A Chemical Probe for Tudor Domain Protein Spin1 To

A CHEMICAL PROBE FOR TUDOR DOMAIN PROTEIN SPIN1 TO INVESTIGATE CHROMATIN FUNCTIONS Vincent Fagan1,2,‡, Catrine Johansson2,3,‡, Carina Gileadi2,3, Octovia Monteiro1,2, James Dunford3, Reshma Nibhani3, Martin Philpott3, Jessica Malzahn3, Graham Wells3, Ruth Farham3, Adam Cribbs3, Nadia Halidi2,3, Fengling Li4, Irene Chau4, Holger Greschik5, Srikannathasan Velupillai2, Abdellalh Al- lali-Hassani4, James Bennett1,2, Thomas Christott1,2, Charline Giroud1,2, Andrew M Lewis1,2, Kilian VM Huber1,2, Nick Athanasou3, Chas Bountra2, Manfred Jung5,6, Roland Schüle5, Masoud Vedadi4, Cheryl Arrowsmith4, Yan Xiong7, Jian Jin7, Oleg Fedorov1,2, Gillian Farnie2,3, Paul E Brennan1,2*, Udo Opper- mann2,3,6* AUTHOR ADDRESS 1 Target Discovery Institute, Nuffield Department of Medicine, University of Oxford, OX3 7FZ, Ox- ford, UK; 2Structural Genomics Consortium, Nuffield Department of Medicine, University of Oxford, OX3 7DQ, Oxford, UK; 3Botnar Research Centre, Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Sciences, NIHR Biomedical Research Centre, University of Oxford, Oxford, OX3 7LD, UK; 4Structural Genomics Consortium, University of Toronto, Canada; 5University of Freiburg, Freiburg, Germany; 6FRIAS - Freiburg Institute of Advanced Studies, 79104, Freiburg, Germany; 7Mount Sinai Center for Therapeutics Discovery, Departments of Pharmacological Sciences and Onco- logical Sciences, Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, United States KEYWORDS epigenetics, chromatin, Tudor domain, SPIN1, chemical probe,

ABSTRACT: Lysine and arginine methylation are amongst the most frequent modifications on unstructured histone tails and in combination with other modifications provide the basis for a combinatorial 'chromatin or histone code'. Recognition of modified histone residues is accomplished in a specific manner by 'reader' domains that recognize chromatin modifications, allowing for association with specific effector complexes that mediate chromatin functions. The methyl-lysine and methyl-arginine reader domain protein SPINDLIN1 (SPIN1) belongs to a family of 5 human genes, and has been identified as a putative oncogene and transcriptional co-activator. It contains three Tudor domains that are able to mediate chromatin binding. Here we report on the discovery of a potent and selective bidentate Tudor domain inhibitor, which simultaneously engages Tudor domains 1 and 2 and effective- ly competes with chromatin binding in cells. Inhibitor, chemoproteomic and knockdown studies in squamous cell carcinoma indi- cate complex SPIN-mediated chromatin interactions leading to transcriptional changes in cellular differentiation processes.

INTRODUCTION harbor reader domains and play critical roles e.g. Covalent chromatin modification is an essential in activation and repression of gene expression. process to orchestrate the dynamic and complex Amongst the multitude of possible PTMs, lysine behavior of gene regulation, chromatin organiza- residues serve as the primary modification sites tion and genome integrity1,2. Multiple posttrans- on histone tails, and they can be acetylated, lational modifications (PTMs) on histone tails methylated and ubiquitinated. Whereas acetylated directly affect chromatin function. The combina- lysine residues are recognized by Bromo and tions of histone PTMs establish a “histone or YEATS domains, methylated lysine residues are chromatin code” 1 which serves as a signalling read by Chromo, PHD, Tudor, MBT and PWWP platform to recruit specific effector molecules to domains 3,4. execute genomic or chromatin templated func- The Tudor domain containing chromatin protein tions. Recognition of histone PTMs is achieved SPINDLIN1 (SPIN1) was originally described as by specialized “reader” domains which mediate an abundant maternal transcript found in mouse chromatin or transcriptional complex formation oocytes 5, and was subsequently characterized in through protein-protein interactions 3,4. There are a screen for genes involved in ovarian cancer 6. hundreds of epigenetic effector molecules that In humans SPIN proteins constitute a small fami-

2 ly of related proteins (SPIN1, 2A, 2B, 3 and 4) as a SPIN1 hit through screening of a focused that all harbor three Tudor domains and are library 25. Using this as a starting template we broadly expressed in different tissues 5,7-11. now set out to develop inhibitors against SPIN1 Chromatin binding of SPIN1 was identified in a chromatin binding, and we here report on the bi- mass spectrometry based screen as a potential ochemical, structural and cellular characterization histone 3- lysine 4 trimethyl (H3K4me3) binding leading to the discovery of a first in class Tudor protein 12, and subsequent structural and func- domain chemical probe with low nanomolar in tional studies identified a direct interaction be- vitro activity, exquisite selectivity over a panel of tween the second Tudor domain of SPIN1 and methyl reader or writer proteins, and with submi- the H3K4me3 mark 13 where methyl-Lys binding cromolar cellular activity. is accomplished through occupation of an 'aromatic cage' 14. The subsequent discovery of an interaction with trimethylated lysine 20 of his- RESULTS and DISCUSSION tone 4 (H4K20me3) via domain 2 suggested a In order to design SPIN1 selective inhibitors (Fig possible role in DNA damage, however little is 1A), we initially utilized X-ray crystallography to known about this interaction 15. Importantly the analyze the binding mode of A366 in SPIN1. first Tudor domain also recognizes the asymmet- This potent methyltransferase inhibitor shows 3- rically dimethylated arginine mark H3R8me2a 14, 10 nM IC values to human G9a and GLP me- making SPIN1 a bidentate reader of the histone 50 thyltransferases 24 and has an IC of 0.2 - 0.8 M code. Binding to these epigenetic marks at gene 50 µ against SPIN1 in an AlphaScreen assay format. promoter regions leads to transcriptional activa- The structure of SPIN1 in complex with A-366 tion of ribosomal proteins13 and a number of re- allowed comparison to the ligand binding mode cent studies have shown that in a number of solid with the methyl transferase G9a (Fig 1B, C). In- tumors including ovarian, colon and breast can- terestingly, we find that A366 occupies the aro- cers as well as sarcomas, SPIN1 promotes cancer matic cages in both domain 1 and domain 2 in cell proliferation by activation of the Wnt/β- SPIN1, where binding is largely dictated by π- catenin or GDNF-RET-MAZ pathways 10, and is stacking interactions in domain 1 involving resi- involved in PI3K/Akt signaling 14,16-18. SPIN1 dues Trp62, Trp72, Tyr91, Phe94 and Tyr98 and knockdown resulted in cancer cell and xenograft in domain 2 through contacts with residues tumor growth inhibition, and such studies suggest His139, Phe141, Trp151, Tyr177 and Tyr179, in that small molecule inhibition of SPIN1 may be a addition to polar interactions identified with viable approach for the treatment of certain can- Asp137 in domain 2 (Fig 1C). In contrast to the- cers10,16,17,19-21. The recent discovery of a regula- se hydrophobic and electrostatic interactions, tory protein C11ORF84, which directly interacts A366 binding in G9a is largely dominated by po- with SPIN1 and thereby attenuates its coactivator lar contacts (Fig 1B). activity, was significant as it provided the first evidence for an additional layer of regulation of Development of monodentate inhibitor series 8 SPIN1 activity . for SPIN1-H3K4me3 interactions Previous Development of suitable chemical tools to selec- studies revealed that H3K4me3-R8me2a peptide tively and potently inhibit any SPIN1 interaction binding is largely driven by binding of the as a means to understand its biological functions K4me3 mark to domain 2 14 which led us to ini- has not been achieved yet. To this end micromo- tially focus on the domain 2 binding pocket for lar inhibitors were previously identified using in inhibitor design. We reasoned that unlike G9a, 22 silico screening and structure guided design domain 2 of SPIN1 could accommodate a larger leading to micromolar bidentate inhibitors that spirocycle and larger substituents in place of the interact with domains 1 and 2 was recently re- methoxy group of A-366 (Fig. 1). Therefore, in 23 ported . The potent G9a methyltransferase in- efforts to gain selectivity towards SPIN1, our ini- 24 hibitor A-366 was previously identified by us tial strategy was to enlarge the spirocycle and

FIGURE 1: Development and characterization of monodentate SPIN1 inhibitors. (a) Design of SPIN inhibitor series 1-3 based on the A366 template. (b) Structural analysis of A366 recognition in methyltransferase G9a. Overall structure of G9a in complex with A366 shown in pink 24 (PDB 4NVQ). (c) Crystal structure of SPIN1 in complex with A366. The domains in SPIN1 are colored in purple, green and yellow, A366 is shown with cyan sticks and details of ligand binding are shown in boxes. (d) Se- lectivity profile of monodentate inhibitor for SPIN1 (AlphaScreen assay) and G9a (Scintillation proximity assay). Abbreviations: nd: not determined; na: no inhibitor activity detected. (e) Full length wild-type SPIN1 and Histone 3.3 Nanobret assay showing in cell target engagement of compounds 2 and 4. Graph showing fold-change in milliBRET Units (mBU) after 24h treatment with 1µM compounds 2, 3, 4 and 5. Mean±SD, n=3 independent experiments, n≥3 internal replicates. One way ANOVA with Dunnetts post-hoc multiple comparison test, ** p<0.005 replace the methoxy group with bulkier alkyl hibition, without significantly impacting SPIN1 groups. The published synthetic route of A-366 24 inhibitory activity. Furthermore, a number of ter- was modified (Supplementary Notes) in order to tiary amines from series 2 resulted in improved allow larger alkyl group incorporation at R1 (Fig. binding to SPIN1, with analogues containing iso- 1A, series 1) and tertiary amines at R2 (Fig. 1A, indolines at R2 consistently showing strongest Series 2). SPIN1 binding affinities. A small library of compounds was prepared which incorporated the op- To monitor improvements in activity and selec- timum substituents from series 1 and 2 leading to tivity of the designed series, we employed an Al- series 3 (Fig 1A). This process resulted in a phaScreen competitive binding assay to assess number of potent inhibitors of SPIN1, which dis- SPIN1 activity, while an enzymatic scintillation played no inhibition of G9a or the related GLP proximity assay (SPA) was used to evaluate their methyltransferases (SI Data 1). Compounds 1 inhibition of G9a (Fig 1D, SI Data 1). First, a and 2 (Fig. 1A, D) were among the most potent, methylcyclopropyl group was found to be opti- with IC50 values of 0.34 and 0.40 µM respective- mal at R1 leading to a significant loss of G9a in-

4 ly. To determine if the spirocycle was required 1 and 2 via H3K4me3 and H3R8me2a modifica- for SPIN1 selectivity, the gem-dimethyl contain- tions resulted in an increase, though modest, in ing compound 4 was prepared. Compared to the peptide binding 14. A recent report provided mi- analogous 2, compound 4 was equipotent as a cromolar inhibitors that engaged both domain 1 SPIN1 inhibitor, and displayed no G9a inhibition, and 2 of SPIN1, and although they had weak af- indicating that the spirocycle was not required for finity, they showed a good degree of selectivity SPIN1 selectivity. A representative ligand struc- 23. We therefore set out to investigate bidentate ture reveals binding of compound 2 to domain 2 inhibitors which can bind simultaneously to do- only and additional π-stacking interactions be- mains 1 and 2 as a means to further increase tween the isoindoline ring and residues Phe141, binding and selectivity. Azide-functionalized 6 Trp151, Tyr170 and Tyr177 in the aromatic cage and 7 (Fig 2A, SI Data 1, Supplementary Ma- may explain the improved binding affinity of iso- terial) were coupled to alkyne-functionalized 8 indoline-containing analogues (SI Fig 1). Taking and 9, via a copper catalyzed “click” reaction, to advantage of earlier failed attempts to modify the provide a small library of molecules, containing alkyl chain, compounds 3 and 5 were prepared, triazole linkers of various lengths (Fig. 2A). The- which possessed a chain elongated by one carbon se bidentate compounds, as well as compound 4 atom (Fig. 1A). This change resulted in a 16- and were evaluated as SPIN1 inhibitors using a range 19-fold decrease in potency for compound 3 and of biophysical assays and X-ray crystallography 5 compared to compound 2 and 4 respectively, (Fig 2B). Three of the four linked compounds and hence compound 3 and 5 were deemed inac- displayed a large increase in potency compared to tive controls for compound 2 and 4. At this point compound 4 (Fig 2) with compound 12 being the it was important to assess cellular target engage- most potent. Analysis of the binding mode of ment and possible toxicity. We developed a compound 12 using X-ray crystallography con- nanoBRET assay26 by overexpressing a halotag- firmed a bidentate inhibitor mode where the histone 3.3 and an N-terminal Nanoluc-SPIN1 ethylpyrrolidine moiety indeed occupies the aro- fusion protein in U2OS cells. Treatment with matic pocket in domain 1 (Fig 2C) while binding compounds 2-5 at a concentration of 1 µM re- of the isoindoline to domain 2 is retained. Since sulted in a significant reduction of the nanoBRET compound 11 displayed significantly lower bind- signal for compound 2 and 4, but not for the inac- ing affinity, an analogue of this (compound 15) tive compounds 3 and 5, indicating the developed was prepared as an inactive control, in which the inhibitors indeed showed cellular target engage- propyl chain was extended to a butyl chain. Iso- ment (Fig 1E). However, at elevated concentra- thermal titration calorimetry (ITC) showed that tions (>3 µM) increased cell death was observed compound 15 was approximately 130 times less in U2OS cells (SI Fig 2), importantly for both the potent than compound 12 and thus, compounds active and inactive compounds, indicating that 12 and 15 represent good candidates for an active off-target activity is likely responsible for cyto- and inactive chemical probe pairing (CPP), to be toxicity. Despite reasonable activity and selec- utilized in molecular biology studies. tivity profiles for these monodentate molecules, further modifications of this series did not result Bidentate SPIN1 inhibitor 12 is selective in desirable and significant activity increases, against methyltransferases and methyl-Lys leading us to pursue a bidentate inhibitor design. binding domains Next, we analyzed in detail the selectivity profile of compound 12. Within Development of bidentate SPIN1 inhibitors the human genome, the Tudor domain family Bidentate histone peptide binding to domains and consists of 43 members (Fig 3A) and the

FIGURE 2: Development of bidentate SPIN1 inhibitors. (a) Overview on synthesis and design routes for bidentate inhibitor analogues. (b) Evaluation of SPIN1 inhibition of bidentate analogues using biophysical assays (AlphaScreen, BioLayer Interferom- etry (BLI), Isothermal Titration Calorimetry (ITC) and Differential Scanning Fluorimetry (DSF). (c) Crystal structure of SPIN1 in complex with compound 12. Left, electrostatic surface potential of SPIN 1 with compound 12 depicted as cyan sticks. Right, details of the interactions between SPIN1 and compound 12. Residues in domain 1 and domain 2 of SPIN1 are shown as sticks colored magenta, green and yellow. reader domain is often found in combination with tivity profiles against a panel of 16 methyl-Lys or catalytic domains such as Jmj-type histone deme- methyl-Arg binding domains (including Chromo, thylases, e.g. found in KDM4 (JmjD2) PHD, Tudor and MBT domains) were determined members27. There are 5 distinct human SPIN for compound 12 and its inactive control 15 using family members which form a distinct branch in a SYPRO Orange thermal shift assay28. In sup- the phylogenetic tree (Fig 3A), and ITC data port of ITC data (Fig 3C, D) compound 12 in- showed that compound 12 binds with varying duced a large shift in the thermal stability of all 4 affinities (KD values ranging from 10 to 130 nM) members of the SPIN subfamily assessed (ΔTm = to SPIN subfamily members (including SPIN1, 6.5-14.1 °C), whereas no other significant shift in SPIN2B, SPIN3 and SPIN4) suggesting similar thermal stability was observed neither for com- domain architectures and binding modes amongst pound 12 nor 15 (Fig 3E) in the off-target panel the SPIN subfamily members (Fig 3B), in line employed. In addition, 12 displayed over 250- with their overall structures and peptide binding fold selectivity over the malignant brain tumor features (SI Fig 3). Interestingly we found that (MBT) methyl reader domains, L3MBTL1 and the construct design of SPIN1 influences ligand L3MBTL3 using a fluorescence polarization as- binding. Whereas a construct ranging from resi- say (SI Figure 4). Using the methyltransferase dues 49-262 in SPIN1 shows a KD ranging from inhibitor A366 as the starting point for SPIN in- 6-10 nM, a 10-15fold increase in affinity was ob- hibitor development demanded an extensive se- served for a construct ranging from residues 26- lectivity assessment of different methyltransfer 262, indicating a significant role for the N- ases. We therefore tested compound 12 against a terminus in ligand binding (Figs 3C, D). Selec- panel of 33 protein, DNA or RNA

FIGURE 3: Selectivity profiles for compounds 12 and 15. (a) Phylogenetic tree for human Tudor domain containing proteins. The SPIN subfamily is highlighted. (b) Average KD values as determined by ITC for compound 12 against human SPIN members. (c) Heatmap of selected methyl-Lys and methyl-Arg binding domains using Differential Scanning Fluorimetry. Scale shows Δ(T). (d) Inhibition data for compound 12 at 10 and 50 µM against a panel of 33 protein, DNA and RNA methyltransferases. Scale shows residual activity in %. (e) IC50 data (in µM) for compound 12 and 15 against selected methyltransferases. methyltransferases using established in-house would have suitable physicochemical properties scintillation proximity assays 29. Compound 12 for cellular penetration. To investigate whether was initially tested at two different concentra- compound 12 could displace the interaction be- tions (10 and 50 µM) against the panel and fur- tween SPIN1 and histone H3 we therefore first ther IC50 determinations were carried out for a tested target engagement in U2OS cells using the select number where significant inhibition oc- NanoBret assay. In this assay compound 12 dis- curred at 50 µM. Compound 12 displayed at least played potent inhibition of the SPIN1-histone 3.3 300-fold selectivity over all methyltransferases interaction, with an EC50 of 270±40 nM (Fig. tested (Fig 3F, G), and combined with its me- 4A), well within the acceptable range of cellular 30 thyl-reader profile, the data suggests an exquisite activity for a chemical probe . The inactive 15 selectivity profile for compound 12 over a large displayed no inhibition (Fig. 4A) and important- set of methyl readers and writers. ly, no unspecific toxic effects were detected for compounds 12 and 15. We next investigated the Compound 12 shows intracellular target en- effects of compound 12 in the squamous carci- gagement - Whilst compound 4 was shown to noma cell line SCC040. In squamous cell carci- disrupt SPIN1-chromatin interactions in a cellular noma, intratumour SPIN1 expression was previ- context (Fig 1E) it was unclear whether com- ously found to be highly correlated with the tu- 31 pound 12, due to its increased molecular mass, mour stroma derived risk factor galectin 1 ,

FIGURE 4: Compound 12 induces keratinization in squamous cell carcinoma and changes in histone composition (a) Full length wild-type SPIN1 and Histone 3.3 Nanobret assay. Graph showing milliBRET Units after 24h treatment with dose response 0.05-15µM to compounds 12 and 15. (b) Results from chemoproteomic analyses using either compound 12 (left panel) or 15 (right panel) as competitors showing specific interactions between compound 12 and SPIN1, SPIN4 and C11ORF84 (SPIN-DOC) in human squamous carcinoma cell line SCC040 lysates. (c) Heatmap of overlapping genes between compound treatment and siRNA knockdown in squamous carinoma cells. (d) Browser tracks (RNAseq) for histone locus for compound 12 (blue) or SPIN1 siRNA (green) treatment and respective controls (red) showing differential changes in histone gene expressions. and it was therefore of interest to investigate the 12 were found to be SPIN1, SPIN4 and the inter- effect of inhibition of SPIN1-chromatin interac- acting protein C11ORF84 8, whereas no signifi- tions in this biological system. We next set out to cant binding partners were identified with 15, identify the cellular interaction partners of com- highlighting the excellent specificity of compound 12 using a chemoproteomic approach 32. pound 12 also in a biological environment (Fig For this experiment, we prepared the affinity 4B). probe 57 (SI Data 1, Supplementary Notes) containing a 4-aminobutyl linker to enable cou- Compound 12 induces a specific transcrip- pling to NHS-activated sepharose beads. Incuba- tional profile in squamous cell carcinoma To tion of the affinity matrix with SCC040 cell ly- further understand the specific perturbations of sates in absence or presence of compounds 12 or SPIN1-chromatin interactions in SCC040 cells 15 allowed for determination of compound- we performed transcriptional analysis by specific interactors using label-free quantification RNAseq upon compound treatment or siRNA- (LFQ) protein mass spectrometry. Notably, the mediated knockdown of SPIN1 (SI Data 2). sole specific interaction partners for compound Cells were treated with compound 12 or 15 at 1

8 µM for 5 or 7 days, or for 2 or 4 days with siR- members and interactors leading to cell-specific NAs 10 before RNA isolation. Considering only changes in transcriptional programs upon inhibition specific changes, either induced by compound 12 suggests a complex role of SPIN members in gene or by specific SPIN1 siRNA knockdown, a list of regulation. 42 overlapping genes emerged. Interestingly, a This discovery provides a useful small-molecule subset of genes was regulated in opposing direc- probe that selectively targets an important aspect of tions. For example, whereas compound 12 induc- "reading" the "histone code". Together with other es >2-fold expression of histone H1 and H2 small-molecule regulators of histone-modifying genes, siRNA knockdown reduced these by enzymes compound 12 should find applications for >50% (Fig 4D). With few exceptions (such as studies of histone methylation dynamics in a wide seen with keratin genes KRT16 or KRT6) this range of biological processes, including embryonic development and differentiation, germline mainte- trend appeared to dominate the overlapping list of nance and meiosis, pertaining to disease processes genes. Reactome analysis of either compound 12 such as those leading to the development of cancer. or siRNA regulated genes showed a set of important pathways significantly affected by SPIN1 ASSOCIATED CONTENT modulation (SI Data 2). These comprised p53 Supporting Information regulated cell death or proliferation, chromatin, The Supporting Information is available free of charge on the matrix organization, cell contacts, immune sig- ACS Publications website. nalling and keratinization pathways. Keratiniza- Materials and Methods (PDF) tion, characterized by expression of several cy- SI Data 1 (list of compounds) (Excel file) tokeratins, is a diagnostic hallmark of differentia- SI Data 2 (RNAseq data) tion for squamous cell carcinoma, and might be Supplementary Notes (PDF) Supplementary Information (PDF) related to metastasis and overall survival e.g. in nasopharyngeal tumors 33. In addition, several of AUTHOR INFORMATION the identified SPIN1 regulated genes have been Corresponding Authors previously identified as differentially expressed [email protected] ; [email protected] when comparing normal tissue to adenocarcino- Author Contributions ma tumours or have been implied in metastasis of 34,35 Experiments: VF‡, CJ‡, CG, OM, JD, RN, MP, JM, GW, RF, head and neck squamous cell carcinoma . Fur- NH, FL, IC, AAH, JB, TC, CG, AL, HG; data analysis VF, CJ, thermore, the overrepresentation of genes in- RN, KH, NA, MJ, SV, RS, MV, CA, JJ, OF, GF, PB, UO. Con- volved in matrix organization such as proteinases cept, supervision, materials, funding: VF, CA, KH, CB, MJ, YX, JJ, RS, GF, PB, UO. Manuscript writing VF, UO with contribu- or inhibitors thereof (eg. members of SERPIN tions from all authors. All authors have given approval to the final and SPINK families), as well as integral mem- version of the manuscript. ‡These authors contributed equally. brane proteins like CLDN8 or UPKB1 involved Funding Sources in tight junction or cell polarity, respectively, The study was supported by project grant C41580/A23900 from suggest that essential processes like epithelial Cancer Research UK, the Oxford NIHR Biomedical Research integrity, deregulation of intercellular adhesions Centre, Myeloma UK and Arthritis Research UK (program grant 36 20522). The SGC is a registered charity (number 1097737) that and cell polarity are in squamous cell carcino- receives funds from AbbVie, Bayer Pharma AG, Boehringer ma in part controlled by SPIN proteins. Ingelheim, Canada Foundation for Innovation, Eshelman Institute for Innovation, Genome Canada, Innovative Medicines Initiative (EU/EFPIA) [ULTRA-DD grant no. 115766], Janssen, Merck CONCLUSIONS We have described a cell-active KGaA Darmstadt Germany, MSD, Novartis Pharma AG, Ontario selective small-molecule inhibitor of the Tudor Ministry of Economic Development and Innovation, Pfizer, São domain containing, chromatin binding protein Paulo Research Foundation-FAPESP, Takeda, and Wellcome [106169/ZZ14/Z]. The research has received funding from the SPIN1. Using a strategy to displace simultaneously People Programme (Marie Curie Actions) of the European Un- histone H3K4me3/H3Arg8me2a binding the biden- ion's Seventh Framework Programme (FP7/2007-2013) under tate compound 12 showed a high degree of selec- REA grant agreement n° [609305]. JJ acknowledges the support by the grants R01CA218600, R01CA230854, R01GM122749 and tive inhibitory activity against chromatin methyl R01HD088626 from the U.S. National Institutes of Health. The marks in vitro and in cells. In addition, the discov- views expressed are those of the author(s) and not necessarily ery of cellular interactions with different SPIN those of the NHS, the NIHR or the Department of Health.

9 (18) Wang, J. X.; Zeng, Q.; Chen, L.; Du, J. C.; Notes Yan, X. L.; Yuan, H. F.; Zhai, C.; Zhou, J. N.; Jia, Y. L.; Yue, W.; Pei, X. T. Molecular Cancer Research 2012, 10, 326. For future work we have named compound 12 VinSpinIn (Vin- (19) Chen, X.; Dong, H.; Liu, S.; Yu, L.; Yan, D.; nie’s Spindlin Inhibitor) and compound 15 VinSpinIC (Vinnie’s Yao, X.; Sun, W.; Han, D.; Gao, G. Am J Transl Res 2017, 9, 90. Spindlin Inactive Control). Data availability. The macromolecu- (20) Drago-Ferrante, R.; Pentimalli, F.; Carlisi, D.; lar structures have been deposited with Protein Data Bank under De Blasio, A.; Saliba, C.; Baldacchino, S.; Degaetano, J.; Debono, J.; the following accession numbers: 6I8Y (SPIN1-A366), 6I8L Caruana-Dingli, G.; Grech, G.; Scerri, C.; Tesoriere, G.; Giordano, (SPIN1-cpd 2), 6I8B (SPIN1-cpd 12). PDB codes for SPIN2, A.; Vento, R.; Di Fiore, R. Oncotarget 2017, 8, 28939. SPIN3 and SPIN4 structures: SPIN2B-H3K4me3 (5LUG), SPIN3 (21) Fang, Z.; Cao, B.; Liao, J. M.; Deng, J.; (5AH1), SPIN4-H3K4me3 (5UY4). RNA-seq data were deposited Plummer, K. D.; Liao, P.; Liu, T.; Zhang, W.; Zhang, K.; Li, L.; with the GEO database with accession number GSE123084. The Margolin, D.; Zeng, S. X.; Xiong, J.; Lu, H. Elife 2018, 7. mass spectrometry data have been deposited to the Proteo- (22) Robaa, D.; Wagner, T.; Luise, C.; Carlino, L.; meXchange Consortium via the PRIDE partner repository with McMillan, J.; Flaig, R.; Schule, R.; Jung, M.; Sippl, W. the dataset identifier PXD011703. Chemmedchem 2016, 11, 2327. (23) Bae, N.; Viviano, M.; Su, X.; Lv, J.; Cheng, D.; ABBREVIATIONS Sagum, C.; Castellano, S.; Bai, X.; Johnson, C.; Khalil, M. I.; Shen, J.; Chen, K.; Li, H.; Sbardella, G.; Bedford, M. T. Nat Chem Biol SPIN1, SPINDLIN1; SPA, scintillation proximity assay; BLI, 2017, 13, 750. BioLayer Interferometry; ITC, Isothermal Titration Calorimetry; (24) Sweis, R. F.; Pliushchev, M.; Brown, P. 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