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Investigating the Molecular Mechanisms of Action of Lenalidomide and Other Immunomodulatory Derivatives

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Investigating the Molecular Mechanisms of Action of Lenalidomide and Other Immunomodulatory Derivatives Investigating the Molecular Mechanisms of Action of Lenalidomide and Other Immunomodulatory Derivatives The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Haldar, Saurav Daniel. 2018. Investigating the Molecular Mechanisms of Action of Lenalidomide and Other Immunomodulatory Derivatives. Doctoral dissertation, Harvard Medical School. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:37006480 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 TABLE OF CONTENTS! ! 1 Abstract……………………………………………………………………………………………..3 2 Glossary……………………………………………………………………………………………..5 3 Introduction………………………………………………………………………………………....6 3.1 A Tragic Beginning…...………………………………………………………………………..6 3.2 Thalidomide: The Comeback Drug…………………………………………………………..9 3.3 Development of Thalidomide Analogs……………………………………………………..12 3.4 iMiD Mechanism of Action…………………………………………………………………..15 3.5 Purpose of Inquiry……………………………………………………………………………22 4 Materials and Methods…………………………………………………...…...…………………24 4.1 Mutant Degron Analysis via a Fluorescent Reporter Assay……….……….…….…......24 4.2 Generation of CRISPR/Cas9 Single Cell Knockout Clones………………….…….……25 4.3 Western Immunoblotting…………………………………………………………………….28 4.4 TNF-α Enzyme-Linked Immunosorbent Assay (ELISA)……………………..………….29 5 Results.……………………………………………………………………………….……………31 5.1 Effects of IKZF3 Degron Mutagenesis on Substrate Recognition by the iMiD-CRBN Complex……...……………………………..………………………………….31 5.2 Validation of ZFP91 as a Novel CRBN-CRL4 E3 Ubiquitin Ligase Substrate.………..33 5.3 Effects of ZFP91 Degron Mutagenesis on Substrate Recognition by the iMiD-CRBN Complex…………………………………………..………………….………...34 5.4 iMiD-Induced TNF-! Downregulation in U937 Monocytic Cells…………..……………36 5.5 Effects of CRISPR/Cas9-Mediated CRBN Inactivation on iMiD-Induced TNF-!"Downregulation………………..………………………………………………….….36 6 Discussion…………………………………………………………………………………………38 6.1 Functional Dissection of CRBN-CRL4 Substrate Degrons……..……………………….38 6.2 Investigation of the Molecular Basis for iMiD-Induced TNF-α Downregulation..........41 7 Conclusions ………………………………………………………………………………………44 8 Summary…………………………………………………………………………………………..45 9 Acknowledgments………………………………………………………………………………..46 10 Tables………………………………………………………………………………………………47 11 Figures……………………………………………………………………………………………..50 References……………………………………………………………………………………………….57 2 1 ABSTRACT Background. Thalidomide was first released as a sedative and anti-emetic by a German pharmaceutical company in the 1950s. After its withdrawal from the market due to severe teratogenicity, thalidomide and its novel iMiD analogs were repurposed decades later for a variety of new clinical indications, including the treatment of erythema nodosum leprosum, multiple myeloma, and del(5q)-MDS. Recent work has demonstrated that iMiDs act by modulating the substrate specificity of the CRBN-CRL4 E3 ubiquitin ligase. Selective degradation of IKZF1/IKZF3 and CK1α by this complex in the presence of iMiDs leads to therapeutic activity against multiple myeloma and del(5q)-MDS, respectively. In spite of these advances, our molecular understanding of the pleiotropic effects of iMiDs remains limited. Here, we address two distinct aspects of iMiD biology that warrant further investigation: (1) substrate recognition by the iMiD-CRBN complex via degron motifs and (2) the molecular mechanism of iMiD-induced TNF-α inhibition. Methods. Characterization of iMiD-sensitive degron motifs was performed using a flow cytometry-based fluorescent reporter assay. In brief, HEK293T cells were transfected with GFP- fusion constructs expressing wild-type and mutant degrons of IKZF3 and ZFP91. Substrate degradation in the presence of iMiD treatment was assessed by quantifying GFP reporter expression via flow cytometry. Comparison of a Drug:DMSO GFP ratio between wild-type and mutant degrons was used to determine the effects of single amino acid substitutions on iMiD- induced substrate degradation. The mechanism of TNF-α inhibition by iMiDs was investigated by generating CRISPR/Cas9-mediated single cell knockout clones in the pro-monocytic U937 cell line. Targeted genes were components of the CRBN-CRL4 complex and its associated substrates 3 including CRBN, IKZF1, IKZF3, CSNK1A1, ZFP91, ZNF692, and RNF166. Knockout clones were analyzed by deep sequencing to confirm the presence of inactivating out-of-frame indels. In parallel with non-targeting sgRNA clones, validated knockout clones were treated with PMA to induce macrophagic differentiation, subjected to iMiD treatment, and then stimulated with LPS. TNF-α levels were measured by ELISA to determine any changes resulting from genetic inactivation of candidate targets. Results. Degron mutagenesis studies revealed the presence of critical amino acid residues located in zinc-finger domain 2 of IKZF3 including Q147, C148, G152, A153, and H164. Similarly, amino acids Q401 and G406 in zinc-finger domain 4 were identified as critical residues for the ZFP91 degron. In addition, ZFP91 was validated as a CRBN-CRL4 substrate by Western immunoblotting. Lastly, preliminary TNF-α studies performed to phenotypically characterize CRBN knockout U937 clones suggested a plausible CRBN-dependent mechanism for this iMiD effect, although the results were limited by the presence of clonal heterogeneity. Conclusions. In this study, degron characterization via a fluorescent reporter assay led to the identification of shared amino acid motifs among CRBN-CRL4 substrates that are necessary for conferring sensitivity to iMiD-inducible degradation. Future work employing structural biology approaches would be informative in elucidating the structural determinants of novel zinc-finger substrate recognition by the iMiD-CRBN complex. On the other hand, efforts to dissect the mechanism of iMiD-induced TNF-α inhibition were not fully conclusive. Although the results suggest a plausible CRBN-dependent mechanism, our experimental model warrants further refinement to overcome the limitations encountered in the present study. As an alternative approach, an unbiased proteomics experiment conducted in a monocytic system should be considered to identify novel lineage-specific targets that may provide insight into this elusive mechanism. 4 2 GLOSSARY CK1α……………………………………………..…………………...………………casein kinase 1A1 CRBN……………………………………………………………………………………………...cereblon CRBN-CRL4…………………………….CUL4–RBX1–DDB1–CRBN E3 ubiquitin ligase complex CUL4……………………………………………………………………………….……………….cullin-4 DDB1…………………………………………………………damage-specific DNA-binding protein 1 del(5q)-MDS………………………...….myelodysplastic syndrome with chromosomal 5q deletion ELISA………………………………………...………………….enzyme-linked immunosorbent assay ENL………………………………………..………………...…………....erythema nodosum leprosum FGF2………………………………………..………………………….……...fibroblast growth factor-2 GFP………………………………………………………………………….…green fluorescent protein IFN-γ…………………………………………..…………………………..………….interferon-gamma IKZF1………………………………………..……………………………................................…..Ikaros IKZF3……………………………….………………………………………..……………………….Aiolos IL-2…………………………………………..……………………………...........................interleukin-2 iMiD………………………………………..……………………………... ……immunomodulatory drug LD50……………………………………………………………………………………median lethal dose LPS………………………………………..……………………………................….lipopolysaccharide MM………………………………………..……………………………...................…multiple myeloma NF-!B…………………………………………………………………………….nuclear factor kappa-B NTG……………………………………………...……………………………...….non-targeting sgRNA PBMC…………………………………………..…………………...peripheral blood mononuclear cell RBX1……………………………………………………… ……………………….RING-box protein 1 RNF166………………………………………..………………………..………...ring finger protein 166 TNF-α………………….………………………………………..…………tumor necrosis factor-alpha ZFP91………………………………………..……………………………............zinc-finger protein 91 ZNF692………………………………………..……………………….………...zinc-finger protein 692 VEGF…………………………………………..…………………….vascular endothelial growth factor 5 3 INTRODUCTION 3.1 A TRAGIC BEGINNING Thalidomide is the first member of a class of compounds known as immunomodulatory drugs (iMiDs) that have pleiotropic biological effects including significant anti-neoplastic and anti-inflammatory activities (Table 1) [1,2]. The history of thalidomide as a drug remains exceptionally unique because its legacy has evolved dramatically over the decades since it was first released in 1954 [3]. Introduced as a novel sedative and anti-emetic, thalidomide was initially considered a miracle drug for the treatment of morning sickness in pregnant women. In a tragic turn of events, the drug was banned within a few years due to the widespread emergence of birth defects in children born to mothers who had taken it during their pregnancies [4,5]. Several decades later, thalidomide and its analogs were repurposed as therapeutic agents for the treatment of hematologic malignancies such as multiple myeloma (MM) and del(5q)- myelodysplastic syndrome (MDS) [6,7]. The tortuous history of thalidomide begins with its development by a family-owned pharmaceutical company named Chemie Grünenthal GmbH based in Stolberg, Germany. Established in 1946, this company was the first to introduce penicillin to the German pharmaceutical market in the post-World War II era. To capitalize on its initial success in the antibiotic niche, Chemie
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