Investigating the Mechanism and Species- Specificity of Thalidomide Derivatives The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Fink, Emma Catherine. 2018. Investigating the Mechanism and Species-Specificity of Thalidomide Derivatives. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:41129136 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Investigating the mechanism and species-specificity of thalidomide derivatives A dissertation presented by Emma Catherine Fink to The Division of Medical Sciences in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the subject of Biological and Biomedical Sciences Harvard University Cambridge, Massachusetts April 2018 © 2018 Emma Catherine Fink All rights reserved. Dissertation Advisor: Dr. Benjamin Ebert Emma Catherine Fink Investigating the mechanism and species-specificity of thalidomide derivatives Abstract Thalidomide, once infamous for its teratogenic effects, and its derivatives lenalidomide and pomalidomide have found new clinical utility as highly effective treatments for multiple myeloma, other B cell neoplasms, and myelodysplastic syndrome (MDS) with del(5q). These drugs bind to cereblon (CRBN), the substrate adaptor of the CRL4CRBN E3 ubiquitin ligase and induce the recruitment and ubiquitination of specific protein targets, ultimately resulting in their degradation by the proteasome. Prior work identified lenalidomide-induced ubiquitination and degradation of the lymphoid transcription factors Ikaros (IKZF1) and Aiolos (IKZF3). Degradation of IKZF1 and IKZF3 kills multiple myeloma cells and increases IL-2 release by T cells, explaining two aspects of lenalidomide’s clinical efficacy, but does not account for lenalidomide’s therapeutic window in MDS with del(5q). Here, we demonstrated that lenalidomide induces the ubiquitination of casein kinase 1A1 (CK1α) by CRL4CRBN, resulting in CK1α degradation. CK1α is encoded by a gene, CSNK1A1, within the common deleted region for del(5q) MDS, and its haploinsufficiency sensitizes cells to lenalidomide therapy. Thus, lenalidomide-induced degradation of CK1α provides a mechanistic basis for lenalidomide’s therapeutic window in del(5q) MDS. Despite their widespread clinical use, studies of thalidomide derivatives have been limited by their lack of effect in mouse models. While characterizing CK1α as a lenalidomide- induced substrate, we found that mouse cells are resistant to thalidomide-derivatives because of species-specific differences in CRBN. To enable in vivo study of this class of drugs, we iii developed a knock-in mouse model with a single amino acid change in Crbn, CrbnI391V, which shows thalidomide-induced degradation of Ikzf1, Ikzf3, and Ck1α. Using this model, we demonstrated that haploinsufficiency for Csnk1a1 confers lenalidomide sensitivity in vivo and that both lenalidomide-induced selection and Trp53-mediated resistance occur at the level of hematopoietic stem cells. We also demonstrated that CrbnI391V is sufficient to confer thalidomide-induced fetal loss in mice. Further study of the CrbnI391V model will provide valuable insights into the in vivo efficacy and toxicity of this class of drugs, as well as the opportunities to identify tissue-specific substrates which may contribute to efficacy in other conditions. iv Table of Contents List of Figures ........................................................................................................................... vii List of Tables .............................................................................................................................. ix Acknowledgements ..................................................................................................................... x Chapter 1 Introduction................................................................................................................. 1 Preface ......................................................................................................................................... 2 A brief history of the thalidomide tragedy .................................................................................. 2 Thalidomide embryopathy .......................................................................................................... 5 The repurposing of thalidomide and development of the IMiDs ................................................ 7 Mechanism of action ................................................................................................................. 10 Therapeutic activity of IMiDs in multiple myeloma and other B cell neoplasms .................... 12 Similar drugs ............................................................................................................................. 14 Pathogenesis of del(5q) MDS ................................................................................................... 15 Lenalidomide in del(5q) MDS .................................................................................................. 17 Mechanism of thalidomide’s teratogenicity .............................................................................. 19 IMiDs in rodent models ............................................................................................................. 20 Aims of the thesis ...................................................................................................................... 22 Chapter 2 Lenalidomide induces the ubiquitination of CKlα by CRL4CRBN ........................ 24 Contributions ............................................................................................................................. 25 Summary ................................................................................................................................... 26 Introduction ............................................................................................................................... 27 Results ....................................................................................................................................... 27 Lenalidomide induces degradation of CK1α ......................................................................... 27 CK1α is a substrate of CRL4CRBN ......................................................................................... 31 Effect of CSNK1A1 expression level ..................................................................................... 36 Species-specific effects of lenalidomide ............................................................................... 42 Differential substrate specificity ............................................................................................ 51 Discussion ................................................................................................................................. 56 Methods ..................................................................................................................................... 58 Chapter 3 Development of a thalidomide-sensitive mouse model via a single amino acid change in Cereblon ..................................................................................................................... 77 Contributions ............................................................................................................................. 78 v Summary ................................................................................................................................... 79 Introduction ............................................................................................................................... 80 Design considerations ............................................................................................................ 80 Results ....................................................................................................................................... 82 Thalidomide derivatives induce degradation of substrates in CrbnI391V knock-in mice ....... 82 Effect of lenalidomide treatment on hematopoiesis .............................................................. 95 In vivo modeling of efficacy of lenalidomide in cells haploinsufficient for Csnk1a1 ........ 101 Thalidomide-induced teratogenicity in CrbnI391V mice ....................................................... 109 Discussion ............................................................................................................................... 118 Methods ................................................................................................................................... 120 Chapter 4 Discussion ................................................................................................................ 131 Drug-induced synthetic haploinsufficiency ............................................................................ 133 Other therapeutic indications for CK1α degradation .............................................................. 134 Contribution of other del(5q) genes to lenalidomide sensitivity in MDS with del(5q) .......... 135 Potential mechanisms of lenalidomide in non-del(5q)