Abstract Purpose of the study: Lung cancer is not a single disease entity; it represents a spectrum of malignancies. Out of these lung adenocarcinoma (LUAD) accounts for about 65% of all lung cancers and shows a strong etiological correlation with smoking. Clinical studies show that about 30% of LUAD patients continue to smoke after being diagnosed with lung cancer. In addition, many others are exposed to nicotine via secondhand smoke and smoking cessation devices. Nicotine, the addictive component of tobacco smoke, accelerates the growth of human LUADs via the by nicotinic acetylcholine receptors (nAChRs) on target cells. The first-line therapy for LUAD patients involves the administration of tyrosine kinase inhibitors (TKIs) targeting EGFR, ALK, MET kinases. Such targeted therapies show potent anti-cancer activity in non-smokers and never smokers but show poor efficacy in patients who continue to smoke after their diagnosis. The primary objective of our research is to evaluate the potential of choline acetyltransferase (ChAT, the enzyme which synthesizes acetylcholine) as a drug target for LUAD in smokers. We evaluated the anti-neoplastic activity of BW813U (small molecule inhibitor of ChAT) in LUADs associated with moderate-heavy smoking history. We also investigated the anti-angiogeneic activity of BW813U (as a molecular mechanism underlying its growth-inhibitory effects) LUAD. Experimental procedures. Primary human microvascular endothelial cells from the lung (HMEC-L) were used for all the experiments. These cells were used between passage 3-7. The anti-angiogenic activity of BW813U was measured in cell culture models by using the “MATRIGEL ASSAY”. The results obtained from the “MATRIGEL ASSAY” were confirmed using chicken chorioallantoic membrane (CAM) angiogenesis experiments. Finally, the anti-tumor activity of BW813U was measured in vivo by using xenograft model systems in SCID mice. Our studies also investigated the signaling pathways underlying the anti-angiogenic activity of BW813U, using chemical inhibitors and siRNA methodology. Results: BW813U robustly suppressed angiogenesis in Matrigel assays and CAM model systems. The administration of BW813U potently decreased the growth rate of H838 tumors xenotransplanted in SCID mice. Immunohistochemical staining experiments revealed that the anti-tumor activity of BW813U was correlated with decrease of CD31 angiogenic biomarker in H838 tumor sections. The anti-angiogenic activity of BW813U was mediated by the alpha7-nAChR pathway and involved the nitric oxide pathway. Taken together, our studies show that ChAT may be a valuable drug target for the therapy of LUAD. Conclusions: ChAT antagonists like BW813U decrease the growth of LUAD by suppressing tumor angiogenesis. Citation Format: Piyali Dasgupta, Eric W. Bow, Krista Denning, Rama S. Gadepalli, John M. Rimoldi, Yi Charlie Chen, Sarah L. Miles. Choline Acetyltransferase: A novel drug target in lung adenocarcinoma patients who are exposed to tobacco smoke [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 587.

of the study: Lung adenocarcinoma (LUAD) accounts for about 60% of all lung cancers and shows a strong etiological correlation with smoking. Nicotine, the addictive component of tobacco smoke is known to accelerate the growth of human lung tumors by stimulating tumor angiogenesis. The pro-angiogenic effects of nicotine are mediated by nicotinic acetylcholine receptors (nAChRs). The endogenous ligand for nicotine is the neurotransmitter acetylcholine (ACh). The primary aim of this study was to determine if disruption of ACh production (via inhibition of the enzyme choline acetyltransferase; ChAT) could inhibit angiogenesis and suppress the growth of human LUADs. We used a small molecule inhibitor of ChAT namely BW813U for our studies. Primary human microvascular endothelial cells from the lung (HMEC-L) were used for all the experiments. These cells were used between passage 3-7. The anti-angiogenic activity of BW813U was measured in cell culture models by using the “MATRIGEL ASSAY”. The results obtained from the “MATRIGEL ASSAY” were confirmed using chicken chorioallantoic membrane (CAM) angiogenesis experiments. Finally, the anti-tumor activity of BW813U was measured in vivo by using xenograft model systems in SCID mice. Our studies also investigated the signaling pathways underlying the anti-angiogenic activity of BW813U, using chemical inhibitors and siRNA methodology. BW813U robustly suppressed angiogenesis in Matrigel assays and CAM model systems. The administration of BW813U potently decreased the growth rate of H838 tumors xenotransplanted in SCID mice. Immunohistochemical staining experiments revealed that the anti-tumor activity of BW813U was correlated with decrease of CD31 angiogenic biomarker in H838 tumor sections. The anti-angiogenic activity of BW813U was mediated by the alpha7-nAChR pathway and involved the nitric oxide pathway. Taken together, our studies show that ChAT may be a valuable drug target for the therapy of LUAD. ChAT antagonists like BW813U decrease the growth of LUAD by suppressing tumor angiogenesis. Support or Funding Information: Funding for our study was supported by a NIH R15-AREA Grant (2R15CA161491-03) and a Center for Natural Products pilot grant from the WV-INBRE Grant Program (P20GM103434, PI:Gary Rankin). Furthermore, this study was supported in part by an Institutional Development Award (IDeA) Grant number P20GM104932 from the National Institute of General Medical Sciences (NIGMS) and the Research Core B of COBRE, a component of the National Institutes of Health (NIH). Piyali Dasgupta, Eric W. Bow, Krista L. Denning, Rama S. Gadepalli, John M. Rimoldi, Yi Charlie Chen, Sarah L. Miles. Disruption of the acetylcholine signaling pathway inhibits angiogenesis and tumor growth in human lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5740.

Low-efficacy mu opioid receptor (MOR) agonists may serve as novel candidate analgesics with improved safety relative to high-efficacy opioids. This study used a recently validated assay of pain-depressed behavior in mice to evaluate a novel series of MOR-selective C9-substituted phenylmorphan opioids with graded MOR efficacies. Intraperitoneal injection of dilute lactic acid (IP acid) served as a noxious stimulus to depress locomotor activity by mice in an activity chamber composed of two compartments connected by an obstructed door. Behavioral measures included (1) crosses between compartments (vertical activity over the obstruction) and (2) movement counts quantified as photobeam breaks summed across compartments (horizontal activity). Each drug was tested alone and as a pretreatment to IP acid. A charcoal-meal test and whole-body-plethysmography assessment of breathing in 5% CO2 were also used to assess gastrointestinal (GI) inhibition and respiratory depression, respectively. IP acid produced a concentration-dependent depression in crosses and movement that was optimally alleviated by intermediate- to low-efficacy phenylmorphans with sufficient efficacy to produce analgesia with minimal locomotor disruption. Follow-up studies with two low-efficacy phenylmorphans (JL-2-39 and DC-1-76.1) indicated that both drugs produced naltrexone-reversible antinociception with a rapid onset and a duration of ∼1 h. Potency of both drugs increased when behavior was depressed by a lower IP-acid concentration, and neither drug alleviated behavioral depression by a non-pain stimulus (IP lithium chloride). Both drugs produced weaker GI inhibition and respiratory depression than fentanyl and attenuated fentanyl-induced GI inhibition and respiratory depression. Results support further consideration of selective, low-efficacy MOR agonists as candidate analgesics. SIGNIFICANCE STATEMENT This study used a novel set of mu opioid receptor (MOR)-selective opioids with graded MOR efficacies to examine the lower boundary of MOR efficacy sufficient to relieve pain-related behavioral depression in mice. Two novel low-efficacy opioids (JL-2-39, DC-1-76.1) produced effective antinociception with improved safety relative to higher- or lower-efficacy opioids, and results support further consideration of these and other low-efficacy opioids as candidate analgesics.

ID 21458 Poster Board 270 Aim: Biased signaling by mu-opioid agonists has been proposed to result in fewer opioid side effects and, consequently, an improved safety profile compared to conventional prescription opioids. We evaluated the acute effects of novel G-protein preferring opioid agonists in an assay of self-administration in nonhuman primates to determine whether G-protein preferring opioid agonists have reduced abuse liability. Methods: Squirrel monkeys (Saimiri sciureus, n=3) surgically implanted with IV catheters were trained to respond on one lever for a food reinforcer (20% sweetened condensed milk) and on another lever for an IV injection of 0.01 mg/kg heroin under concurrent fixed ratio schedules. Once trained, heroin dose-effect functions were established in each subject by varying the unit dose of heroin available for injection, from 0.001 to 0.032 mg/kg/inj, under a modified double alternation schedule. Next, under similar conditions, the reinforcing effects of the novel mu-opioid agonists EWB-3-1, EWB-2-189 and EG-1-203 were evaluated, as were the effects of the rapid acting opioid agonist, remifentanil. The reinforcing effects of heroin were periodically redetermined to ensure stability of performance. Results: Under the concurrent schedules of reinforcement, responding was primarily distributed to the food-associated lever when saline was available and was increasingly allocated to the injection-associated lever with increasing doses of heroin. Heroin maintained self-administration over the dose range of 0.0032 to 0.032 mg/kg/inj, with peak number of injections, 26±13, obtained at 0.0032 mg/kg/inj heroin. Higher doses maintained fewer injections, resulting in an inverted-U shaped dose response function. Remifentanil had qualitatively and quantitatively similar effects to heroin, although it was more potent with peak effects occurring at the unit dose of 0.00032 mg/kg/inj. The three novel opioids also maintained self-administration behavior: responding on the injection-associated lever increased with the unit dose of each novel compound, the peak number of injections was similar across drugs, ranging from 22±4 (EWB-1-203) to 29±3 (EWB-2-189), and inverted U-shaped functions were obtained with EWB-3-1 and EG-2-189. Solubility limits precluded testing doses of EWB-1-203 on the descending portion of the dose-effect curve. Conclusion: EWB-3-1, EG-1-203, and EWB-2-189 all had reinforcing effects in a self-administration procedure, indicative of potential abuse liability. These data demonstrate that the absence of β-arrestin recruitment by mu-opioid agonists does not necessarily predict lower abuse liability of opioid agonists. Funded by NIH/NIDA DA047574

All possible diastereomeric C9-hydroxymethyl-, hydroxyethyl-, and hydroxypropyl-substituted 5-phenylmorphans were synthesized to explore the three-dimensional space around the C9 substituent in our search for potent MOR partial agonists. These compounds were designed to lessen the lipophilicity observed with their C9-alkenyl substituted relatives. Many of the 12 diastereomers that were obtained were found to have nanomolar or subnanomolar potency in the forskolin-induced cAMP accumulation assay. Almost all these potent compounds were fully efficacious, and three of those chosen for in vivo evaluation, 15, 21, and 36, were all extremely G-protein biased; none of the three compounds recruited beta-arrestin2. Only one of the 12 diastereomers, 21 (3-((1S,5R,9R)-9-(2-hydroxyethyl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol), was a MOR partial agonist with good, but not full, efficacy (Emax = 85%) and subnanomolar potency (EC50 = 0.91 nM) in the cAMP assay. It did not have any KOR agonist activity. This compound was unlike morphine in that it had a limited ventilatory effect in vivo. The activity of 21 could be related to one or more of three well-known theories that attempt to predict a dissociation of the desired analgesia from the undesirable opioid-like side-effects associated with clinically used opioids. In accordance with the theories, 21 was a potent MOR partial agonist, it was highly G-protein biased and did not attract beta-arrestin2, and it was found to have both MOR and DOR agonist activity. All the other diastereomers that were synthesized were either much less potent than 21 or had either too little or too much efficacy for our purposes. It was also noted that a C9-methoxymethyl compound with 1R,5S,9R stereochemistry (41) was more potent than the comparable C9-hydroxymethyl compound 11 (EC50 = 0.65 nM for 41 vs. 2.05 nM for 11). Both 41 and 11 were fully efficacious.

Low-efficacy mu-opioid receptor (MOR) agonists represent promising therapeutics, but existing compounds (e.g., buprenorphine, nalbuphine) span a limited range of low MOR efficacies and have poor MOR selectivity. Accordingly, new and selective low-efficacy MOR agonists are of interest. A novel set of chiral C9-substituted phenylmorphans has been reported to display improved MOR selectivity and a range of high-to-low MOR efficacies under other conditions; however, a full opioid receptor binding profile for these drugs has not been described. Additionally, studies in mice will be useful for preclinical characterization of these novel compounds, but the pharmacology of these drugs in mice has also not been examined. Accordingly, the present study characterized the binding selectivity and in vitro efficacy of these compounds using assays of opioid receptor binding and ligand-stimulated [35 S]GTPɣS binding. Additionally, locomotor effects were evaluated as a first step for in vivo behavioral assessment in mice. The high-efficacy MOR agonist and clinically effective antidepressant tianeptine was included as a comparator. In binding studies, all phenylmorphans showed improved MOR selectivity relative to existing lower-efficacy MOR agonists. In the ligand-stimulated [35 S]GTPɣS binding assay, seven phenylmorphans had graded levels of sub-buprenorphine MOR efficacy. In locomotor studies, the compounds again showed graded efficacy with a rapid onset and ≥1 h duration of effects, evidence for MOR mediation, and minor sex differences. Tianeptine functioned as a high-efficacy MOR agonist. Overall, these in vitro and in vivo studies support the characterization of these compounds as MOR-selective ligands with graded MOR efficacy and utility for further behavioral studies in mice.

Angiogenesis refers to the development of new blood vessels from preexisting blood vessels. The angiogenic pathway is essential for the growth and progression of almost every kind of human cancer. Nicotine, the addictive component of tobacco smoke is known to accelerate the growth of human lung tumors by stimulating tumor angiogenesis. The pro‐angiogenic effects of nicotine are mediated by nicotinic acetylcholine receptors (nAChRs). The endogenous ligand for nicotine is the neurotransmitter acetylcholine (ACh). Published reports show that ACh acts as growth factor for human lung cancers. Almost all lung tissues (including fetal lung) express signaling proteins involved in the synthesis, transport of the ACh‐signaling pathway. These include choline acetyl transferase enzyme (ChAT), vesicular acetylcholine transporter (VAChT), acetylcholine esterase (AChE) and choline transporter (ChT). The primary objective of our studies was to determine if disruption of ChAT could inhibit angiogenesis and suppress the growth of human LACs. We used a small molecule inhibitor of ChAT namely BW813U for our studies. BW813U robustly suppressed angiogenesis in Matrigel assays and chicken chorioallantoic membrane (CAM) assays. The administration of BW813U potenetly decreased the growth rate of H838 tumors xenotransplanted in SCID mice. Immunohistochemical staining experiments revealed that the anti‐tumor activity of BW813U was correlated with decrease of CD31 angiogenic biomarker in H813 tumor sections. The anti‐angiogenic activity of BW813U was mediated by the alpha7‐nAChR pathway and involved the nitric oxide pathway. Taken together, our studies show that ChAT antagonists like BW813U may have applications in the treatment of LAC.

Opioid use disorders and fatal overdose due to consumption of fentanyl-laced heroin remain a major public health menace in the United States. Vaccination may serve as a promising potential remedy to combat accidental overdose and to mitigate the abuse potential of opioids. We previously reported the heroin and fentanyl monovalent vaccines carrying, respectively, a heroin hapten, 6-AmHap, and a fentanyl hapten, para-AmFenHap, conjugated to tetanus toxoid (TT). Herein, we describe the mixing of these antigens to formulate a bivalent vaccine adjuvanted with liposomes containing monophosphoryl lipid A (MPLA) adsorbed on aluminum hydroxide. Immunization of mice with the bivalent vaccine resulted in IgG titers of >105 against both haptens. The polyclonal sera bound heroin, 6-acetylmorphine, morphine, and fentanyl with dissociation constants (Kd) of 0.25 to 0.50 nM. Mice were protected from the anti-nociceptive effects of heroin, fentanyl, and heroin +9% (w/w) fentanyl. No cross-reactivity to methadone and buprenorphine was observed in vivo. Naloxone remained efficacious in immunized mice. These results highlighted the potential of combining TT-6-AmHap and TT-para-AmFenHap to yield an efficacious bivalent vaccine that could ablate heroin and fentanyl effects. This vaccine warrants further testing to establish its potential translatability to humans.

Lung adenocarcinoma (LACs) accounts for about 60% of all lung cancer cases in the United States. Epidemiological studies show that the development of lung adenocarcinoma is strongly correlated to smoking habits. Although, cigarette smoke is a complex mixture of over 4000 chemicals, nicotine is the active and addictive component of cigarette smoke. Our previous data show that nicotine accelerates the growth of human lung cancers by promoting angiogenesis. The endogenous ligand for nicotine in lung cancer cells is the neurotransmitter ACh. The enzyme choline acetyltransferase (ChAT) synthesizes ACh in mammalian cells. The present study aims to determine the anti-tumor and anti-angiogenic activity of BW813U in human LACs. Human microvascular endothelial cells from the lung (HMEC-L) robustly express enzymatically active ChAT. BW813U robustly blocked the proliferation and angiogenic tubule formation of HMEC-Ls. These experiments were repeated in lung adenocarcinoma-associated endothelial cells (LAAECs) and similar results were obtained. The anti-angiogenic activity of BW813 was confirmed in rat retinal explants assay and chicken chorioallantoic membrane (CAM) assays. Finally, the administration of BW813U potently suppressed the growth of human LAC tumors xenotransplanted in athymic mice. Immunoshistochemical experiments revealed that BW813U-treated human LAC tumors (isolated from athymic mice) contained fewer number of blood vessels than vehicle-treated mice. The anti-angiogenic activity of BW813U was mediated by nicotinic acetylcholine receptors on HMEC-Ls and required the Akt pathway. The administration of BW813U did not cause any discomfort or toxicity in mice. Therefore ChAT blockers (like BW813U) may be useful in suppressing angiogenesis and growth of human LACs.

The need for safer pain-management therapies with decreased abuse liability inspired a novel drug design that retains μ-opioid receptor (MOR)-mediated analgesia, while minimizing addictive liability. We recently demonstrated that targeting the dopamine D3 receptor (D3R) with highly selective antagonists/partial agonists can reduce opioid self-administration and reinstatement to drug seeking in rodent models without diminishing antinociceptive effects. The identification of the D3R as a target for the treatment of opioid use disorders prompted the idea of generating a class of ligands presenting bitopic or bivalent structures, allowing the dual-target binding of the MOR and D3R. Structure-activity relationship studies using computationally aided drug design and in vitro binding assays led to the identification of potent dual-target leads (23, 28, and 40), based on different structural templates and scaffolds, with moderate (sub-micromolar) to high (low nanomolar/sub-nanomolar) binding affinities. Bioluminescence resonance energy transfer-based functional studies revealed MOR agonist-D3R antagonist/partial agonist efficacies that suggest potential for maintaining analgesia with reduced opioid-abuse liability.

Active immunization is an emerging potential modality to combat fatal overdose amid the opioid epidemic. In this study, we described the design, synthesis, formulation, and animal testing of an efficacious vaccine against fentanyl. The vaccine formulation is composed of a novel fentanyl hapten conjugated to tetanus toxoid (TT) and adjuvanted with liposomes containing monophosphoryl lipid A adsorbed on aluminum hydroxide. The linker and hapten N-phenyl-N-(1-(4-(3-(tritylthio)propanamido)phenethyl)piperidin-4-yl)propionamide were conjugated sequentially to TT using amine-N-hydroxysuccinimide-ester and thiol-maleimide reaction chemistries, respectively. Conjugation was facile, efficient, and reproducible with a protein recovery of >98% and a hapten density of 30-35 per carrier protein molecule. In mice, immunization induced high and robust antibody endpoint titers in the order of >106 against the hapten. The antisera bound fentanyl, carfentanil, cyclopropyl fentanyl, para-fluorofentanyl, and furanyl fentanyl in vitro with antibody-drug dissociation constants in the range of 0.36-4.66 nM. No cross-reactivity to naloxone, naltrexone, methadone, or buprenorphine was observed. In vivo, immunization shifted the antinociceptive dose-response curve of fentanyl to higher doses. Collectively, these preclinical results showcased the desired traits of a potential vaccine against fentanyl and demonstrated the feasibility of immunization to combat fentanyl-induced effects.

A series of compounds have been synthesized with a variety of substituents based on a three-carbon chain at the C9-position of 3-hydroxy-N-phenethyl-5-phenylmorphan (3-(2-phenethyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol). Three of these were found to be μ-opioid receptor agonists in the inhibition of forskolin-induced cAMP accumulation assay and they did not recruit β-arrestin at all in the PathHunter assay and in the Tango assay. Compound 12 (3-((1S,5R,9R)-2-phenethyl-9-propyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol), 13 (3-((1S,5R,9R)-9-((E)-3-hydroxyprop-1-en-1-yl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol), and 15a (3-((1S,5R,9R)-9-(2-hydroxypropyl)-2-phenethyl-2-azabicyclo[3.3.1]nonan-5-yl)phenol) were partial μ-agonists. Two of them had moderate efficacies (E MAX ca. 65%) and one had lower efficacy, and they were ca. 5, 3, and 4 times more potent, respectively, than morphine in vitro. Computer simulations were carried out to provide a molecular basis for the high bias ratios of the C9-substituted 5-phenylmorphans toward G-protein activation.

The clinicopathological properties of lung adenocarcinoma (LAC) in smokers is divergent from LAC in non‐smokers. One of the distinctive features of LAC in smokers is that the disease is relatively resistant to targeted molecular therapies. For example, targeted therapeutic agents like EGFR‐inhibitors (erlotinib and gefitinib) are highly effective in LACs in non‐smokers. However, these agents display much lower anti‐tumor activity in LACs in patients who are active smokers. Similarly, lung cancer patients (who are active smokers) show a lower response to chemotherapy than those who are non‐smokers. However, the majority of LAC patients are smokers. This underlines the need to identify viable drug targets for LAC therapy in patients who are exposed to cigarette smoke. A survey of literature reveals that nicotine (the addictive component of cigarette smoke) accelerates the growth of lung cancers, as well as confers resistance to chemotherapy. One of the mechanisms underlying the mitogenic activity of nicotine is that it promotes the production of the neurotransmitter acetylcholine (ACh) from LAC cells. ACh is known to be an autocrine growth factor for LAC cells and is synthesized by the enzyme choline acetyltransferase (ChAT). The present study investigates the feasibility of ChAT as a molecular target for LAC in smokers. We find that ChAT levels are upregulated in human LAC cell lines and tissues in analogous to their smoking history. Finally, the ChAT inhibitor BW813U causes robust apoptosis in human LAC cell lined and LAC cell lines isolated from patients. The magnitude of BW813U‐induced apoptosis is similar across LAC cell lines irrespective of smoking history; however, the concentration of BW813U which causes apoptosis is lower in LAC cell lines belonging to heavy smokers. The anti‐tumor activity of BW813U is also observed in H838 cells (belonging to an 80 pack‐year smoker) xenografted on athymic mice. Our studies show that antagonists of ChAT like BW813U may have therapeutic applications in the majority of the population of LAC who are smokers.Support or Funding InformationFunding for our study was supported by a NIH R15‐AREA Grant (2R15CA161491‐02). Furthermore, this study was supported in part by an Institutional Development Award (IDeA) Grant number P20GM104932 from the National Institute of General Medical Sciences (NIGMS) and the Research Core B of COBRE, a component of the National Institutes of Health (NIH).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The clinicopathological features of lung cancer in smokers is completely different from never‐smokers. Whereas lung cancer patients (who are never smokers) respond well to targeted therapies involving EGFR inhibitors and ALK kinase inhibitors, the response of these drugs in lung cancer patients who are smokers is poor. Nicotine is the addictive component of cigarette smoke. Nicotine (at concentrations present in the plasma of moderate smokers) accelerates the growth, angiogenesis and metastasis of lung cancers. The biological activity of nicotine is mediated by nicotinic acetylcholine receptors (nAChRs). The endogenous ligand for nAChRs in the lung is the neurotransmitter acetylcholine (ACh). ACh is known to be an autocrine growth factor for both SCLC and NSCLC. One of the ways that nicotine and cigarette smoke promote the growth of lung cancers is by promoting the production of ACh, which binds to its cognate receptor (on lung cancer cells) in an autocrine manner and induces the proliferation of lung cancer cells. The objective of the present study is to investigate the whether the ACh‐mitogenic signaling pathway are altered in human lung cancer. We show that levels three acetylcholine‐signaling proteins namely ChAT, VAChT and AChE are significantly altered in lung cancer occurring in active smokers. Small‐molecule regulators of these proteins suppress the growth of human lung cancer cells. Our studies will facilitate the identification of targeted therapies for the majority of lung cancer patients who are exposed to tobacco smoke.Support or Funding InformationFunding for our study was supported by an NIH R15‐AREA Grant (2R15CA161491‐02) and the West Virginia IDeA Network of Biomedical Research Excellence (WV‐INBRE) grant GM103434 (PI: Dr. G. Rankin). Furthermore, this study was supported in part by an Institutional Development grant (IDeA) Grant number P20GM104932 from the National Institute of General Medical Sciences (NIGMS) and the Research Core B of COBRE, a component of the National Institutes of Health (NIH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The pathophysiological characteristics of lung adenocarcinoma (LAC) in smokers is divergent from LAC in non‐smokers. One of the distinctive features of LAC in smokers is that the disease is relatively resistant to targeted molecular therapies. For example, targeted therapeutic agents like EGFR‐inhibitors (erlotinib and gefitinib) are highly effective in LACs in non‐smokers. However, these agents display much lower anti‐tumor activity in LACs in patients who are active smokers. Similarly, lung cancer patients (who are active smokers) show a lower response to chemotherapy than those who are non‐smokers. However, the majority of LAC patients are smokers. This underlines the need to identify viable drug targets for LAC therapy in patients who are exposed to cigarette smoke. A survey of literature reveals that nicotine (the addictive component of cigarette smoke) accelerates the growth of lung cancers, as well as confers resistance to chemotherapy. One of the mechanisms underlying the mitogenic activity of nicotine is that it promotes the production of the neurotransmitter acetylcholine (ACh) from LAC cells. ACh is known to be an autocrine growth factor for LAC cells and is synthesized by the enzyme choline acetyltransferase (ChAT). The present study investigates the feasibility of ChAT as a molecular target for LAC in smokers. We find that ChAT levels are upregulated in human LAC cell lines and tissues in analogous to their smoking history. Finally, the ChAT inhibitor BW813U causes robust apoptosis in human LAC cells. The magnitude of BW813U‐induced apoptosis is similar across LAC cell lines (irrespective of smoking history); however, the concentration of BW813U which causes apoptosis is lower in LAC cell lines belonging to heavy smokers. The anti‐tumor activity of BW813U is also observed in H838 cells (belonging to an 80 pack‐year smoker) xenografted on athymic mice. Our studies show that antagonists of ChAT like BW813U may have therapeutic applications in the majority of the population of LAC who are smokers. Support or Funding Information Funding for our study was supported by a NIH R15‐AREA Grant (2R15CA161491‐02). Furthermore, this study was supported in part by an Institutional Development Award (IDeA) Grant number P20GM104932 from the National Institute of General Medical Sciences (NIGMS) and the Research Core B of COBRE, a component of the National Institutes of Health (NIH). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

C1 Disruption of Choline Acetyltransferase Activity Suppresses Lung Adenocarcinoma Growth in Smokers A.T. Akers1, K.W. Colclough1, J.R. Friedman1, E.W. Bow2, J.M. Rimoldi2, S.J. Cutler3, E.W. Hardman1, P. Dasgupta1, J.M. Seidler1 1Joan C. Edwards School of Medicine, Huntington, West Virginia, USA; 2The University of Mississippi, Oxford, Mississippi, USA; 3The University of South Carolina, Columbia, South Carolina, USA The clinicopathological profile of lung adenocarcinoma (LAC) differs in smokers and non-smokers. LAC in smokers is relatively resistant to therapies. For example, targeted therapeutic agents like EGFR-inhibitors (erlotinib and gefitinib) are highly effective in LACs which develop in nonsmokers. However, such targeted therapies display much lower anti-tumor activity in LACs in patients who are active smokers. Similarly, lung cancer patients (who are active smokers) show a lower response to chemotherapy than those who are non-smokers. However, the majority of LAC patients are smokers. This underlines the need to identify novel molecular targets relevant for LAC therapy in patients who are exposed to cigarette smoke via active smoking or exposure to secondhand smoke. Several convergent studies show that nicotine (the addictive component of cigarette smoke) accelerates the growth of lung cancers, as well as confers resistance to chemotherapy. One of the mechanisms underlying the biological activity of nicotine is that it promotes the secretion of the neurotransmitter acetylcholine (ACh) from LAC cells. ACh is known to be an autocrine growth factor for LAC cells and is synthesized by the enzyme choline acetyltransferase (ChAT). We investigated the feasibility of ChAT as a molecular target for LAC in smokers. We find that ChAT levels are upregulated in human LAC cell lines and tissues in a smoking history– dependent manner. Finally, the ChAT inhibitor BW813U causes robust apoptosis in human LAC cells. The magnitude of BW813U-induced apoptosis is similar across LAC cell lines (irrespective of smoking history); however, the concentration of BW813U that causes apoptosis is lower in LAC cell lines belonging to heavy smokers. Our studies validate choline acetyltransferase (ChAT) as a viable drug target for the majority of the population of lung cancer patients who are smokers.

The clinicopathological profile of lung adenocarcinoma (LAC) in smokers is completely different from those in non‐smokers. One of the unique features of LAC in smokers is that it is relatively resistant to therapies. For example, targeted therapeutic agents like EGFR‐inhibitors (erlotinib and gefitinib) are highly effective in LACs which develop in non‐smokers. However, these EGFR‐inhibitors display much lower anti‐tumor activity in LACs in patients who are active smokers. Similarly, lung cancer patients (who are active smokers) show lower response to chemotherapy than those who are non‐smokers. However, majority of LAC patients are smokers. This underlines the need to identify novel molecular targets relevant for LAC therapy in patients who are exposed to cigarette smoke via active smoking or exposure to secondhand smoke. Several convergent studies show that nicotine (the addictive component of cigarette smoke) accelerates the growth of lung cancers, as well as confers resistance to chemotherapy. One of the mechanisms underlying these biological activity of nicotine is that it promotes the secretion of the neurotransmitter acetylcholine (ACh) from LAC cells. ACh is known to be an autocrine growth factor for LAC cells and is synthesized by the enzyme choline acetyltransferase (ChAT). The present study investigates the feasibility of ChAT as a molecular target for LAC in smokers. We find that ChAT levels are upregulated in human LAC cell lines and tissues in a manner dependent on the smoking history. Finally, the ChAT inhibitor BW813U causes robust apoptosis in human LAC cells. The magnitude of BW813U‐induced apoptosis is similar across LAC cell lines (irrespective of smoking history); however, the concentration of BW813U which causes apoptosis is lower in LAC cell lines belonging to heavy smokers. Our studies validate choline acetyltransferase (CHAT) as a viable drug target for the majority of population of lung cancer patients who are smokers.Support or Funding InformationFunding for our study was supported by the an NIH R15‐AREA Grant (2R15CA161491‐02), WVU‐MU Health Partnership Grant and by ACTSI Grant from Marshall University. Furthermore, this study was supported in part by an Institutional Development Award (IDeA) Grant number P20GM104932 from the National Institute of General Medical Sciences (NIGMS) and the Research Core B of COBRE, a component of the National Institutes of Health (NIH).

The cannabinoids are members of a deceptively simple class of terpenophenolic secondary metabolites isolated from Cannabis sativa highlighted by (-)-Δ 9 -tetrahydrocannabinol (THC), eliciting distinct pharmacological effects mediated largely by cannabinoid receptor (CB1 or CB2) signaling. Since the initial discovery of THC and related cannabinoids, synthetic and semisynthetic classical cannabinoid analogs have been evaluated to help define receptor binding modes and structure–CB1/CB2 functional activity relationships. This perspective will examine the classical cannabinoids, with particular emphasis on the structure–activity relationship of five regions: C3 side chain, phenolic hydroxyl, aromatic A-ring, pyran B-ring, and cyclohexenyl C-ring. Cumulative structure–activity relationship studies to date have helped define the critical structural elements required for potency and selectivity toward CB1 and CB2 and, more importantly, ushered the discovery and development of contemporary nonclassical cannabinoid modulators with enhanced physicochemical and pharmacological profiles.

Our research focuses on the molecular architecture of biomolecules to obtain insight into the folding and catalytic behavior of the Anabaena group I intron in order to formulate a model structure. Group I introns are RNA sequences found in many organisms, which are capable of self‐splicing and removal from neighboring RNA sequences. The Anabaena group I intron itself comes from a cyanobacteria. To study the Anabaena ribozyme we are using both chemical methods coupled with computer bioinformatics. The folding and catalytic behavior of the ribozyme can be studied using fluorescent‐based assays to observe structural changes in the ribozyme. JalView sequence alignment software has also been adapted to analyze and annotate specific sequences of this Group I catalytic RNA. We can use this to map both chemical and genetic studies of this RNA molecule. As the structure of Anabaena itself has not yet been determined, it is our goal to use computation software to develop to an accurate three‐dimensional representation of the Anabaena ribozyme. Describing the three dimensional structure and dynamic of this model biomolecule system will help us gain a broader understanding of their function in living systems. The research was supported by the Albion College Foundation for Undergraduate Research, Scholarly and Creative Activity.