Activity of the Second Generation BTK Inhibitor Acalabrutinib in Canine and Human B-cell Non-Hodgkin Lymphoma Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Bonnie Kate Harrington Graduate Program in Comparative and Veterinary Medicine The Ohio State University 2018 Dissertation Committee John C. Byrd, M.D., Advisor Amy J. Johnson, Ph.D. Krista La Perle, D.V.M., Ph.D. William C. Kisseberth, D.V.M., Ph.D. 1 Copyrighted by Bonnie Kate Harrington 2018 2 Abstract Acalabrutinib (ACP-196) is a second-generation inhibitor of Bruton’s Tyrosine Kinase (BTK) with increased target selectivity and potency compared to ibrutinib. In these studies, we evaluated acalabrutinib in spontaneously occurring canine lymphoma, a model of B-cell malignancy reported to be similar to human diffuse large B-cell lymphoma (DLBCL), as well as primary human chronic lymphocytic leukemia (CLL) cells. We demonstrated that acalabrutinib potently inhibited BTK activity and downstream B-cell receptor (BCR) effectors in CLBL1, a canine B-cell lymphoma cell line, primary canine lymphoma cells, and primary CLL cells. Compared to ibrutinib, acalabrutinib is a more specific inhibitor and lacked off-target effects on the T-cell and NK cell kinase IL-2 inducible T-cell kinase (ITK) and epidermal growth factor receptor (EGFR). Accordingly, acalabrutinib did not antagonize antibody dependent cell cytotoxicity mediated by NK cells. Finally, acalabrutinib inhibited proliferation and viability in CLBL1 cells and primary CLL cells and abrogated chemotactic migration in primary CLL cells. To support our in vitro findings, we conducted a clinical trial using companion dogs with spontaneously occurring B-cell lymphoma. Twenty dogs were enrolled in the clinical trial and treated with acalabrutinib at dosages of 2.5 to 20mg/kg every 12 or 24 hours. Acalabrutinib was generally well tolerated, with ii adverse events consisting primarily of grade 1 or 2 anorexia, weight loss, vomiting, diarrhea and lethargy. Overall response rate (ORR) was 25% (5/20) with a median progression free survival (PFS) of 22.5 days. Clinical benefit was observed in 30% (6/20) of dogs. These findings suggest that acalabrutinib is safe and exhibits activity in canine B-cell lymphoma patients and support the use of canine lymphoma as a relevant model for human non-Hodgkin lymphoma (NHL). iii Dedication This work is dedicated to my father, Jeff Harrington, a survivor of both CLL and renal carcinoma; to my mother, Sandy Harrington; to my husband, Alan Flechtner; and to my son, Blaise Flechtner. Thank you for supporting me. iv Acknowledgments I would like to acknowledge my mentors Dr. John Byrd and Amy Johnson for providing the scholarly yet nurturing environment for my intellect to grow; the funding sources that provided my stipend during training, the National Institutes of Health Oncology T32 and the Pelotonia Graduate Fellowship; and all of those in the Experimental Hematology Lab or elsewhere that supported me in graduate school and contributed to my development as an independent investigator. v Vita December 27, 1984……………………………………….Born – North Canton, Ohio 2003……………..……………………..…………………………GlenOak High School 2007………………………………….…B.S. Psychology, The Ohio State University 2012……………………………..…………………D.V.M., The Ohio State University 2012-Present…………..………………Ph.D. Candidate, The Ohio State University PUBLICATIONS Ozer H.G., El-Gamal D., Ben Powell B., Hing Z.A., Blachly J.S., Harrington B.K., Mitchell S., Grieselhuber N.R.,Williams K., Lai T., Alinari L., Baiocchi R.A., Brinton L., Baskin E., Cannon M., Beaver L., Goettl, V.M., Lucas D.M., Woyach J.A., Sampath D., Lehman A.M., Yu L.,Zhang J., Ma Y., Zhang Y., Spevak W., Shi S., Severson P., Shellooe R., Carias H., Tsang G., Dong K., Ewing T., Marimuthu A., Tantoy C., Walters L., Sanftner L., Rezaei H., Nespi M., Matusow B., Habets G., Ibrahim P., Zhang C., Mathé1 E.A., Bollag G., Byrd J.C., Lapalombella R.(2018) BRD4 profiling identifies critical chronic lymphocytic leukemia oncogenic circuits and reveals sensitivity to PLX51107, a novel structurally distinct BET inhibitor. Cancer Discov. 4, 458-477. vi Tsai Y.T., Lakshmanan A., Lehman A., Harrington B.K., McClanahan Lucas F., Tran M., Sass E.J., Long M., Flechtner A.D., Jaynes F., La Perle K., Coppola V., Lozanski G., Muthusamy N., Byrd J.C., Grever M., Lucas D.M. (2017) BRAFV600E accelerates disease progression and enhances immune suppression in a mouse model of B-cell leukemia. Blood Adv. 24, 2147-2160. Chen T.L., Tran M., Lakshmanan A., Harrington B.K., Goettl V.M., Lehman A.M., Trudeau S., Lucas D.M., Johnson A.J., Byrd J.C., Hertlein E. (2017). NF-κB p50 (nfkb1) contributes to pathogenesis in the Eμ-TCL1 mouse model of chronic lymphocytic leukemia. Blood 130, 376-379. Herman S.E.M., Montraveta A., Niemann C.U., Mora-Jensen H., Gulrajani M., Krantz F., Mantel R., Smith L.L., McClanahan F., Harrington B.K., Colomer D., Covey T., Byrd J.C., Izumi R., Kaptein A., Ulrich R., Johnson A.J., Lannutti B.J., Wiestner A., Woyach J.A. (2017). The Bruton Tyrosine Kinase (BTK) Inhibitor Acalabrutinib Demonstrates Potent On-Target Effects and Efficacy in Two Mouse Models of Chronic Lymphocytic Leukemia. Clin. Cancer Res. 23, 2831-284. Harrington, B.K., Gardner, H.L., Izumi, R., Hamdy, A., Rothbaum, W., Coombes, K., Kaptein, A., Covery, T., Gulrajani, M., Van Lith, B., Krejsa, C., Russell, D., Zhang, X., Urie, B., London,C.A., Byrd, J.C., Johnson, A.J., Kisseberth, W.C. vii (2016). ACP-196: A second generation BTK inhibitor in a canine model of B-cell non-Hodgkin lymphoma. PLoS One. 11, 7. Byrd, J.C.*, Harrington, B.*, O’Brien, S., Jones, J.A., Schuh, A., Devereux, S., Chaves, J., Wierda, W.G., Awan, F.T., Brown, J.R., et al. (2016). Acalabrutinib (ACP-196) in Relapsed Chronic Lymphocytic Leukemia. N. Engl. J. Med. 374, 323–332. * Indicates these authors contributed equally Stiff A., Trikha P., Wesolowski R., Kendra K., Hsu V., Uppati S., McMichael E., Duggan M, Campbell A, Keller K., Landi I., Zhong Y., Dubovsky J., Howard J.H., Yu L, Harrington B., Old M., Reiff S., Mace T., Tridandapani S., Muthusamy N., Caligiuri M.A., Byrd J.C., Carson W.E. 3rd. (2016). Myeloid-Derived Suppressor Cells Express Bruton's Tyrosine Kinase and Can Be Depleted in Tumor-Bearing Hosts by Ibrutinib Treatment. Cancer Res. 76, 2125-2136. Coble, D.J., Shoemaker, M., Harrington, B., Dardenne, A.D., and Bolon, B. (2015). Histiocytic Sarcoma and Bilateral Facial Vein Thrombosis in a Siberian Hamster (Phodopus sungorus). Comp. Med. 65, 127–132. Dubovsky, J.A., Flynn, R., Du, J., Harrington, B.K., Zhong, Y., Kaffenberger, B., Yang, C., Towns, W.H., Lehman, A., Johnson, A.J., et al. (2014). Ibrutinib viii treatment ameliorates murine chronic graft-versus-host disease. J. Clin. Invest. 124, 4867–4876. Woyach, J.A., Bojnik, E., Ruppert, A.S., Stefanovski, M.R., Goettl, V.M., Smucker, K.A., Smith, L.L., Dubovsky, J.A., Towns, W.H., MacMurray, J., Harrington, B.K. et al. (2014). Bruton’s tyrosine kinase (BTK) function is important to the development and expansion of chronic lymphocytic leukemia (CLL). Blood 123, 1207–1213. Fenger, J.M., Bear, M.D., Volinia, S., Lin, T.-Y., Harrington, B.K., London, C.A., and Kisseberth, W.C. (2014). Overexpression of miR-9 in mast cells is associated with invasive behavior and spontaneous metastasis. BMC Cancer 14, 84. Zhong, Y., El-Gamal, D., Dubovsky, J.A., Beckwith, K.A., Harrington, B.K., Williams, K.E., Goettl, V.M., Jha, S., Mo, X., Jones, J.A., et al. (2014). Selinexor suppresses downstream effectors of B-cell activation, proliferation and migration in chronic lymphocytic leukemia cells. Leukemia 28, 1158–1163. Dubovsky, J.A., Chappell, D.L., Harrington, B.K., Agrawal, K., Andritsos, L.A., Flynn, J.M., Jones, J.A., Paulaitis, M.E., Bolon, B., Johnson, A.J., et al. (2013). ix Lymphocyte cytosolic protein 1 is a chronic lymphocytic leukemia membrane- associated antigen critical to niche homing. Blood 122, 3308–3316. Fields of Study Major Field: Comparative and Veterinary Medicine x Table of Contents Abstract .......................................................................................................................ii Dedication ..................................................................................................................iv Acknowledgments ..................................................................................................... v Vita .............................................................................................................................vi Table of Contents ......................................................................................................xi List of Tables............................................................................................................ xiii List of Figures .......................................................................................................... xiv Chapter 1. Introduction.............................................................................................. 1 1.1 Chronic Lymphocytic Leukemia ...................................................................... 1 1.2 Traditional Therapies for Lymphoid Malignancies ......................................... 3 1.3 Targeting Bruton’s Tyrosine Kinase (BTK) in B-cell Malignancies ............... 4 1.4 Exploiting Canine Lymphoma as a Model for Human Disease..................... 6 Chapter 2. Preclinical Evaluation of the Novel BTK Inhibitor