to eliminate off-target inhibitors and reanalyzed to prioritize compounds with IC50 values below 10 μM. The CML cell line K562 was then used to identify five compounds that decrease MAPK1/3 phosphorylation, which we determined to be an indicator of RIN1-dependent ABL signaling. One of these compounds is a thiadiazole, and the other four are structurally related acyl piperidine amides. Notably, these five compounds lower cellular BCR-ABL1 kinase activity by blocking a positive regulatory interaction rather than directly inhibiting ABL catalytic function.

Background: Approximately 20% of melanomas contain a mutation in NRAS. However no direct inhibitor of NRAS is available. One of the main signaling pathways downstream of NRAS is the MAPK pathway. In this study we investigated the possibility of blocking oncogenic signaling of NRAS by inhibiting two signaling points in the MAPK pathway., Methods: Fourteen NRAS mutated human melanoma cell lines were treated with a pan-RAF inhibitor (PRi, Amgen Compd A), a MEK inhibitor (MEKi, trametinib) or their combination and the effects on proliferation, cell cycle progression, apoptosis, transcription profile and signaling of the cells were investigated., Results: The majority of the cell lines showed a significant growth inhibition, with high levels of synergism of the PRi and MEKi combination. Sensitive cell lines showed induction of apoptosis by the combination treatment and there was a correlation between p-MEK levels and synergistic effect of the combination treatment. Proliferation of sensitive cell lines was blocked by the inhibition of the MAPK pathway, which also blocked expression of cyclin D1. However, in resistant cell lines, proliferation was blocked by combined inhibition of the MAPK pathway and cyclin D3, which is not regulated by the MAPK pathway. Resistant cell lines also showed higher levels of p-GSK3β and less perturbation of the apoptotic profile upon the treatment in comparison with the sensitive cell lines., Conclusions: The combination of PRi + MEKi can be an effective regimen for blocking proliferation of NRAS mutant melanomas when there is higher activity of the MAPK pathway and dependence of proliferation and survival on this pathway.

Canonical Wnt signaling plays an important role in development and disease, regulating transcription of target genes and stabilizing many proteins phosphorylated by glycogen synthase kinase 3 (GSK3). We observed that the MiT family of transcription factors, which includes the melanoma oncogene MITF (micropthalmia-associated transcription factor) and the lysosomal master regulator TFEB, had the highest phylogenetic conservation of three consecutive putative GSK3 phosphorylation sites in animal proteomes. This finding prompted us to examine the relationship between MITF, endolysosomal biogenesis, and Wnt signaling. Here we report that MITF expression levels correlated with the expression of a large subset of lysosomal genes in melanoma cell lines. MITF expression in the tetracycline-inducible C32 melanoma model caused a marked increase in vesicular structures, and increased expression of late endosomal proteins, such as Rab7, LAMP1, and CD63. These late endosomes were not functional lysosomes as they were less active in proteolysis, yet were able to concentrate Axin1, phospho-LRP6, phospho-β-catenin, and GSK3 in the presence of Wnt ligands. This relocalization significantly enhanced Wnt signaling by increasing the number of multivesicular bodies into which the Wnt signalosome/destruction complex becomes localized upon Wnt signaling. We also show that the MITF protein was stabilized by Wnt signaling, through the novel C-terminal GSK3 phosphorylations identified here. MITF stabilization caused an increase in multivesicular body biosynthesis, which in turn increased Wnt signaling, generating a positive-feedback loop that may function during the proliferative stages of melanoma. The results underscore the importance of misregulated endolysosomal biogenesis in Wnt signaling and cancer.

Unlabelled: Members of the Ewing sarcoma family of tumors (ESFT) contain tumor-associated translocations that give rise to oncogenic transcription factors, most commonly EWS/FLI1. EWS/FLI1 plays a dominant role in tumor progression by modulating the expression of hundreds of target genes. Here, the impact of EWS/FLI1 inhibition, by RNAi-mediated knockdown, on cellular signaling was investigated using mass spectrometry-based phosphoproteomics to quantify global changes in phosphorylation. This unbiased approach identified hundreds of unique phosphopeptides enriched in processes such as regulation of cell cycle and cytoskeleton organization. In particular, phosphotyrosine profiling revealed a large upregulation of STAT3 phosphorylation upon EWS/FLI1 knockdown. However, single-cell analysis demonstrated that this was not a cell-autonomous effect of EWS/FLI1 deficiency, but rather a signaling effect occurring in cells in which knockdown does not occur. Conditioned media from knockdown cells were sufficient to induce STAT3 phosphorylation in control cells, verifying the presence of a soluble factor that can activate STAT3. Cytokine analysis and ligand/receptor inhibition experiments determined that this activation occurred, in part, through an IL6-dependent mechanism. Taken together, the data support a model in which EWS/FLI1 deficiency results in the secretion of soluble factors, such as IL6, which activate STAT signaling in bystander cells that maintain EWS/FLI1 expression. Furthermore, these soluble factors were shown to protect against apoptosis., Implications: EWS/FLI1 inhibition results in a novel adaptive response and suggests that targeting the IL6/STAT3 signaling pathway may increase the efficacy of ESFT therapies., (©2014 American Association for Cancer Research.)

Introduction: (131)I-CLR1404 is a small molecule that combines a tumor-targeting moiety with a therapeutic radioisotope. The primary aim of this phase 1 study was to determine the administered radioactivity expected to deliver 400 mSv to the bone marrow. The secondary aims were to determine the pharmacokinetic (PK) and safety profiles of (131)I-CLR1404., Methods: Eight subjects with refractory or relapsed advanced solid tumors were treated with a single injection of 370 MBq of (131)I-CLR1404. Whole body planar nuclear medicine scans were performed at 15-35 minutes, 4-6, 18-24, 48, 72, 144 hours, and 14 days post injection. Optional single photon emission computed tomography imaging was performed on two patients 6 days post injection. Clinical laboratory parameters were evaluated in blood and urine. Plasma PK was evaluated on (127)I-CLR1404 mass measurements. To evaluate renal clearance of (131)I-CLR1404, urine was collected for 14 days post injection. Absorbed dose estimates for target organs were determined using the RADAR method with OLINDA/EXM software., Results: Single administrations of 370 MBq of (131)I-CLR1404 were well tolerated by all subjects. No severe adverse events were reported and no adverse event was dose-limiting. Plasma (127)I-CLR1404 concentrations declined in a bi-exponential manner with a mean t½ value of 822 hours. Mean Cmax and AUC(0-t) values were 72.2 ng/mL and 15753 ng • hr/mL, respectively. An administered activity of approximately 740 MBq is predicted to deliver 400 mSv to marrow., Conclusions: Preliminary data suggest that (131)I-CLR1404 is well tolerated and may have unique potential as an anti-cancer agent., Trial Registration: ClinicalTrials.gov NCT00925275.

Current toxicology studies frequently lack measurements at molecular resolution to enable a more mechanism-based and predictive toxicological assessment. Recently, a systems toxicology assessment framework has been proposed, which combines conventional toxicological assessment strategies with system-wide measurement methods and computational analysis approaches from the field of systems biology. Proteomic measurements are an integral component of this integrative strategy because protein alterations closely mirror biological effects, such as biological stress responses or global tissue alterations. Here, we provide an overview of the technical foundations and highlight select applications of proteomics for systems toxicology studies. With a focus on mass spectrometry-based proteomics, we summarize the experimental methods for quantitative proteomics and describe the computational approaches used to derive biological/mechanistic insights from these datasets. To illustrate how proteomics has been successfully employed to address mechanistic questions in toxicology, we summarized several case studies. Overall, we provide the technical and conceptual foundation for the integration of proteomic measurements in a more comprehensive systems toxicology assessment framework. We conclude that, owing to the critical importance of protein-level measurements and recent technological advances, proteomics will be an integral part of integrative systems toxicology approaches in the future.

Purpose: PD-L1 is the main ligand for the immune inhibitory receptor PD-1. This ligand is frequently expressed by melanoma cells. In this study, we investigated whether PD-L1 expression is controlled by melanoma driver mutations and modified by oncogenic signaling inhibition., Experimental Design: Expression of PD-L1 was investigated in a panel of 51 melanoma cell lines containing different oncogenic mutations, including cell lines with innate and acquired resistance to BRAF inhibitors (BRAFi). The effects of targeted therapy drugs on expression of PD-L1 by melanoma cells were investigated., Results: No association was found between the level of PD-L1 expression and mutations in BRAF, NRAS, PTEN, or amplification of AKT. Resistance to vemurafenib due to the activation of alternative signaling pathways was accompanied with the induction of PD-L1 expression, whereas the resistance due to the reactivation of the MAPK pathway had no effect on PD-L1 expression. In melanoma cell lines, the effects of BRAF, MEK, and PI3K inhibitors on expression of PD-L1 were variable from reduction to induction, particularly in the presence of INFγ. In PD-L1-exposed lymphocytes, vemurafenib paradoxically restored activity of the MAPK pathway and increased the secretion of cytokines., Conclusions: In melanoma cell lines, including BRAFi-resistant cells, PD-L1 expression is variably regulated by oncogenic signaling pathways. PD-L1-exposed lymphocytes decrease MAPK signaling, which is corrected by exposure to vemurafenib, providing potential benefits of combining this drug with immunotherapies., (©2014 American Association for Cancer Research.)

Many solid tumors contain an overabundance of phospholipid ethers relative to normal cells. Capitalizing on this difference, we created cancer-targeted alkylphosphocholine (APC) analogs through structure-activity analyses. Depending on the iodine isotope used, radioiodinated APC analog CLR1404 was used as either a positron emission tomography (PET) imaging ((124)I) or molecular radiotherapeutic ((131)I) agent. CLR1404 analogs displayed prolonged tumor-selective retention in 55 in vivo rodent and human cancer and cancer stem cell models. (131)I-CLR1404 also displayed efficacy (tumor growth suppression and survival extension) in a wide range of human tumor xenograft models. Human PET/CT (computed tomography) and SPECT (single-photon emission computed tomography)/CT imaging in advanced-cancer patients with (124)I-CLR1404 or (131)I-CLR1404, respectively, demonstrated selective uptake and prolonged retention in both primary and metastatic malignant tumors. Combined application of these chemically identical APC-based radioisosteres will enable personalized dual modality cancer therapy of using molecular (124)I-CLR1404 tumor imaging for planning (131)I-CLR1404 therapy., (Copyright © 2014, American Association for the Advancement of Science.)