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Cytochrome P450 2W1 (Cyp2w1) As a Novel Drug Target in Colon Cancer Therapy Cyp2w1 As a Novel Drug Target in Colon Cancer Therapy

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Cytochrome P450 2W1 (Cyp2w1) As a Novel Drug Target in Colon Cancer Therapy Cyp2w1 As a Novel Drug Target in Colon Cancer Therapy Thesis for doctoral degree (Ph.D.) 2013 Thesis for doctoral degree (Ph.D.) 2013 CYTOCHROME P450 2W1 (CYP2W1) AS A NOVEL DRUG TARGET IN COLON CANCER THERAPY CYP2W1 AS A NOVEL DRUG TARGET IN COLON CANCER THERAPY CANCER IN COLON TARGET DRUG A NOVEL CYP2W1 AS Sandra Travica Sandra Travica From the Department of Physiology and Pharmacology Section of Pharmacogenetics Karolinska Institutet, Stockholm, Sweden CYTOCHROME P450 2W1 (CYP2W1) AS A NOVEL DRUG TARGET IN COLON CANCER THERAPY Sandra Travica Stockholm 2013 All previously published papers were reproduced with permission from the publisher. Cover artwork – DNA bound to anticancer drug, copyright by Martin McCarthy. Printed by Reproprint AB, Sweden Published by Karolinska Institutet © Sandra Travica, 2013 ISBN 978‐91‐7549‐222‐3 Printed by 2013 Gårdsvägen 4, 169 70 Solna TO MY LOVING FAMILY ABSTRACT Cytochrome P450 2W1 (CYP2W1), is a monooxygenase enzyme endogenously expressed in fetal colon and normally silenced after birth. In adult life, the expression of CYP2W1 occurs exclusively in tumor cells yielding high amounts of the enzyme in 30% of human colon cancers. Remaining the third most commonly diagnosed malignancy in the world, the colon cancer requires new chemotherapeutic strategies that would provide higher selectivity towards transformed cells. The unique confinement of CYP2W1 to tumor tissue opens the possibility for its targeting in colon cancer therapy. The studies in this thesis have focused on characterization of the CYP2W1 enzyme and the development of novel anticancer prodrugs designed for CYP2W1 ‐ specific activation. In cancer tissues and transfected cells CYP2W1 gives multiple immunoreactive bands, suggesting that the protein might be subjected to posttranslational modifications. Our in vitro and in vivo studies reveal that CYP2W1 undergoes glycosylation at Asn177, which is enabled by the unique inverted topology of the protein in the ER membrane. Regardless of the reversed emplacement, CYP2W1 retains its catalytic function in cell systems and is capable of converting inactive substrates to potent cytotoxic species, demonstrating the potential for prodrug activation. With the goal to develop antitumor prodrugs for specific activation by CYP2W1, we have employed a library of novel duocarmycin‐based compounds and identified ICT2705 and ICT2706 as the first molecules whose cytotoxic activity is dependent on CYP2W1 enzyme. Our studies reveal the results of the first attempt of CYP2W1 targeting in cancer, both in vitro and in vivo. We show that the colon cancer cell lines expressing CYP2W1 suffer substantial DNA damage and rapid loss of viability following incubation with ICT2705 and ICT2706. Moreover, we demonstrate that the CYP2W1‐positive human xenografts undergo complete growth inhibition in mice dosed with ICT2706, with no apparent deleterious effects detected in any of the treated animals. In addition, we present in vitro evidence that the potent cytotoxic effect is most likely propagated by a bystander killing mechanism. Polymorphisms in CYP2W1 gene yield two alleles of significant frequencies, designated CYP2W1*2 and CYP2W1*6. The resulting variant enzymes carry missense mutations that might affect the therapeutic outcome of prodrug targeting as well as the course of the metastatic disease. A recent study has associated CYP2W1*2 with a decreased risk for colon cancer, suggesting the altered catalytic activity of this enzyme variant. Using the novel duocarmycin substrates, we were able to evaluate the catalytical properties of the variant enzymes for the first time. In contrast to previous findings, our studies show comparable catalytic capacity for all CYP2W1 enzymes, and no association between CYP2W1*2 genotype and colon cancer risk based on 10‐fold larger patient cohorts. In conclusion, we have found that CYP2W1 is a glycosylated enzyme with reversed ER topology, whose catalytic activity is sustained in an intact cell system and unaffected by the most frequent polymorphic changes. We have developed first prodrugs for CYP2W1 targeting in cancer and demonstrated their antitumor efficacy in a preclinical setting. The work presented herein provides the basis for a novel therapeutic approach in colon cancer chemotherapy. LIST OF PUBLICATIONS This thesis is based on the following papers which are referred to in the text by their Roman numerals: I. Alvin Gomez, Jana Nekvindova, Sandra Travica, Mi‐young Lee, Inger Johansson, David Edler, Souren Mkrtchian and Magnus Ingelman‐ Sundberg (2009). Colorectal cancer‐specific cytochrome P450 2W1: intracellular localization, glycosylation, and catalytic activity. Mol Pharmacol, 78(6):1004‐11 II. Helen M. Sheldrake*, Sandra Travica*, Inger Johansson, Paul M. Loadman, Mark Sutherland, Lina Elsalem, Nicola Illingworth, Alexander J. Cresswell, Tristan Reuillon, Steven D. Shnyder, Souren Mkrtchian, Mark Searcey, Magnus Ingelman‐Sundberg, Laurence H. Patterson, Klaus Pors (2013). Re‐engineering of the duocarmycin structural architecture enables bioprecursor development targeting CYP1A1 and CYP2W1 for biological activity. J Med Chem, 56(15):6273–6277 III. Sandra Travica*, Klaus Pors*, Paul M. Loadman, Steven D. Shnyder, Inger Johansson, Mohammed N. Alandas, Helen M. Sheldrake, Souren Mkrtchian, Laurence H. Patterson, Magnus Ingelman‐Sundberg (2013). Colon cancer‐specific cytochrome P450 2W1 converts duocarmycin analogues into potent tumor cytotoxins. Clin Cancer Res, 19(11):2952‐2961 IV. Kristina Stenstedt*, Sandra Travica*, Jia Guo, Isabel Barragan, Klaus Pors, Laurence Patterson, David Edler, Souren Mkrtchian, Inger Johansson, Magnus Ingelman‐Sundberg (2013). Cytochrome P450 2W1 polymorphism: functional aspects and relation to risk for colorectal cancer. Pharmacogenomics, accepted for publication July 15th 2013. *Equal contribution CONTENTS 1. INTRODUCTION ............................................................................................... 1 1.1. CANCER .......................................................................................................... 1 Colon cancer .............................................................................................. 2 1.2. CANCER THERAPY ........................................................................................ 4 Colon cancer therapy ....................................................................... 4 Prodrug strategies ................................................................................... 6 ADEPT ............................................................................................... 6 GDEPT ............................................................................................... 6 PMT ................................................................................................... 7 Alkylating agents in anticancer therapy ........................................ 8 Antitumor antibiotics ............................................................................. 9 Duocarmycins ...................................................................................... 9 Duocarmycin prodrugs .................................................................. 11 1.3. THE CYTOCHROME P450 ENZYMES .......................................................... 12 CYP2W1 ........................................................................................... 13 1.4. CYPs AND CANCER .............................................................................................. 15 Cancer predisposition and CYPs ................................................... 15 Metabolism of chemotherapeutic drugs ...................................... 15 Targeting of cancer‐specific CYP enzymes ..................................... 16 2. AIMS ............................................................................................................ 18 3. RESULTS AND DISCUSSION .............................................................................. 19 3.1. CYP2W1 glycosylation, localization, and catalytic activity ‐ Paper I ........... 19 3.2. CYP2W1‐mediated activation of novel duocarmycin prodrugs as a new approach in colon cancer therapy – Papers II and III ................... 21 3.3. CYP2W1 polymorphism: functional aspects and relation to risk for colorectal cancer – Paper IV ............................................................................. 26 4. CONCLUSIONS ........................................................................................... 28 5. SUMMARY AND FUTURE PERSPECTIVES ................................................. 29 6. ACKNOWLEDGEMENTS ............................................................................ 31 7. REFERENCES ............................................................................................... 34 LIST OF ABBREVIATIONS 5‐FU 5‐flourouracil ADEPT Antibody‐directed enzyme prodrug therapy AEC Antibody‐enzyme conjugate AhR Aryl hydrocarbon receptor APC Adenomatous polyposis coli Asn Asparagine CD20 B‐lymphocyte antigen CD20 CPA Cyclophosphamide CRC Colorectal cancer CSC Cancer stem cell CYP Cytochrome P450 (also P450) EGFR Epidermal growth factor receptor ENDO H Endoglycosidase H ER Endoplasmic reticulum GDEPT Gene‐directed enzyme prodrug therapy H2AX H2A histone family member X Her‐2/NEU Human Epidermal Growth Factor Receptor 2 IFA Ifosfamide MGMT O‐6‐methylguanine‐DNA methyltransferase MLH1 MutL homolog 1 MSH2 MutS homolog 2 MSH6 MutS homolog 6 MYH MUTYH (mutY Homolog) NADPH Nicotinamide adenine dinucleotide phosphate P450 Cytochrome P450 (also CYP) PAH Polycyclic aromatic
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