Marie Nlend

Alternative Signaling Pathways as Potential Therapeutic Targets for Overcoming EGFR and c-Met Inhibitor Resistance in Non-Small Cell Lung Cancer

The use of tyrosine kinase inhibitors (TKIs) against EGFR/c-Met in non-small cell lung cancer (NSCLC) has been shown to be effective in increasing patient progression free survival (PFS), but their efficacy is limited due to the development of resistance and tumor recurrence. Therefore, understanding the molecular mechanisms underlying development of drug resistance in NSCLC is necessary for developing novel and effective therapeutic approaches to improve patient outcome. This study aims to understand the mechanism of EGFR/c-Met tyrosine kinase inhibitor (TKI) resistance in NSCLC. H2170 and H358 cell lines were made resistant to SU11274, a c-Met inhibitor, and erlotinib, an EGFR inhibitor, through step-wise increases in TKI exposure. The IC50 concentrations of resistant lines exhibited a 4–5 and 11–22-fold increase for SU11274 and erlotinib, respectively, when compared to parental lines. Furthermore, mTOR and Wnt signaling was studied in both cell lines to determine their roles in mediating TKI resistance. We observed a 2–4-fold upregulation of mTOR signaling proteins and a 2- to 8-fold upregulation of Wnt signaling proteins in H2170 erlotinib and SU11274 resistant cells. H2170 and H358 cells were further treated with the mTOR inhibitor everolimus and the Wnt inhibitor XAV939. H358 resistant cells were inhibited by 95% by a triple combination of everolimus, erlotinib and SU11274 in comparison to 34% by a double combination of these drugs. Parental H2170 cells displayed no sensitivity to XAV939, while resistant cells were significantly inhibited (39%) by XAV939 as a single agent, as well as in combination with SU11274 and erlotinib. Similar results were obtained with H358 resistant cells. This study suggests a novel molecular mechanism of drug resistance in lung cancer.

Mechanism of Resistance and Novel Targets Mediating Resistance to EGFR and c-Met Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer

Tyrosine kinase inhibitors (TKIs) against EGFR and c-Met are initially effective when administered individually or in combination to non-small cell lung cancer (NSCLC) patients. However, the overall efficacies of TKIs are limited due to the development of drug resistance. Therefore, it is important to elucidate mechanisms of EGFR and c-Met TKI resistance in order to develop more effective therapies. Model NSCLC cell lines H1975 and H2170 were used to study the similarities and differences in mechanisms of EGFR/c-Met TKI resistance. H1975 cells are positive for the T790M EGFR mutation, which confers resistance to current EGFR TKI therapies, while H2170 cells are EGFR wild-type. Previously, H2170 cells were made resistant to the EGFR TKI erlotinib and the c-Met TKI SU11274 by exposure to progressively increasing concentrations of TKIs. In H2170 and H1975 TKI-resistant cells, key Wnt and mTOR proteins were found to be differentially modulated. Wnt signaling transducer, active β-catenin was upregulated in TKI-resistant H2170 cells when compared to parental cells. GATA-6, a transcriptional activator of Wnt, was also found to be upregulated in resistant H2170 cells. In H2170 erlotinib resistant cells, upregulation of inactive GSK3β (p-GSK3β) was observed, indicating activation of Wnt and mTOR pathways which are otherwise inhibited by its active form. However, in H1975 cells, Wnt modulators such as active β-catenin, GATA-6 and p-GSK3β were downregulated. Additional results from MTT cell viability assays demonstrated that H1975 cell proliferation was not significantly decreased after Wnt inhibition by XAV939, but combination treatment with everolimus (mTOR inhibitor) and erlotinib resulted in synergistic cell growth inhibition. Thus, in H2170 cells and H1975 cells, simultaneous inhibition of key Wnt or mTOR pathway proteins in addition to EGFR and c-Met may be a promising strategy for overcoming EGFR and c-Met TKI resistance in NSCLC patients.

Photophysical properties of a new DyLight 594 dye

We describe spectral properties of novel fluorescence probe DyLight 594. Absorption and fluorescence spectra of this dye are in the region of Alexa 594 fluor spectra. The quantum yield of DyLight 594 in conjugated form to IgG is higher than corresponding quantum yield of Alexa 594 by about 50%. The new DyLight dye also shows slightly longer lifetime and photostability. These favorable properties and high anisotropy value, as well as a high cross-section for two-photon excitation, make this fluorophore attractive as a fluorescence probe in biochemical/biological studies involving fluorescence methods.

Development and evaluation of a fluorescent antibody-drug conjugate for molecular imaging and targeted therapy of pancreatic cancer

Antibodies are widely available and cost-effective research tools in life science, and antibody conjugates are now extensively used for targeted therapy, immunohistochemical staining, or in vivo diagnostic imaging of cancer. Significant advances in site-specific antibody labeling technologies have enabled the production of highly characterized and homogenous conjugates for biomedical purposes, and some recent studies have utilized site-specific labeling to synthesize bifunctional antibody conjugates with both imaging and drug delivery properties. While these advances are important for the clinical safety and efficacy of such biologics, these techniques can also be difficult, expensive, and time-consuming. Furthermore, antibody-drug conjugates (ADCs) used for tumor treatment generally remain distinct from conjugates used for diagnosis. Thus, there exists a need to develop simple dual-labeling methods for efficient therapeutic and diagnostic evaluation of antibody conjugates in pre-clinical model systems. Here, we present a rapid and simple method utilizing commercially available reagents for synthesizing a dual-labeled fluorescent ADC. Further, we demonstrate the fluorescent ADC’s utility for simultaneous targeted therapy and molecular imaging of cancer both in vitro and in vivo. Employing non-site-specific, amine-reactive chemistry, our novel biopharmaceutical theranostic is a monoclonal antibody specific for a carcinoembryonic antigen (CEA) biomarker conjugated to both paclitaxel and a near-infrared (NIR), polyethylene glycol modified (PEGylated) fluorophore (DyLight™ 680-4xPEG). Using in vitro systems, we demonstrate that this fluorescent ADC selectively binds a CEA-positive pancreatic cancer cell line (BxPC-3) in immunofluorescent staining and flow cytometry, exhibits efficient internalization kinetics, and is cytotoxic. Model studies using a xenograft of BxPC-3 cells in athymic mice also show the fluorescent ADC’s efficacy in detecting tumors in vivo and inhibiting tumor growth more effectively than equimolar amounts of unconjugated drug. Overall, our results demonstrate that non-selective, amine-targeting chemistry is an effective dual-labeling method for synthesizing and evaluating a bifunctional fluorescent antibody-drug conjugate, allowing concurrent detection, monitoring and treatment of cancer.

Abstract 4305: Biodistribution and clearance of non-targeted DyLight dyes in tumor-free nude mice for in vivo imaging

Far red to near infra red (NIR) fluorophores are commonly used in cell-based assays and for deep tissue in vivo imaging. The light emitting and physico-chemical characteristics of these fluorophores, e.g. excitation and emission spectra, relative hydrophilicity/hydrophobicity and molecule size, can significantly influence their performance in these applications. Currently there are no guidelines for selection of fluorophores optimal for targeted in vivo imaging. To address this issue we have designed a panel of DyLight dyes with maximum excitation wavelength in the range of 650 -785 nm, maximum emission wavelength in the range of 670 - 800 nm and different levels of sulfonation and/or PEGylation. We studied the kinetic of tissue distribution and clearance of these fluorophores as well as their effect on morphology of different tissues in mice after intravenous injection. Briefly, nude mice were anesthetized, injected with a dye (100 µL of dye at 0.5mg/ml in PBS) into retro-orbital plexus, and imaged using Carestream MSFX or Carestream XTREME Imager. Images were generated before dye injection, immediately after injection (0 hours) and 3, 6, 12 and 24 hours post injection. At the end of the experiment, animals were sacrificed; internal organs were collected and used for ex vivo analysis. The results of in vivo imaging experiments demonstrated that: hydrophobic fluorophores have a relatively slow kinetic of distribution and clearance, and often cleared via a hepatic pathway. Introduction of the negatively charged groups and or PEG chains increased dye solubility affected the kinetics of both fluorophore distribution and its clearance. In addition, negatively charged fluorophores had a tendency to be cleared predominantly via renal pathway. A histological analysis of the internal organs showed no detectable tissue damage. These results provide guidelines for the selection of optimal NIR dyes for in vivo imaging. Citation Format: Marie C. NLEND, Surbhi Desai, Suk J. Hong, Mary Beth Anderson, Georgyi V. Los, Greg T. Hermanson, Justin M. Diener, Warren M. Leevy, Peter A. Bell. Biodistribution and clearance of non-targeted DyLight dyes in tumor-free nude mice for in vivo imaging. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4305. doi:10.1158/1538-7445.AM2014-4305

Abstract 1599: Epithelial mesenchymal transition and its role in TKI resistant NSCLC cell lines

NSCLC cells acquire resistance to EGFR and c-Met TKIs after prolonged use. Our studies indicate that resistance maybe due to upregulation of alternative signaling pathways such as Wnt and mTOR. We have found that activation of Wnt/β-Catenin is also associated with EMT which results in loss of cell adhesion properties and gain of motility and invasiveness. To understand the mechanism of TKI resistance in NSCLC cells with wild type EGFR, we have developed and used H2170-P (parental) cells and the TKI resistant H2170-ER (erlotinib resistant) and H2170-SR (SU11274 resistant) cells. We aim to study EMT and determine if inhibition of β-Catenin, a key regulator of transcription by siRNA or inhibition of ZEB-1 a transcriptional repressor of cell adhesion proteins by inducing mir-200a will help overcome the TKI resistance in NSCLC cells. Using immunoblotting, we observed modulations in key EMT-related proteins in H2170-ER cells which showed upregulation of ZEB-1, N-cadherin, active beta-catenin, Vimentin, PRMT1 and ZO1 by 1.8, 2.4, 2, 1.8, 2.6 and 2 fold, respectively, and downregulation of E-cadherin (1.8 fold) as compared to H2170-P cells. Similar results were observed for H2170-SR cells. These results were verified using qPCR where we show that β-catenin (3.4-3.2 fold) and N-cadherin (2-1.9 fold) have increased gene expression while E-cadherin (1.7-2.4 fold) has a decreased gene expression in TKI resistant H2170-ER and SR cells when compared to H2170-P cells. miR-200a induction in H2170-ER cells showed significant downregulation of ZEB-1 (3 fold) at 72 hr and an upregulation of E-cadherin (2 fold) when compared to the mock transfected cells. Morphological changes indicative of EMT were detected using immunofluorescence with Vimentin and E-Cadherin antibodies, which displayed upregulation of Vimentin filaments (2 fold) and downregulation of E-Cadherin (3 fold) in H2170-ER and H2170-SR cells when compared to H2170-P cells. We then conducted experiments where we suppressed ZEB-1 by inducing miR-200a in TKI resistant cells. The immunoblotting results suggested recovery of E-Cadherin, and downregulation of ZEB-1 and N-Cadherin in TKI resistant cells. We also observed increased sensitivity towards erlotinib and SU11274 by 20-25%. Additionally, we observed decrease in levels of β-Catenin and upon siRNA knockdown of β-Catenin, suppression of levels of ZEB-1. This indicates a direct correlation between nuclear accumulation of β-Catenin and occurrence of EMT in TKI resistant cells by increase in expression of ZEB-1. Our results indicate that increased activation of Wnt/β-Catenin pathway in the TKI resistant NSCLC cells is due to the EMT. In NSCLC patients, L858R and T790M mutations are associated with TKI resistance which could be responsible for inducing EMT. We are further studying cell lines with EGFR mutations to determine their role in induction of EMT this may provide clinicians with novel targets to overcome TKI resistance in NSCLC patients. Citation Format: Ichwaku Rastogi, Tsatsral Iderzorig, Gagan Chhabra, Gregory M. Botting, Andrew Webb, Brad Foster, Brian Webb, Marie Nlend, Neelu Puri. Epithelial mesenchymal transition and its role in TKI resistant NSCLC cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1599.

Abstract 337: Mechanism of TKI resistance and role of epithelial mesenchymal transition in NSCLC

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Current EGFR/c-Met tyrosine kinase inhibitor (TKI) combination therapies are shown to be susceptible to acquired resistance in the majority of NSCLC patients. To understand how cells develop EGFR/c-Met TKI resistance, we used three model NSCLC cell lines: H2170, H3255 and H1975. H2170 cell line express high levels of EGFR and c-Met and are EGFR wild-type. The H3255 cell line is positive for an EGFR TKD mutation, L858R, and is sensitive to initial TKI treatment. The H1975 cell line is positive for two EGFR TKD mutations, T790M and L858R, which confer resistance to erlotinib. Previous studies in our lab have shown that activation of alternative signaling pathways lead to the development of TKI resistance. In the wild type TKI resistant cells, H2170 erlotinib resistant (ER) and H2170 SU11274 resistant(SR), we observed activation of Wnt and mTOR pathway along with increased accumulation of β-catenin in the nucleus. However, in H1975 cells with mutated EGFR (L858R and T790M), we observed activation of only mTOR pathway. β-catenin is associated with Epithelial-Mesenchymal Transition (EMT), during which cells lose tight junction proteins such E-Cadherin and gain expression of transcriptional proteins such as β-catenin and ZEB1, which may lead to aberrant activation of Wnt signaling pathway and increased motility and invasion. Activation of mTOR pathway leads to increased expression of p70S6K and 4E-BP1 which are key translational regulators. We found increased accumulation of active β-catenin in the nucleus by 1.9-2.5 folds and 2.9-3.1 folds in H2170 ER and SR cells, respectively, compared to H2170 parental cells. In H2170 ER cells we observed modulations in key EMT-related proteins, N-Cadherin (2-3.3 fold) and Claudin-1 (2.5-14 fold) were upregulated and Snail was downregulated 1.7-2.5 fold in the presence and absence of erlotinib and EGF, when compared to H2170 parental cells. Similarly in H2170 SR cells we observed upregulation of N-Cadherin (1.3-2.3 fold) and Zeb-1 (1.2-1.4 fold) and downregulation of ZO-1 (1.4-1.9 fold) and E-Cadherin (1.5-2.0 fold) in the presence and absence of HGF and SU11274 when compared to H2170 parental cells. Morphological changes indicative of EMT were detected using immunostaining with Vimentin and E-Cadherin antibodies, which displayed increased Vimentin filaments (2-fold) and loss of E-Cadherin (5-10 fold) in H2170 ER cells when compared to H2170 parental cells. In H1975 cells we observed that proteins related to mTOR signaling pathway such as p-mTOR, p-GSK3-β, p-4EBP1 and p-p70S6K were upregulated 2.5 fold, 4-5 fold, 2.2-2.4 fold, and 4.4-10.4 fold, respectively, when compared to H3255 cells in the presence of erlotinib. Based on these results, we treated H1975 cells with combination of EGFR and mTOR inhibitors. We observed that 1 μM everolimus with 2.5 μM erlotinib synergistically inhibited cell growth by 53%. These studies may provide clinicians with novel targets for improving treatment options for future NSCLC patients. Citation Format: Ichwaku Rastogi, Gregory M. Botting, Andrew Webb, Brian L. Webb, Marie C. Nlend, Neelu Puri. Mechanism of TKI resistance and role of epithelial mesenchymal transition in NSCLC. [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 337. doi:10.1158/1538-7445.AM2015-337

Abstract LB-001: Development and evaluation of a fluorescent antibody-drug conjugate for molecular imaging and targeted therapy of pancreatic cancer

Developing new strategies to effectively diagnose and treat various types of cancer is paramount to increasing patient survival rates. Although chemotherapeutic small molecules are effective for some cancer types, they often have harmful side effects, resulting in significant damage to healthy tissue. Targeted therapy, where anti-cancer drugs are more precisely delivered to specific cells, has the potential to revolutionize chemotherapy, through increased localized effective doses with minimized systemic toxicity. Antibody-drug conjugates (ADCs) are one such class of targeted therapy biopharmaceuticals, employing the inherent specificity of antibodies as a targeting mechanism to yield a potent drug delivery system. In addition to being used as ADCs, antibody-conjugates are also commonly used as a highly effective tool for cancer typing and longitudinal treatment monitoring by immunohistochemistry staining or diagnostic imaging. However, antibody-conjugates used for tumor diagnosis and treatment are different molecules. Fluorescent dye labeling of therapeutic antibodies has been previously demonstrated for cancer imaging. However, therapeutic antibodies dual-labeled with both chemotherapeutic small molecules and fluorescent dyes has not been reported. Here, we demonstrate the development of a directly-labeled, fluorescent antibody-drug conjugate for simultaneous targeted drug delivery and in vivo molecular imaging of cancer. Our novel biopharmaceutical entity is a monoclonal antibody specific for a carcinoembryonic antigen (CEA) biomarker conjugated to an average of one molecule of paclitaxel and two molecules of a near-infrared fluorophore (DyLight 680-4xPEG). Preliminary data show that this fluorescent ADC selectively binds CEA positive cells and is cytotoxic using in vitro model systems. Ongoing studies using an in vivo mouse xenograft cancer model will demonstrate the utility of this fluorescent ADC as a new concurrent pancreatic cancer detection, monitoring and treatment technology. Citation Format: Steve Knutson, Erum Raja, Ryan Bomgarden, Marie Nlend, Aoshuang Chen, Ramaswamy Kalyanasundaram, Surbhi Desai. Development and evaluation of a fluorescent antibody-drug conjugate for molecular imaging and targeted therapy of pancreatic cancer. [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 LB-001. doi:10.1158/1538-7445.AM2015-LB-001