Adam Stewart

Measuring behavioral and endocrine responses to novelty stress in adult zebrafish

Several behavioral assays are currently used for high-throughput neurophenotyping and screening of genetic mutations and psychotropic drugs in zebrafish (Danio rerio). In this protocol, we describe a battery of two assays to characterize anxiety-related behavioral and endocrine phenotypes in adult zebrafish. Here, we detail how to use the novel tank test to assess behavioral indices of anxiety (including reduced exploration, increased freezing behavior and erratic movement), which are quantifiable using manual registration and computer-aided video-tracking analyses. In addition, we describe how to analyze whole-body zebrafish cortisol concentrations that correspond to their behavior in the novel tank test. This protocol is an easy, inexpensive and effective alternative to other methods of measuring stress responses in zebrafish, thus enabling the rapid acquisition and analysis of large amounts of data. As will be shown here, fish anxiety-like behavior can be either attenuated or exaggerated depending on stress or drug exposure, with cortisol levels generally expected to parallel anxiety behaviors. This protocol can be completed over the course of 2 d, with a variable testing duration depending on the number of fish used.

Analyzing habituation responses to novelty in zebrafish (Danio rerio)

Analysis of habituation is widely used to characterize animal cognitive phenotypes and their modulation. Although zebrafish (Danio rerio) are increasingly utilized in neurobehavioral research, their habituation responses have not been extensively investigated. Utilizing the novel tank test, we examine intra- and inter-session habituation and demonstrate robust habituation responses in adult zebrafish. Analyzing the intra-session habituation to novelty further, we also show that selected anxiogenic drugs (caffeine, pentylenetetrazole), as well as stress-inducing alarm pheromone, attenuated zebrafish habituation. Some acute anxiolytic agents, such as morphine and ethanol, while predictably reducing zebrafish anxiety, had no effects on habituation. Chronic ethanol and fluoxetine treatments improved intra-session habituation in zebrafish. In general, our study parallels literature on rodent habituation responses to novelty, and reconfirms zebrafish as a promising model for cognitive neurobehavioral research.

Three-Dimensional Neurophenotyping of Adult Zebrafish Behavior

The use of adult zebrafish (Danio rerio) in neurobehavioral research is rapidly expanding. The present large-scale study applied the newest video-tracking and data-mining technologies to further examine zebrafish anxiety-like phenotypes. Here, we generated temporal and spatial three-dimensional (3D) reconstructions of zebrafish locomotion, globally assessed behavioral profiles evoked by several anxiogenic and anxiolytic manipulations, mapped individual endpoints to 3D reconstructions, and performed cluster analysis to reconfirm behavioral correlates of high- and low-anxiety states. The application of 3D swim path reconstructions consolidates behavioral data (while increasing data density) and provides a novel way to examine and represent zebrafish behavior. It also enables rapid optimization of video tracking settings to improve quantification of automated parameters, and suggests that spatiotemporal organization of zebrafish swimming activity can be affected by various experimental manipulations in a manner predicted by their anxiolytic or anxiogenic nature. Our approach markedly enhances the power of zebrafish behavioral analyses, providing innovative framework for high-throughput 3D phenotyping of adult zebrafish behavior.

A Phase I Pharmacokinetic and Pharmacodynamic Study of CHR-3996, an Oral Class I Selective Histone Deacetylase Inhibitor in Refractory Solid Tumors

Purpose: This clinical trial investigated the safety, tolerability, pharmacokinetic (PK), and pharmacodynamic (PD) profile of CHR-3996, a selective class I histone deacetylase inhibitor. Patients and Methods: CHR-3996 was administered orally once a day. This phase I trial used a 3+3 dose-escalation design. PK profiles were analyzed by liquid chromatography–tandem mass spectroscopic methods and PD studies were conducted using ELISA studying histone H3 acetylation in peripheral blood mononuclear cells. Results: Thirty-nine patients were treated at dose levels of 5 mg (n = 3), 10 mg (n = 4), 20 mg (n = 3), 40 mg (n = 10), 80 mg (n = 10), 120 mg (n = 4), and 160 mg (n = 5) administered orally once daily. The dose-limiting toxicities seen were thrombocytopenia (160 mg), fatigue (80 and 120 mg), plasma creatinine elevation (80 and 120 mg), and atrial fibrillation (40 mg). The area under the curve was proportional to the administered dose and a maximal plasma concentration of 259 ng/mL at a dose of 40 mg exceeded the concentrations required for antitumor efficacy in preclinical models. Target inhibition measured by quantification of histone acetylation was shown at doses of 10 mg/d and was maximal at 40 mg. A partial response was seen in one patient with metastatic acinar pancreatic carcinoma. Conclusions: Taking the toxicity and PK/PD profile into consideration, the recommended phase II dose (RP2D) is 40 mg/d. At this dose, CHR-3996 has a favorable toxicologic, PK, and PD profile. CHR-3996 has shown preliminary clinical activity and should be evaluated in further clinical trials. Clin Cancer Res; 18(9); 2687–94. ©2012 AACR.

Anxiogenic-like effects of chronic nicotine exposure in zebrafish

Nicotine is one of the most widely used and abused legal drugs. Although its pharmacological profile has been extensively investigated in humans and rodents, nicotine CNS action remains poorly understood. The importance of finding evolutionarily conserved signaling pathways, and the need to apply high-throughput in vivo screens for CNS drug discovery, necessitate novel efficient experimental models for nicotine research. Zebrafish (Danio rerio) are rapidly emerging as an excellent organism for studying drug abuse, neuropharmacology and toxicology and have recently been applied to testing nicotine. Anxiolytic, rewarding and memory-modulating effects of acute nicotine treatment in zebrafish are consistently reported in the literature. However, while nicotine abuse is more relevant to long-term exposure models, little is known about chronic effects of nicotine on zebrafish behavior. In the present study, chronic 4-day exposure to 1-2mg/L nicotine mildly increased adult zebrafish shoaling but did not alter baseline cortisol levels. We also found that chronic exposure to nicotine evokes robust anxiogenic behavioral responses in zebrafish tested in the novel tank test paradigm. Generally paralleling clinical and rodent data on anxiogenic effects of chronic nicotine, our study supports the developing utility of zebrafish for nicotine research.

A study of PD-L1 expression in KRAS mutant non-small cell lung cancer cell lines exposed to relevant targeted treatments

We investigated PD-L1 changes in response to MEK and AKT inhibitors in KRAS mutant lung adenocarcinoma (adeno-NSCLC). PD-L1 expression was quantified using immunofluorescence and co-culture with a jurkat cell-line transfected with NFAT-luciferase was used to study if changes in PD-L1 expression in cancer cell lines were functionally relevant. Five KRAS mutant cell lines with high PD-L1 expression (H441, H2291, H23, H2030 and A549) were exposed to GI50 inhibitor concentrations of a MEK inhibitor (trametinib) and an AKT inhibitor (AZD5363) for 3 weeks. Only 3/5 (H23, H2030 and A549) and 2/5 cell lines (H441 and H23) showed functionally significant increases in PD-L1 expression when exposed to trametinib or AZD5363 respectively. PD-L1 overexpression is not consistent and is unlikely to be an early mechanism of resistance to KRAS mutant adeno-NSCLC treated with MEK or AKT inhibitors.

Abstract 5343: The effect of horizontal and vertical inhibition of nodes within the MAPK and PI3K-AKT pathways in NSCLC models

Introduction and Aims Efficacy of targeted anticancer drugs is limited by de novo resistances related to cross talk within and between complex signal transduction networks. Rational combinatorial targeted therapies are one of the main solutions to increase activity or overcome resistance. We aimed to investigate the effects of inhibiting different nodes within the MAPK pathway (RAF and MEK) and PI3K-AKT pathway (PI3K, AKT and m-TORC1/2) either alone or in different combinations, on cell growth in a panel of KRAS mutant and KRAS wt NSCLC cell lines. Material & Methods We used dabrafenib, trametinib, GDC-0941, MK2206 and AZD2014 to inhibit RAF, MEK, PI3K, AKT and m-TORC1/2 respectively. Inhibition of signalling output in MEK, PI3K, AKT and m-TORC1/2 was assessed by maximal reduction of p-ERK, p-AKT (Thr308), p-AKT (Ser473) and p-S6 by trametinib, GDC-0941, MK2206 and AZD2014 respectively. In keeping with its mechanism of action, maximal induction of p-MEK was considered as inhibition of RAF by dabrafenib in the cell line panel (none had mutations in BRAF). Quantification of phosphoproteins was done using MesoScale Discovery ELISA. Using concentrations of drugs shown to inhibiting signalling though these nodes, the effects of inhibiting the nodes alone or in combination on cell growth was studied using WST-1 assays. The NSCLC cell line panel included KRAS wild-type (H522; H1838; H1651) or KRAS mutant (A-549; Calu-6; H23) cells. Results The concentration range of dabrafenib, trametinib, GDC-0941, MK2206 and AZD2014 to inhibit RAF, MEK, PI3K, AKT and m-TORC1/2 related to the different cell lines was [40-1000 nM], [40-200 nM], [1000 - 10 000 nM], >50 000 nM, and [500-1500 nM] respectively. The degree of growth inhibition following inhibition of an individual node varied between 5% and 75%. Highest inhibitions of cell proliferation by drugs alone were obtained with PI3K-AKT pathways compared to the MAPK pathway inhibitors for 6/6 cell lines. Drug combinations improved inhibition of cell proliferation in all cell lines. In 3/3 KRAS mutant cells, best combinations included MEK inhibitor and any other of the PI3K-AKT pathway as compared to inhibition of different nodes within the PI3K-AKT or MAPK pathways. In 3/3 KRAS wt cell lines, vertical combinations within the PI3K-AKT pathway induced highest inhibitions of cell proliferation compared to vertical combinations within the MAPK pathway or horizontal combinations between the PI3K-AKT and MAPK pathways. Discussion/Conclusions In the cell line panel tested, KRAS mutant cell lines were more sensitive to horizontal combinations than vertical combinations with the MAPK and PI3K-AKT pathways. KRAS wt cells were more sensitive to vertical combinations within the PI3K-AKT pathway. This preclinical study has clinical implications while planning combinations of targeted agents to treat NSCLC. Citation Format: Sophie Broutin, Adam Stewart, Parames Thavasu, Angelo Paci, Jean-Michel Bidart, Udai Banerji. The effect of horizontal and vertical inhibition of nodes within the MAPK and PI3K-AKT pathways in NSCLC models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5343. doi:10.1158/1538-7445.AM2015-5343

Abstract 2627: A study of dynamic changes in PD-L1 expression inKRASmutant adenocarcinoma of the lung exposed to signal transduction inhibitors

Aim MEK and AKT inhibitors inhibit signaling down steam of KRAS and are being evaluated as single agents or in combination with other anticancer drugs for the treatment of KRAS mutant adenocarcinoma of the lung (adeno-NSCLC). We investigated whether increased PD-L1 expression with functional T cell inhibitory consequences were a common mechanism of resistance to these drugs in this setting. Material and Methods A panel of 10 KRAS mutant adeno-NSCLC cell lines were studied. Cells were exposed to GI50 concentrations of a MEK (trametinib) and AKT (AZD5363) inhibitor for 6hrs, 24hrs and 3 weeks. PD-L1 expression on cell lines was studied by immunofluorescence. Immunofluorescence at various time points were expressed as a ratio of values in drug treated cells compared to untreated control. Functional consequences of PD-L1 expression were studied using a T cell cancer cell line (Jurkat) transfected with a luciferase reporter where co-culture of cancer cells expressing PD-L1 led to reduction in luminescence. Luminescence at various time points were expressed as ratio of luminescence values of drug treated cells compared to untreated controls. Results Following characterization of expression of PD-L1 in 10 KRAS mutant adeno-NSCLC cell lines, 5 cell lines with the highest expression of PD-L1 was chosen for functional assays (H441, H2291, H23, H2030 and A549). When exposed to trametinib for 3 weeks, 3/5 cell lines (H23, H2030 and A549) showed an statistically significant increase in expression of PD-L1 (range 1.1-2.4) however significant reduction of luciferase activity was only observed in H23 and H2030, 0.93 and 0.75, p= 0.018 and p=0.01 respectively. When exposed to AZD5363 for 3 weeks, 3/5 cell lines (H441, H23 and H2030) showed a statistically significant increase in PD-L1 expression (range 1.3-1.9) however significant reduction in luciferase reduction was seen in only H441 and H23, 0.86 and 0.76, p= 0.03 and p= 0.04 respectively. Conclusion Chronic exposure of KRAS mutant cell lines to MEK and AKT inhibitors cause minor increases in PD-L1 expression but this does not result in functional inhibition of T cells in all instances. Consequently, a functional increase in PD-L1 expression is unlikely to be a common mechanism of resistance to MEK and AKT inhibitors in KRAS mutant adeno-NSCLC. 1 Citation Format: Anna R. Minchom, Parames Thavasu, Zai Ahmad, Adam Stewart, Alexandros Georgiou, Mary ER O9Brien, Sanjay Popat, Jaishree Bhosle, Timothy A. Yap, Johann de Bono, Udai Banerji. A study of dynamic changes in PD-L1 expression in KRAS mutant adenocarcinoma of the lung exposed to signal transduction inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2627. doi:10.1158/1538-7445.AM2017-2627

Abstract 2441: Evaluation of combination of an EGFR and AKT inhibitor in EGFR mutant and EGFR wild type non-small cell lung cancer cells lines.

Background: Epidermal growth factor receptor (EGFR) inhibition with tyrosine kinase inhibitors (TKIs) has proven successful in the management of patients with non small cell lung cancer (NSCLC). However, clinical benefit is predominantly limited to the subgroup of patients with an activating EGFR mutation (10%). Treating the remaining 90% of patients with wild type (wt) EGFR is an unmet clinical need. Aim: To evaluate the effect of an allosteric AKT inhibitor (AKTi 1/2) in combination with the EGFR inhibitor gefitinib on cell proliferation, apoptosis and signalling output in EGFR mutant/wt NSCLC cell lines. Methods: Four NSCLC cell lines were selected: NCI-H522 and NCI-H1651 (EGFR wt and K-RAS wt), PC9 and HCC827 (EGFR mutant). The 96hr sulphorhodamine assays was used to assess the effect of gefitinib and AKTi 1/2 on growth inhibition, and median effect analysis to calculate combination indices (CIs) to assess the effect of both drugs in combination. The expression of p-EGFR, p-AKT, p-S6, p-ERK and cleaved PARP were studied by Western blotting in the EGFR wt NCI-H522 and the EGFR mutant PC9 cell lines. Results: EGFR wt cell lines NCI-H522 and NCI-H1651 were relatively resistant to gefitinib (IC50 values of 7.0 ± 2.5 uM and 8.8 ± 0.9 uM, respectively) compared with the EGFR mutant cell lines PC9 and HCC827 (IC50 values of 0.07 ± 0.03 uM and 0.004 ± 0.0005 uM, respectively). The combination of gefitinib and AKTi 1/2 produced synergistic inhibition of growth in both EGFR wild type and mutant cell lines. Synergism was more marked in EGFR wt cell lines with CI values (ED50) of 0.53 ± 0.28 and 0.49 ± 0.17 for NCI-H522 and NCI-H1651 respectively, compared with 0.74 ± 0.11 and 0.76 ± 0.14 for the EGFR mutant cell lines PC9 and HCC827, respectively. Concomitant exposure of cells to gefitinib and AKTi 1/2 for 24 hrs caused incremental inhibition of p-AKT, p-S6 and p-ERK in both the EGFR wt (NCI-H522) and EGFR mutant (PC9) cell lines. Gefitinib alone resulted in partial inhibition of AKT phosphorylation in PC9 cells and up-regulation in NCI-H522 cells. Incremental induction of cleaved PARP, a marker of apoptosis, was seen in PC9 cells treated with the combination of gefitinib and AKTi 1/2. Conclusions: These results demonstrate synergistic growth inhibition of EGFR wt and mutant NSCLC cell lines by the combination of gefitinib with AKTi 1/2. Additional pre-clinical studies are investigating this further. Clinical studies of the combination doublet are warranted to expand the utility of EGFR TKI in EGFR wt NSCLC. Citation Format: Martina Puglisi, Parames Thavasu, Adam Stewart, Jaishree Bhosle, Sanjay Popat, Mary E R O9Brien, Udai Banerji. Evaluation of combination of an EGFR and AKT inhibitor in EGFR mutant and EGFR wild type non-small cell lung cancer cells lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2441. doi:10.1158/1538-7445.AM2013-2441