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Biologic Activity of the Novel SINE Compound KPT-335 Against Canine Melanoma Cell Lines THESIS

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Biologic Activity of the Novel SINE Compound KPT-335 Against Canine Melanoma Cell Lines THESIS Biologic Activity of the Novel SINE Compound KPT-335 Against Canine Melanoma Cell Lines THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Megan Nicole Breit, BS, DVM Graduate Program in Comparative and Veterinary Medicine The Ohio State University 2014 Master's Examination Committee: William C. Kisseberth, DVM, PhD, Co-Advisor Cheryl A. London, DVM, PhD, Co-Advisor Emma E. Warry, BVSc (Hons) Copyrighted by Megan Nicole Breit 2014 Abstract Background: Exportin 1 (XPO1, also known as CRM1), is a protein responsible for the export of over 200 target proteins out of the nucleus. XPO1 is upregulated in several human cancers and its expression is also linked to the development of chemotherapy resistance. Recent studies using both human and murine cancer cell lines have demonstrated that XPO1 is a relevant target for therapeutic intervention. The present study sought to characterize the biologic activity of an orally bioavailable selective inhibitor of nuclear export (SINE), KPT-335, against canine melanoma cell lines as a prelude to future clinical trials in dogs with melanoma. Results: We evaluated the effects of KPT-335 on 4 canine malignant melanoma cell lines and found that KPT-335 inhibited proliferation and induced apoptosis of treated cells. Additionally, KPT-335 downregulated XPO1 protein while inducing a concomitant increase in XPO1 messenger RNA. Lastly, KPT-335 treatment of cell lines induced the expression of the tumor suppressors p53 and p21, with enhanced nuclear localization. Conclusion: KPT-335 demonstrates biologic activity against canine melanoma cell lines at physiologically relevant doses, suggesting that KPT-335 may represent a viable treatment option for dogs with malignant melanoma. ii Acknowledgments I would like to thank my advisors, Cheryl London, William Kisseberth, Sandra Barnard and Emma Warry for their support and guidance during my residency program. I would also like to thank Michael S. Kent for providing the melanoma cell lines. Karyopharm INC. provided the drug for this experiment and also providing experimental support. In particular Dr. Yosef Landesman provided guidance on experimental design and his lab technician Trinayan Kashyap performed the real time for p21 and MIC1. None of the experiments would have been possible without the guidance and patient teaching of Misty D. Bear. I am grateful to Joelle Fenger for assistance with real time PCR as well as other aspects of the study design and research methods. Thank you to Timothy Vojt for his hard work preparing the figures. Also thank you to Jerry R. Harvey in the College of Veterinary Medicine Biomedical Media and Instructional Technologies department for the images of the clonogenic assays. I am grateful for the training and technical support provided by The Ohio State University campus microscopy and imaging facility. iii Vita 2005 ...............................................................Bachelors of Science Neuroscience, Washington State University, Pullman, WA 2009 ...............................................................Doctor of Veterinary Medicine, Washington State University, Pullman, WA 2009-2010 .....................................................Small Animal Medical and Surgical Rotating Internship, Animal Hospital Specialty Center, Highlands Ranch, CO 2010-2011 .....................................................Oncology Specialty Internship, Blue Pearl Veterinary Partners, Tampa, FL 2011-present ..................................................Medical Oncology Resident and Graduate Research Associate, Department of Veterinary Clinical Sciences, The Ohio State University Fields of Study Major Field: Comparative and Veterinary Medicine iv Table of Contents Abstract ............................................................................................................................... ii Acknowledgments.............................................................................................................. iii Vita ..................................................................................................................................... iv Fields of Study ................................................................................................................... iv Table of Contents ................................................................................................................ v List of Figures ................................................................................................................... vii Chapter 1. Literature Review ............................................................................................. 1 1.1 XPO1 and Cancer ...................................................................................................... 1 1.2 Human Melanoma ..................................................................................................... 5 1.3 Canine Malignant Melanoma .................................................................................... 7 1.4 Molecular Abnormalities in Melanoma .................................................................... 9 1.5 Summary ................................................................................................................. 11 Chapter 2. Materials and Methods .................................................................................... 12 2.1 Cell Lines and Reagents .......................................................................................... 12 v 2.2 Cell Proliferation Assay .......................................................................................... 12 2.3 Assessment of Apoptosis ........................................................................................ 13 2.4 Immunoblotting ....................................................................................................... 13 2.5 Quantitative RT-PCR .............................................................................................. 14 2.6 Immunofluorescence ............................................................................................... 14 2.7 Clonogenic Assay .................................................................................................... 15 2.8 Statistics .................................................................................................................. 16 Chapter 3. Results ............................................................................................................. 17 3.2 XPO1 inhibition induces apoptosis of canine melanoma cell lines. ....................... 18 3.3 Colony formation is inhibited by KPT-335............................................................. 20 3.4 KPT-335 downregulates XPO1 protein expression while inducing a concomitant increase in XPO1 mRNA. ............................................................................................. 23 3.5 KPT-335 modulates the expression of p53 and its downstream targets and localization of p53 and p21 in canine melanoma cell lines........................................... 24 Chapter 4. Discussion ....................................................................................................... 28 4.1 Limitations .............................................................................................................. 33 4.2 Future Directions ..................................................................................................... 33 4.3 Conclusions ............................................................................................................. 34 5. References ..................................................................................................................... 35 vi List of Figures Figure 1 KPT-335 inhibits proliferation of canine melanoma cells. ................................ 18 Figure 2 KPT-335 induces apoptosis of canine melanoma cells. ..................................... 20 Figure 3 Effect of KPT-335 on colony formation. ........................................................... 22 Figure 4 Impact of KPT-335 on XPO1 message and protein expression in melanoma cell lines. .................................................................................................................................. 24 Figure 5 Evaluation of p53 and p21 expression and localization in canine melanoma cell lines after KPT-335 treatment. .......................................................................................... 26 Figure 6 Confocal microscopy to detect p21 and p53 cellular localization following KPT- 335 treatment of melanoma cell lines. .............................................................................. 27 vii Chapter 1. Literature Review 1.1 XPO1 and Cancer The nuclear export protein exportin 1(XPO1, also called Chromosome Region Maintenance protein 1 [CRM1]) was first identified in yeast.[1] The function of this protein was initially delineated by showing that the antifungal/antibiotic drug leptomycin B (LMB), caused the nuclear accumulation of multiple proteins and was covalently bound to XPO1.[2, 3] XPO1 is one of seven known nuclear export proteins responsible for shuttling cargo proteins from the nucleus to the cytoplasm [4-6]. It is a member of the karyopherin β family of transport receptors that binds over 200 target proteins through a hydrophobic leucine-rich nuclear export signal (NES) present in the cargo [7]. XPO1 has many important roles including
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