International Journal of Clinical and Therapeutics, Vol. 52 – No. 1/2014 (89-91)

A systems pharmacology approach to improve drug therapy in NSCLC: Establishing a CESAR network Extended Abstract Ganna V. Kalayda1, Martin Michaelis2, Jindrich Cinatl jr.3, Robert M. Mader4, 5 1 6 7 8 ©2014 Dustri-Verlag Dr. K. Feistle Holger Fröhlich , Navin Sarin , Johanna Melin , Florian Engel , Walter Jäger , ISSN 0946-1965 Roland Frötschl7, Ulrich Jaehde1, Charlotte Kloft6, and Christoph A. Ritter9 DOI 10.5414/CPXCES13EA07 e-pub: November 11, 2013 1Institute of Pharmacy, Clinical Pharmacy, University of Bonn, Bonn, Germany, 2Centre for Molecular Processing and School of Biosciences, University of Kent, Canterbury, UK, 3Institut für Medizinische Virologie, Klinikum der Goethe- Universität, Frankfurt/Main, Germany, 4Department of Medicine I, Comprehensive Cancer Center of the Medical University of Vienna, Austria, 5Algorithmic , University of Bonn, Bonn, 6Department of Clinical Pharmacy and Biochemistry, Freie Universität Berlin, 7Bundesinstitut für Arzneimittel und Medizinprodukte, Bonn, Germany, 8Department of Clinical Pharmacy and Diagnostics, University of Vienna, Austria, and 9Clinical Pharmacy, Institute of Pharmacy, University of Greifswald, Germany

Key words Introduction ficial combination therapy to an individual pa- lung cancer – EGFR – tient, knowledge on potential drug resistance drug resistance – sys- tems pharmacology – The epidermal growth factor receptor mechanisms and respective biological mark- network (EGFR) plays an important role in activation ers in a global scale are required. Systems of signaling pathways responsible for prolifer- pharmacology presents an innovative disci- ation of non-small cell lung cancer (NSCLC) pline which combines systematic collections cells. However, EGFR targeting therapy is of experimental data from whole cells, tissues only effective in a subset of EGFR-positive or organisms with the mathematical models of tumors that harbour activating mutations in drug pharmacokinetics and pharmacodynam- the receptor kinase domain occurring in a ics and natural disease progression models frequency of 5 – 20% of EGFR-positive tu- to understand drug action at a global scale mors in Caucasian lung tumor populations. As [3]. Within CESAR a research network was several studies showed that EGFR mutation- established to set up systems pharmacology negative tumors responded better to classical approaches to unravel EGFR-associated drug chemotherapy, regimens based on platinum resistance in NSCLC. complexes or taxanes are still the treatment of choice for those patients [1]. A major draw- back in both regimens, targeted as well as Models and methods chemotherapy treatment, is the development of drug resistance. Mechanisms of drug re- Cell lines of non-small-cell lung can- sistance include alterations in target structure cer origin were selected that express either and function, compensating signaling activa- wild-type EGFR or EGFR harboring ac- Correspondence to tion as well as changes in drug delivery and tivating mutations. The NSCLC cell lines Prof. Dr. Christoph Ritter accumulation within the tumor cell. Typically, HCC4006 and A549 were obtained from Ernst-Moritz-Arndt- Universität Greifswald drug resistance is not restricted to a single ATCC (Manassas, VA, USA). Sub-lines with Institut für Pharmazie, resistance mechanism but is the result of an acquired resistance to cisplatin, gefitinib, or Klinische Pharmazie, accumulation of several mechanisms. There- erlotinib were established as described pre- Friedrich-Ludwig- fore, strategies of combination therapy are viously [4]. The cell lines were designated Jahn-Str. 17, 17487 r 2000 Greifs­wald, Germany increasingly investigated to overcome drug as A549 CDDP (cisplatin-resistant), [email protected] resistance [2]. In order to tailor the most bene- HCC4006rGEFI1 (gefitinib-resistant), and Kalayda, Michaelis, Cinatl jr., et al. 90

NSCLC will be studied at three levels of complexity: –– 1) The EGFR function will be studied at the molecular level. The EGFR-mediated signaling, the EGFR , and the intracellular localization of EGFR in time and space will be analyzed in de- pendence on the receptor genetics, the activation state, and the response to anti- cancer drugs. These data will be used to develop computer-based models of the EGFR subcellular kinetics and the dy- namic EGFR signaling and interaction at a systems level. –– 2) Cellular aspects of drug response and drug resistance that are not directly re- lated to EGFR function are studied. This includes the studying of the intracellular accumulation and distribution of anti- cancer drugs, since cancer cell drug re- sistance mechanisms include decreased cellular drug uptake, increased drug ef- flux, and changes in sub-cellular drug Figure 1. Structure of the CESAR research net- location. In addition, transcriptomics work on systems pharmacology approach to im- analyses will reveal regulatory changes prove drug therapy in NSCLC. in response to drug resistance. –– 3) A third aspect addresses molecular HCC4006rERLO1 (erlotinib-resistant) and changes outside of the cells, i.e., secreted are part of the Resistant Cancer Cell Line species such as exosomes as well as the (RCCL) collection. The A549 cell lines metabolome. These secreted species play were cultivated in MEM, the HCC4006 in a pivotal role as markers to be extrapo- DMEM/HAM’s F12 medium supplemented lated to the patient situation. Differential with 10% fetal bovine serum at 37 °C in a secretion of metabolites and exosome humidified 5% carbon dioxide atmosphere. content, particularly microRNAs will Data collection will consist of investigating be investigated based on EGFR recep- the EGFR interactome (affinity purification tor genetics, activation state, and drug and high-resolution mass spectrometry), response phenotype, characteristics of characterizing exosomes (microRNA con- exosomes will be correlated with whole tent, protein content, EGFR status), analyz- tumor cells, and specifically identified ing the cellular transcriptomes (microarray metabolites and microRNA species will technologies) and metabolomes (mass spec- be correlated to transcriptome and drug trometry), and determining uptake and dis- response phenotype. tribution of cisplatin, erlotinib, and gefitinib Finally, the data will be integrated into (intracellular accumulation and transwell ex- a multi-level network modeling approach periments) in the investigated cell lines. The to develop predictive models for drug resis- experimental data will be used to develop tance depending on EGFR status and drug predictive, coherent in silico models of the treatment by the core bioinformatics and resistance-associated signaling networks. pharmacometrics units.

Resulting concept Conclusions The structure of the research consortium Here, we will develop a systems pharma- is shown in Figure 1. The role of EGFR in cology approach in order to understand the A systems pharmacology approach to improve drug therapy in NSCLC: Establishing a CESAR network 91 role of EGFR in NSCLC 1) as a drug target and 2) in the response to anti-cancer therapy in general which will provide the basis for the development of improved therapies.

Acknowledgments The work was supported by the Hilfe für krebskranke Kinder Frankfurt e.V., the Frankfurter Stiftung für krebskranke Kinder, the Kent Cancer Trust, and the Royal Society.

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