Novel YAP1 Activator, Identified by Transcription-Based Functional Screen, Limits Multiple Myeloma Growth

Published OnlineFirst October 23, 2017; DOI: 10.1158/1541-7786.MCR-17-0382

Signal Transduction Molecular Cancer Research Novel YAP1 Activator, Identiﬁed by Transcription- Based Functional Screen, Limits Multiple Myeloma Growth Junichi Maruyama1, Kazutoshi Inami1, Fumiyoshi Michishita1, Xinliang Jiang1, Hiroaki Iwasa1, Kentaro Nakagawa1, Mari Ishigami-Yuasa2, Hiroyuki Kagechika2,3, Norio Miyamura4, Jun Hirayama4, Hiroshi Nishina4, Daichi Nogawa5, Kouhei Yamamoto5, and Yutaka Hata1,6

Abstract

Yes-associated protein 1 (YAP1) interacts with numerous this in our mind, we screened for YAP1 activators by using transcription factors, including TEA-domain family proteins human retinal pigment epithelial ARPE-19 cells expressing the (TEAD) and p73. YAP1 is negatively regulated by the tumor TEAD-responsive fluorescence reporter under the coexpression suppressor Hippo pathway. In human cancers, the deregulation of YAP1. From an extensive chemical compound library (n ¼ of the Hippo pathway and YAP1 gene amplification lead to the 18,606) 47 candidate YAP1 activators were identified. These activation of YAP1, which induces epithelial–mesenchymal compounds were characterized to determine whether this assay transition (EMT) and drug resistance. YAP1 inhibitors are provides bona fide YAP1 activators. Importantly, one YAP1 expected to be useful in cancer therapy. On the other hand, activator was effective against the human multiple myeloma in certain cancers, YAP1 upregulates p73-dependent gene tran- IM-9 cells and chronic myeloid leukemia K562 cells. scription and behaves as a tumor suppressor. Moreover, as YAP1 regulates self-renewal and differentiation of tissue stem Implications: YAP1 activation limits growth, induces apoptosis, cells and plays an important role in tissue homeostasis, YAP1 and may be useful at suppressing hematological cancers. activators may contribute to the regenerative medicine. With Mol Cancer Res; 1–15. 2017 AACR.

Introduction to the cytoplasm and undergoes degradation. In human cancers, the Hippo pathway is frequently deregulated and the YAP1 gene is Yes-associated protein 1 (YAP1) was identified as a protein amplified, so that YAP1 activity is enhanced (9). YAP1 upregulates interacting with yes tyrosine kinase (1). Thereafter, many YAP1- cell cycle–promoting and antiapoptotic genes (10). Cancer cells interacting proteins, such as ErbB4, p73, TEAD, SMAD, and with hyperactive YAP1 undergo epithelial–mesenchymal transi- Runx2, were reported (2–6). YAP1 does not directly bind to DNA tion and acquire drug resistance (10). The activity of YAP1 but regulates gene transcription through the interaction with these correlates with short survival in cancer patients. Hence, YAP1 is molecules. YAP1 is negatively regulated by the tumor suppressor regarded as a therapeutic target in cancer therapy (11). Among Hippo pathway (7, 8). YAP1 is phosphorylated by large tumor transcription factors interacting with YAP1, TEAD is the most suppressor kinase (LATS) 1 and 2, the core kinases of the Hippo important in the induction of epithelial–mesenchymal transition pathway. The phosphorylated YAP1 is recruited from the nucleus and drug resistance (12, 13). Accordingly, verteporfin and syn- thetic peptides that block the interaction between YAP1 and TEAD are shown to suppress cancer growth (14–16). 1Department of Medical Biochemistry, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan. 2Chemical Biology The role of YAP1 in cancer is twisting. YAP1 cooperates with Screening Center, Tokyo Medical and Dental University, Tokyo, Japan. 3Institute p73, upregulates proapoptotic genes, and suppresses certain of Biomaterials and Bioengineering, Tokyo Medical and Dental University, cancers (17). How YAP1 determines which fate as an oncopro- Tokyo, Japan. 4Department of Developmental and Regenerative Biology, Med- tein or as a tumor suppressor to adopt is not fully understood, ical Research Institute, Tokyo Medical and Dental University, Tokyo, Japan. but the accumulating evidence suggests that Abelson murine 5Department of Comprehensive Pathology, Graduate School of Medical and leukemia viral oncogene homolog 1 (ABL1) is a key determi- Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan. 6Center for Brain Integration Research, Tokyo Medical and Dental University, Tokyo, Japan. nant (18). Upon DNA damage, ABL1 phosphorylates YAP1 at tyrosine 357 and promotes the interaction with p73. A recent Note: Supplementary data for this article are available at Molecular Cancer Research Online (http://mcr.aacrjournals.org/). study has revealed that multiple myeloma (MM) cells show DNA damage response but escape cell death, because YAP1 J. Maruyama, K. Inami, and F. Michishita contributed equally to this article. activity is low (19). When YAP1 is exogenously expressed or the Corresponding Author: Yutaka Hata, Tokyo Medical and Dental University, 1-5- Hippo pathway is suppressed to enhance YAP1 expression, MM 45 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan. Phone: 81358035164; Fax: cells die via the ABL1–YAP1–p73 axis. We can surmise that in 81358030121; E-mail: [email protected] cancers with the high expression of ABL1 and with the low doi: 10.1158/1541-7786.MCR-17-0382 expression of YAP1, YAP1 activators trigger apoptosis and are 2017 American Association for Cancer Research. therapeutically effective.

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YAP1 plays a crucial role in the regulation of tissue stem cells from H2B-mCherry (a gift from Robert Benezra; Addgene (20). YAP1 is essential for tissue repair and protection in intestine, plasmid, 20972; ref. 34) were ligated into NotI/XbaI sites liver, heart, and skin (21–26). YAP1 is also necessary to maintain of pLL3.7 K122 FH-YAP1 and pLL3.7 K122 to obtain neural stem cells in the brain (27). Based on these properties of pLL3.7 K122 FH-YAP1-ires-GFP-TEAD-responsive-promoter- YAP1, we infer that YAP1 activators are useful in the regenerative H2B-mCherry reporter (Fig. 1A) and pLL3.7 K122 control- medicine. TEAD-responsive-promoter-H2B-mCherry, respectively. pCMV We previously searched for YAP1 modulators by means of FLAG-human YAP1 was described previously (35). pCIneoMyc- human osteosarcoma U2OS cells expressing green fluorescent and pCIneoFLAG-LATS1 were generated from pCGN HA-Warts protein (GFP)-tagged YAP1 (28). We used the subcellular (a gift from Hiroyuki Saya) using PCR with the primers (H1921, localization of GFP-YAP1 as the readout and found that 50-acgcgtatgaggcctaagacctttcc-30 and H1922, 50-gtcgactaaacatatac- dobutamine inhibits YAP1 through b-adrenergic receptor. tagatcgcga-30), while pCIneoMyc-LATS2 was generated from However, we have not yet found the compounds that activate pcDNA LATS2-FLAG (a gift from Tadashi Yamamoto) YAP1. In this study, we established a new cell-based assay, in with the primers (H1923, 50-acgcgtatgaggccaaagacttttcc-30 and which the YAP1-dependent TEAD-responsive reporter activity H1924, 50-gtcgactacacgtacacaggctggc-30). pCIneoLuc-PP1A and is monitored. We performed a small chemical compound pCIneoLuc-PP2A were described previously (30). pCMV SPORT library screening with the use of this assay and obtained human TEAD4 (MHS1010-58163) was purchased from Open candidate YAP1 activators. We characterized these compounds Biosystems. PCR was performed with the primers (H2710, to examine whether they indeed activate YAP1 and tested the 50-gaattcgagggcacggccggcaccat-30 and H2710, 50-gtcgactcattcttt- idea that YAP1 activator can be used against MM and other caccagcctg-30). The PCR product was ligated into EcoRI/Sall sites blood cancer cells. of pCIneoLuc to generate pCIneoLuc-TEAD4. pCMV alkaline phosphatase is a gift from Sumiko Watanabe. PCR was performed with the primers (H3159, 50-gcggccgcttaagtgaacaactagtgcca-30 and Materials and Methods H3160, 50-caattgagatctttcacaaattttgtaatc-30) on piLenti-siRNA- DNA constructions and virus productions GFP (Applied Biological Materials Inc.) to amplify GFP-2A-puro- pCIneoFLAG-His6 (pCIneoFH), pClneoFLAG-His6-FLAG mycin. The product was digested with SpeI/MfeI and ligated into (pClneoFHF), pCIneoMyc, pCIneoLuc, pCIneomCherry, and NheI/EcoRI of pLL3.7 to generate pLL3.7-GFP-2A-puro. PCR was pQCXIP-EF were described previously (28–32). NheI/EcoRI frag- performed with the primers (H3161, 50-gcggccgcccccttcacc- ment was isolated from pEGFP-C2 (Clontech Laboratories) and gagggcctattt-30 and H3162, agatctagactattctttcccctgcactgt-30)on ligated into NheI/EcoRI sites of pCIneo (Promega) to generate pLKO1-shYAP2 (a gift from Kunliang Guan; Addgene plasmid, pCIneoEGFPC2. pIRES2-EGFP (Clontech Laboratories) was cut 27369; ref. 6). The product was digested with NotI/XbaI and with NotI, filled in, and digested with NheI. pLL3.7 vector was ligated into the same sites of pLL3.7-GFP-2A-shYAP2. Human cut with EcoRI, filled in, and digested with NheI. The fragment p73 cDNA was obtained from Open Biosystem (40125802). from pIRES-EGFP was ligated into pLL3.7 to replace GFP with The coding region was amplified by PCR with the primers IRES-GFP and the resulting vector was named pLL3.7 K122. PCR (H2074, 50-acgcgtatggcccagtccaccgccac-30 and H2075, 50-gtcgact- 0 was performed with the primers (H3244, 5 -caattggcagaaatcgg- cagtggatctcggcctcc-30), digested by MluI/Sall, and ligated 0 0 tactggctttccatc-3 and H3245, 5 -acgcgtgaattccgcgttatcgctct- into pCIneoFHF to generate pCIneoFHF-p73. pGL2 MDM2 0 gaaagta-3 ) on pHTN-Halo-tag vector (Promega) to amplify reporter was a gift of Hitoshi Okazawa (Tokyo Medical and Dental Halo-tag. The PCR product was digested with MfeI/MluI and University; ref. 36). PCR was performed with the primers (H2781, ligated into EcoRI/MluI of pCIneomCherry to generate pCIneom- 50-gacgtcgacgtgcggtctctctctgtt-30 and H2782, 50-gcggccgctattcta- Cherry-Halo. pCMV SPORT human YAP (MHS1010-7508628) gaaattcagggccgggattctc-30) on piPSC-Nanog (SBI System Bios- was purchased from Open Biosystems. The coding region was ciences) to amplify 2A self-cleaving peptide and with the primers 0 amplified by PCR with the primers (H2130, 5 -acgcgtcccgggcag- (H2862, 50-gctagccccaacatgcctgaaccctctaagtct-30 and H2863, 0 0 0 cagccgccgcctcaa-3 and H2352, 5 -gatatcataaccatgtaagaaagctt-3 ), 50-gtcgaccttgtacagctcgtccatgccgcc-3) on H2B-mCherry. The for- digested by MluI/EcoRV and ligated into MluI/Smal sites of mer product was digested with SalI/NotI and ligated into the pCIneoFH to generate pCIneoFH-YAP1. MluI/NotI fragment same sites of pCIneoFH to generate pCIneoFH-2A, and the later from pCIneoFH-YAP1 was ligated into MluI/NotI sites of product was digested with NheI/Sall and ligated into the same pCIneoLuc and pCIneomCherry-Halo to generate pCIneoLuc- sites of pCIneoFH-2A to generate pCIneo-H2B-mCherry-2A. YAP1 and pCIneomCherry-Halo-YAP1. NheI/NotI fragment SmaI/NotI fragment from pDsRed-N1 (Clontech Laboratories) from pCIneomCherry-Halo-YAP1 was ligated into XbaI/NotI was ligated into the same sites of pIRES (Clontech Laborato- of pQCXIP-EF to generate pQCXIP-mCherry-Halo-YAP1. ries) to generate pIRES-DsRedN1. NheI/PmlI fragment from pCIneoFH-YAP1 was digested with NotI, filled in, and cut with pLL3.7 K122 and PmlI/MfeI fragment from pIRES-DsRedN1 NheI. The isolated fragment was ligated into NheI/SmaI sites of were ligated into NheI/EcoRI sites of pLL3.7 to generate pLL3.7 K122 to generate pLL3.7 K122 FH-YAP1. NcoI/BglII from pLL3.7-ires-DsRedN1. NheI/NotI fragment from pCIneo- pEYFP-C1 (Clontech Laboratories) was ligated into NcoI/BamHI H2B-mCherry-2A was ligated into the same sites of pLL3.7- of pGL3 to replace luciferase with YFP and to generate ires-DsRedN1 to generate pLL3.7-H2B-mCherry-2A. PCR was YFP reporter. BamHI/Sall and Sall/HindIII fragments from performed with the primers (H3112, 50-tctagactagttggtaaagc- 8xGT-IIC-d51LucII luciferase reporter (a gift from Hiroshi Sasaki; caccatggaa-30 and H3113, 50-ctagtcgacggcgatctttccgcccttctt-30) RIKEN RDB08067; ref. 33) were ligated into BglII/HindIII sites on pGL3 (Promega) to amplify luciferase. The product was of the YFP reporter to generate pTEAD-responsive-promoter digested with XbaI/NotI and ligated into the same sites of YFP, which was digested with NotI and partially digested pLL3.7-H2B-mCherry-2A to generate pLL3.7-H2B-mCherry- with HindIII. The resulting fragment and HindIII/XbaI fragment 2A-luciferase.

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Figure 1. A, Schematic of the reporter construct used in this study. FLAG-His6-YAP1 (FH-YAP1) gene followed by internal ribosomal entry sites (IRES) and GFP gene are cloned under CMV promoter. Histone 2B-mCherry (H2B-mCherry) gene is regulated under the TEAD-responsive element harboring eight repeats of gacacacattccacagctg. B, H2B-mCherry and GFP in ARPE-19 cells. H2B-mCherry and GFP are visible at the basal condition. When YAP1 is knocked down, GFP expression is suppressed, because the GFP gene is linked to the YAP1 gene by the IRES sequence. At the same time, the H2B-mCherry signal is also reduced. H2B-mCherry signal was measured by ArrayScan VTI in 100 cells and demonstrated as the histograms. YAP1 knockdown shifted the distribution of H2B-mCherry signals to the leftward. Bars, 200 mm. C, 18,606 compounds were applied to the reporter cells in 96-well plates and H2B-mCherry signals were measured in 3,500 cells for each compound. Z-scores were calculated among the compounds in each plate, in which 80 compounds were tested. D, The representative image of the ARPE-19 reporter cells treated with YAP1 activator. When the reporter cells were exposed to 10 mmol/L IBS003031, H2B-mCherry signals were enhanced, but YAP1 knockdown canceled the effect of the compound. Bars, 100 mm.

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Antibodies and reagents Scientific). The ratio of mCherry intensity over GFP intensity Rat monoclonal anti-YAP1 was described previously (28). was calculated by using BioApplication Compartment Anal- Other antibodies and reagents were obtained from commercial ysis V4 (Thermo Fisher Scientific). The formula for Z-score sources: mouse anti-PARP (51-6639GR) and mouse anti-BAX calculations is as follows: Z-score ¼ (the result obtained for (610982) (BD Pharmingen); mouse anti-a-tubulin (T9026), each cell the median result obtained for each assay plate) / mouse anti-b-actin (A1978), Hoechst 33342, propidium iodide, normalized interquartile range (NIQR, namely 0.7413 (the trypan blue solution, thiazolyl blue tetrazolium bromide, and result corresponding to the first 25% when ranked in order— SIGMAFAST 3,30-diaminbenzidine tablets (D4293) (Sigma- the result corresponding to the first 75% when ranked in Aldrich); anti-DYKDDDDK-tag antibody (014-22383), anti- order) of each assay plate. Each assay plate contained 80 DYKDDDDK-tag beads (016-22784), Phos-tag acrylamide samples. In the first screening, we selected 124 compounds and imatinib (Wako Pure Chemical Industries); mouse anti- that gave Z-scores larger than 3.5 as positive samples. We phospho-Histone H2A.X (Ser139) (05-636) (Merck Millipore); subsequently applied 124 compounds to ARPE-19 cells and rabbit monoclonal anti-YAP1 (14074), rabbit monoclonal anti- reanalyzed them. In the second screening, we used the same YAP1/TAZ (D24E4; 8418), rabbit polyclonal anti-phospho-S127 experimental method as in the first screening but compared YAP1 (4911), rabbit polyclonal anti-phospho-S909/S871 LATS1/ mCherry/GFPratioofeachsamplewiththatoftheDMSO- 2 (9157), and rabbit monoclonal anti-phospho-T1079/T1041 treated control sample to select 47 compounds that augment- LATS1/2 (D57D3) (8654) (Cell Signaling Technology); and ed mCherry/GFP ratio by more than 1.5-fold as positive D-luciferin (Summit Pharmaceuticals International). samples. In the third screening, we evaluated the effects of 47 compounds on a YAP1-depedent TEAD reporter assay in Cell cultures and transfection HEK293FT cells and YAP1 phosphorylation in ARPE-19 HEK293FT and ARPE-19 cells were cultured in Dulbecco's reporter cells. The overview of the screenings is shown in Modified Eagle Medium containing 10% fetal bovine serum (FBS) Supplementary Fig. S1. and 10 mmol/L Hepes-NaOH pH7.4 under 5% CO2 at 37 C. Human MM IM-9 and human chronic myeloid leukemia K562 Quantitative reverse transcription-PCR (qRT-PCR) cells were cultured in RPMI1640 medium supplemented with qRT-PCR analysis was performed using SYBR Green (Roche) 10% FBS, and 10 mmol/L Hepes-NaOH pH7.4. HEK293FT cells and ABI7500 Real-Time PCR system (Applied Biosystems). The were purchased from Invitrogen. ARPE-19 and IM-9 cells were used primers are as follows: 50-cagcacagcaaattctccaa-30 and 50- obtained from Department of Ophthalmology and Department tggattttgagtcccaccat-30 for YAP1;50-ggcaggggagagtgatacaga-30 and of Hematology, Tokyo Medical and Dental University, respec- 50-gaagccaattctcacgaaggg-30 for MDM2;50-gggcatctggaccctcctac-30 tively. Cell authentication was performed in 2016 using the short and 50-tcctttcacctggaggacag-30 for FAS;50-atgttttctgacggcaacttc-30 tandem repeat analysis by JCRB Cell Bank (National Institutes and 50-atcagttccggcaccttg-30 for BAX;50-gtccttcgtgtgggctacat-30 and of Biomedical Innovation, Health and Nutrition). Mycoplasma 50-cgaggatcttcggttgacat-30 for LATS1;50-ttcatccaccgagacatcaa-30 contamination was tested by use of e-Myco Mycoplasma PCR and 50-ctccatgctgtcctgtctga-30 for LATS2;50-ccaatgacaacgcctcctg- Detection Kit (iNtRON Biotechnology). DNA transfection was 30 and 50-tggtgcagccagaaagctc-30 for CTGF;50-agcctcgcatcctata- performed using Lipofectamine 2000 (Invitrogen) and polyethy- caacc-30 and 50-ttctttcacaaggcggcactc-30 for CYR61; and 50- lenemine "Max" (Polysciences). ARPE-19 reporter cells and con- ccactcctccacctttgac-30 and 50-accctgttgctgtagcca-30 for GAPDH. trol reporter cells were prepared using pLL3.7 K122 FH-YAP1- TEAD-responsive-promoter-H2B-mCherry and pLL3.7 K122 con- RNA interferences trol-TEAD-responsive-promoter-H2B-mCherry. HEK293FT cells RNA interferences were performed by use of Lipofectamine expressing mCherry-Halo-YAP1 was generated by use of the RNAiMAX (Life Technologies). The double-strand RNAs retrovirus vector, pQCXIP-mCherry-Halo-YAP1, and puromycin. (dsRNAs) used are as follows: human YAP1#1, s20366, human YAP1-depleted and luciferase-expressing IM-9 cells were prepared YAP1#2, s20367, human LATS1, s17392, human LATS2, s25505, by use of pLL3.7-GFP2A-shYAP2 and pLL3.7-H2B-mCherry-2A- and Silencer Select Negative Control No. 2 siRNA (Ambion). luciferase, respectively. Reporter assay Chemical library screening A total of 4 106 HEK293FT cells and HEK293FT cells For the first screening, we used 18,606 chemical compounds expressing mCherry-Halo-YAP1 were plated on the collagen- owned by the Tokyo Medical and Dental University Chemical coated 6-cm dish and transfected 0.6 mg 8xGT-IIC-d51LucII Biology Screening Center. ARPE-19 reporter cells were plated at luciferase reporter and 0.6 mg pCMV alkaline phosphatase. 3.5 103 cells/well in a 96-well plate. Each plate contained 90 Twenty-four hours later, the cells from one dish were replated mL medium. 24 hours later, each compound was applied to the into 24 wells in 24-well plate, treated with 10 mmol/L each cells to a final concentration of 10 mmol/L. The original stock, compound for 18 hours, and harvested. The cells were lysed in whose concentration was 10 mmol/L in DMSO, was diluted at 200 mL of the lysis buffer (25 mmol/L Tris-HCl pH7.4, 1:100 by the medium and 10 mL of the diluted sample was 2 mmol/L DTT, 2 mmol/L EDTA, 4 mmol/L EGTA, added to each plate. As a negative control, DMSO was similarly 4 mmol/L MgCl2, 0.1%(w/v) Triton X-100, and 10 mg/L diluted and added to the cells. Seventy-two hours later, the (Amidinophenyl)methanesulfonyl fluoride (APMSF). The cells were washed with PBS and fixed with 4% formaldehyde/ luciferase activity and the alkaline phosphatase activity were PBS for 15 min. Then, the formaldehyde/PBS was changed to assayed with PicaGene (Toyo Ink) and CDP-Star (Roche) and PBS containing 1 g/L Hoechst 33342 to visualize the nuclei. measured by ARVO MX (PerkinElmer). The alkaline phospha- Green and red fluorescence intensities of 200 cells in each tase assay was performed in 90 mmol/L glycine buffer pH10.5 well were measured by use of ArrayScan VTI (Thermo Fisher with 0.1 mmol/L ZnCl2.

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Subcellular fractionation 50 mL DMSO, and the absorbance at 570 nm was measured with ARPE-19 reporter cells were plated at 2 105 cells/6-cm dish. SmartSpec 3000 (Bio-Rad). Forty-eight hours later, the cells were harvested, washed with ice- cold PBS twice, suspended in 200 mL of the extraction buffer Lactate dehydrogenase (LDH) assay K562 cells were plated at 3 106 cells/dish in a 6-cm dish and (10 mmol/L Hepes-NaOH pH8.0, 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.34 mol/L sucrose, 10% (v/v) glycerol, 0.05% (v/v) NP40, transfected with control siRNA or YAP1#1 siRNA. Forty-eight 4 and 10 mg/L APMSF), kept on ice for 5 minutes, and suspended hours later, the cells were replated at 3 10 cells/well in a up-down with 200 mL yellow tip ﬁve times. Suspension (66.6 mL) 96-well plate. The cells were treated with either control DMSO or was saved as the total lysate. The remaining sample was centri- 10 mmol/L IBS003031 for 48 hours. As K562 cells, although fuged at 800 g for 5 minutes at 4C. The supernatant was further hematopoietic cells, attached to the plate, 50 mL cell-free medium centrifuged at 20,000 g for 10 minutes and the ﬁnal supernatant was collected and LDH activity was assayed using 2-p-iodophe- was used as the cytosol fraction. The pellet was resuspended in nyl-3-p-nitrophenyl tetrazolium chloride, N-methylphenazo- 133.3 mL of the extraction buffer and centrifuged again at 800 g nium methyl sulfate, nicotinamide adenine nucleotide, and lactic for 4 minutes. The pellet was resuspended in 133.3 mL of the acid (37). All these reagents were obtained from Tokyo Chemical extraction buffer and used as the nuclear fraction. Industry Co., Ltd.

Detection of sub-G population Phosphate-afﬁnity SDS-PAGE 1 IM-9 cells were harvested and ﬁxed in ice-cold 70% (v/v) ARPE-19 reporter cells were plated at 1.5 105 cells/well in a 6- ethanol, washed with PBS, and resuspended in PBS containing well plate and treated with 10 mmol/L each compound. Twenty- 10 mg/L propidium iodide and 1 g/L RNaseA. The sub-G pop- four hours later, the cells were harvested, and the cell lysates were 1 ulation was evaluated with FACS Calibur (BD Biosciences). The analyzed by use of Phos-tag acrylamide and PolyVinylidene data were analyzed with BD CellQuest Pro Software. DiFluoride membranes. The signals were measured with ImageJ.

Animal experiments LATS kinase assay All animal experiments were approved by the Tokyo Medical A total of 2 106 HEK293FT cells were plated in a 6-cm dish. and Dental University Animal Care and Use Committee. A total of Twenty-four hours later the cells were transfected with either 2 106 IM-9 cells carrying luciferase were injected intraperito- pCIneoMyc-LATS1 or pCIneoMyc-LATS2. Twenty-four hours after neally into female C.B17/Icr-scidJcl mice (5 weeks old) (Clea the transfection, the cells were replated at 4 105 cells/well in a Japan. Inc.). Tumor progression was monitored by biolumines- 12-well plate and treated with 10 mmol/L IBS003031. Twenty-four cence imaging on IVIS Lumina system (PerkinElmer). Control hours later, the cells were harvested and the cell lysates were DMSO or IBS003031 (5 mg/kg body weight) was intraperitone- immunoblotted with the indicated antibodies. The sequences ally daily. To evaluate the effect of IBS003031 on YAP1 in vivo, around Ser909 of LATS1 and Ser871 of LATS2 and around 3.5 106 IM-9 cells were subcutaneously transplanted with Thr1079 of LATS1 and Thr1041 of LATS2 are identical, and the Matrigel (BD Biosciences). Twenty-eight days later, after tumor phosphorylated LATS1 and LATS2 are detected with the same formation, DMSO or IBS003031 (5 mg/ kg body weight) was antibodies. intraperitoneally injected. One day later, mice were sacrificed and tumors were excised. Tumors were fixed with 4% (w/v) formal- Coimmunoprecipitation and LUMIER assay dehyde in PBS for overnight and dehydrated in 70% (v/v) ethanol 5 HEK293FT cells were plated at 8 10 cells/well in a 6-well for another overnight. After embedded in paraffin blocks and plate and were transfected with various combinations of expres- sliced into 4-mm slices, the samples were deparaffined and heated sion vectors. After overnight culture, IBS003031 was added to a in citrate buffer to activate antigens. After endogenous peroxidase final concentration of 10 mmol/L. Twenty-four hours later, the was inactivated, the samples were blocked with 3% (w/v) BSA in cells were harvested and lysed in the lysis buffer (25 mmol/L Tris- PBS and immunostained with rabbit monoclonal anti-YAP1 HCl pH7.4, 150 mmol/L NaCl, 10 mmol/L MgCl2, 2 mmol/L antibody. Signals were labeled by using R.T.U. VECTASTAIN kit EDTA, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 10 mg/L (Vector Laboratories Inc.) and SIGMAFAST 3,30-diaminobenzi- APMSF, 10 mg/L leupeptin, and 10 mg/L pepstatin A). After dine tablets. centrifugation at 20,000 g for 10 minutes, the supernatant was collected and the immunoprecipitation was performed with anti- Statistical analysis DYKDDDDK-tag beads. For LUMIER assay, HEK293FT cells were Statistical analyses were performed with the Student t test for plated at 8 105 cells/well in a 6-well plate and were transfected the comparison between two samples and analysis of variance with various combinations of the expression vectors. Twenty-four with the Dunnett test for the multiple comparison using the hours later, the cells from each well were replated into 6 wells of a GraphPad Prism 5.0 (GraphPad Software). 24-well plate and treated with DMSO or 10 mmol/L IBS003031 for 24 hours. The immunoprecipitation was performed with anti- Results DYKDDDDK tag beads. The immunoprecipitates were washed The generation of ARPE-19 cells stably expressing the TEAD- vigorously and the luciferase activity was measured as described responsive reporter for the reporter assay. YAP1 interacts with various transcription factors including TEAD, SMAD, Runx2, and p73. In the in vitro reporter assays, Cell proliferation assay YAP1 enhances the reporter activities that respond to these tran- The colorimetric MTT assay was performed with thiazolyl blue scription factors. However, the recent study using ChIP-seq tetrazolium bromide. The insoluble formazan was dissolved with demonstrates that TEAD is associated with 75% of YAP1 peaks

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and that TEAD is the most prominent partner of YAP1 (38). anti-FLAG antibody but not with anti-phospho-S127 YAP1 anti- Moreover, the TEAD reporter assay exhibits the most remarkable body (Supplementary Fig. S3A). The upper three bands were activity and is the easiest to monitor. We expressed mCherry-fused detected with both the antibodies. Moreover, LATS1/2 silencing histone2B (H2B-mCherry) under the TEAD-responsive promoter increased the lowest band (Supplementary Fig. S3A, an arrow- and FLAG-tagged YAP1 linked to the internal ribosome entry site head). These findings support that the lowest band corresponds to (IRES) and GFP under the cytomegalovirus promoter (CMV) in the unphosphorylated YAP1. We quantified signals by use of immortalized human retinal pigment epithelial ARPE-19 cells ImageJ and calculated the relative amount of the unphosphory- (Fig. 1A). We evaluated mCherry signals driven by the TEAD- lated YAP1. Among 6 TRE compounds (Fig. 3A and B, arrows), responsive promoter. YAP1 knockdown suppressed mCherry IBS014765 did not increase the unphosphorylated YAP1 (Fig. 3A signals, supporting that mCherry signals reflect YAP1 activity and and B, stars). Other 5 TRE compounds did increase unphosphory- that the ARPE-19 reporter cells can be used for the screening of lated YAP1 (Fig. 3A and B). IBS003031 and IBS012851 remark- YAP1 activators (Fig. 1B). ably decreased the phosphorylated YAP1 signal (Fig. 3A, white arrowheads). These compounds were speculated to activate YAP1 The identification of the compounds that enhance the H2B- via the canonical Hippo pathway, which increases phosphorylat- mCherry expression ed YAP1 and decreases unphosphorylated YAP1. Interestingly, the We applied 18,606 chemical compounds to ARPE-19 reporter structures of IBS003031 and IBS012851 are similar (Fig. 3C). We cells and cultured them for 72 hours to select the compounds that have recently reported that ethacridine, which was originally changed the expression of H2B-mCherry (Supplementary Fig. identified as a TAZ activator, reduces phosphorylated YAP1 and S1). We obtained 124 putative enhancers in the initial screening, increases unphosphorylated YAP1 (30). Ethacridine also has an which gave Z-scores higher than 3.5 (Fig. 1C). For the second acridine backbone. We speculate that these compounds activate selection, we directly compared these compounds with the con- YAP1 through the common target molecule. After we confirmed trol and selected 47 compounds that augmented the signals by that IBS003031 enhanced the expression of YAP1 target genes, more than 1.5-fold. To confirm that the compounds enhanced the CTGF and CYR61, in U2OS cells, we decided to focus on signal via YAP1, we applied the compounds to ARPE-19 control IBS003031 (Supplementary Fig. S4). reporter cells expressing only the TEAD-responsive reporter. The effects of the compounds were remarkably attenuated, supporting The effect of IBS003031 on LATS1/2 activities that the exogenously expressed YAP1 is required for the com- We postulated that IBS003031 might activate YAP1 through the pounds to enhance H2B-mCherry signal (Supplementary Fig. inhibition of the phosphorylation by LATS kinases or the facil- S2A). Considering the risk that the concentration employed might itation of the dephosphorylation. We first tested whether be too high, we applied representative enhancers at the lower IBS003031 could inhibit LATS1/2 activities. We expressed Myc- dose. IBS003031 upregulated H2B-mCherry signals at 1 mmol/L LATS1 and Myc-LATS2 in HEK293FT cells and treated the cells and 3 mmol/L, whereas IBS012851 was active at 3 mmol/L (Sup- with IBS003031. The phosphorylation at S909/S871 and T1079/ plementary Fig. S2B). Moreover, when YAP1 was knocked down T1041 of LATS1/2 is essential for the activities of LATS1/2 (39). in the reporter cells, the effects of the compounds were abolished We immunoblotted the cells lysates with the antibodies against (Fig. 1D). These findings corroborate that the compounds phosphorylated-S909/S871 and -T1079/T1041 of LATS1/2. enhance H2B-mCherry signal via YAP1. IBS003031 increased the expression of Myc-LATS1 and augmented the phosphorylation at S909 and T1079 (Fig. 4A, left). The effect on the TEAD reporter activity IBS003031 slightly increased the expression of Myc-LATS2 and We next confirmed the effects of these candidate compounds enhanced the phosphorylation at S871 and T1041 (Fig. 4A, right). on the conventional TEAD-responsive reporter activity in We next tested whether IBS003031 blocked the binding of YAP1 HEK293FT cells. We prepared HEK293FT cells stably expressing to LATS1/2. We expressed luciferase-fused YAP1 with FLAG- YAP1 and after the confirmation that YAP1 overexpression did not LATS1 and LATS2-FLAG, conducted the immunoprecipitation show any significant effect on the cell viability (data not shown), with anti-DYKDDDDK-tag antibody beads, and measured the performed the reporter assay. 6 compounds (IBS003031, coimmunoprecipitated luciferase activity (Fig. 4B). Consistent IBS012851, CBI001316, IBS002470, IBS007607, and with the result in Fig. 3A, IBS003031 enhanced the expression IBS014765) enhanced the reporter activity more than 1.75-fold of FLAG-LATS1 and LATS2-FLAG, and consequently increased the (Fig. 2A, black columns; Fig. 2B, black dots). We designated these amount of FLAG-LATS1 and LATS2-FLAG in the immunopreci- compounds as TEAD reporter enhancers (TRE). 6 compounds pitates (Fig. 4B, the bottom immunoblottings). Luc-YAP1 that (IBS011345, IBS006077, IBS011342, IBS006808, IBS004557, was coimmunoprecipitated with FLAG-LATS1 and LATS2-FLAG and IBS001781) rather suppressed the reporter activity (Fig. was also higher under the treatment with IBS003031. Based on 2A, white columns; Fig. 2B, white dots). We speculate that the these findings, we conclude that it is unlikely that IBS003031 discrepancy between the ARPE-19-based assay and the conven- decreases YAP1 phosphorylation through inhibiting the activa- tional reporter assay might be caused by the differences of cells tion of LATS kinases or blocking the interaction of YAP1 and LATS and the duration of the treatment of the compounds. kinases.

The effects on the phosphorylation of YAP1 The effect of IBS003031 on the dephosphorylation of YAP1 YAP1 is negatively regulated by the canonical Hippo pathway We next hypothesized that IBS003031 might promote the via the phosphorylation (8). To directly address whether and how dephosphorylation of YAP1. We treated ARPE-19 reporter compounds affect the phosphorylation of YAP1, we treated APRE- cells expressing FLAG-YAP1 with IBS003031 and analyzed the 19 reporter cells with the compounds and ran the cell lysates on cell lysates on the Phos-tag SDS-PAGE (Fig. 5A). Under the the Phos-tag SDS-PAGE. The lowest bands were detected with treatment of IBS003031, phosphorylated YAP1 decreased and

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Figure 2. The effect of the compounds on YAP1-depedent TEAD-responsive reporter activity in HEK293FT cells. 8xGT-IIC-d51LucII luciferase reporter (0.6 mg) and pCMV alkaline phosphatase (0.6 mg) were transfected into 4 106 HEK293FT cells stably expressing YAP1 in a 6-cm dish. Twenty-four hours later, the cells were replated at 2 105 cells/well in a 24-well plate. The cells were cultured with 10 mmol/L each compound for 18 hours. The luciferase activity was determined and calibrated by alkaline phosphatase activity. A, Black columns show the compounds (IBS003031, IBS012851, CBI001316, IBS002470, IBS007607, and IBS014765) that enhanced the activity more than 1.75-fold, while white columns demonstrate the compounds that suppressed the activity. Other compounds were depicted as gray bars. B, The results of the reporter assay are shown in descending order of magnitude. Black dots represent the compounds that enhance the reporter activity more than 1.75-fold.

concomitantly unphosphorylated YAP1 increased in a time- phosphorylation of YAP1 by LATS kinases but is likely to promote dependent manner. We also performed the subcellular fraction- the dephosphorylation of YAP1. ation. IBS003031 increased unphosphorylated YAP1 in both the cytoplasm and the nucleus, supporting that YAP1 is depho- The effect of IBS003031 on MM cells in vitro sphorylated in the cytoplasm and then transported into the YAP1 induces apoptosis in MM cells (19). In normal cells, upon nucleus (Fig. 5B). DNA damage, ABL1 is released from 14-3-3 and enters the nucleus (42). The nuclear ABL1 phosphorylates YAP1 at tyrosine, The effect of IBS003031 on the interaction of YAP1 with protein enhances the association of YAP1 with p73, and upregulates phosphatase (PP) 1A and PP2A p73-dependent proapoptotic gene transcription (18). In MM PP1A and PP2A are involved in the dephosphorylation of YAP1 cells, due to the low expression of YAP1, the ABL1–YAP1–p73 (40, 41). We examined whether IBS003031 could strengthen the axis is compromised (19). Certain MM cells express phosphory- interaction of YAP1 with PP1A and PP2A. We expressed luciferase- lated H2A.X termed as g-H2A.X, which is a hallmark of DNA fused PP1A and PP2A with FLAG-YAP1 in HEK293FT cells and damage response, but escape apoptosis because of the low measured the luciferase activity coimmunoprecipitated with expression of YAP1 (43). However, when YAP1 is exogenously FLAG-YAP1. IBS003031 slightly enhanced the interaction of YAP1 expressed or YAP1 is activated by the suppression of the Hippo with PP1A but the difference was not signiﬁcant, while it signif- pathway, MM cells begin to die. We presumed that YAP1 activa- icantly increased the interaction between YAP1 and PP2A (Fig. tors would trigger apoptosis in MM cells. In this study, we used IM- 5C). We further conﬁrmed that IBS003031 enhanced the inter- 9 cells and treated them with various doses of IBS003031 and action between YAP1 and PP2A in U2OS cells (Supplementary performed the MTT assay. IBS003031 decreased the viability in Fig. S5). In conclusion, IBS003031 is supposed not to suppress the the dose-dependent manner (Fig. 6A, left). IM-9 cells expressed

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Figure 3. The effect of the compounds on YAP1 phosphorylation. ARPE-19 reporter cells were plated at 1.5 105 cells/well in a 6-well plate and treated with 10 mmol/L each compound for 24 hours. The cell lysates were analyzed on Phos-tag gels and immunoblotted with anti-FLAG-antibody. The samples were also run on the conventional SDS-PAGE gels and immunoblotted with anti-FLAG and anti- b-actin antibodies. The lysates of LATS1/2- depleted cells were charged on the last lane in each gel, in which the lowest band (unphosphorylated YAP1) increased (arrowheads). Six compounds (IBS003031, IBS012851, CBI001316, IBS002470, IBS007607, and IBS014765) that enhanced the TEAD reporter activity were indicated with arrows. IBS003031, IBS012851, CBI001316, IBS002470, and IBS007607 increased unphosphorylated YAP1, while IBS014765 did not increase the unphosphorylated YAP1 (a star). IBS003031 and IBS012851 reduced YAP1 phosphorylation (white arrowheads). B, The signals in Phos-tag gels were measured with ImageJ. The relative intensity of the lowest band over the sum of all the bands was calculated for each compound. Arrows indicate 6 compounds that enhanced the TEAD-responsive reporter activity in Fig. 2A. C, Structures of IBS003031, IBS012851, and ethacridine.

g-H2A.X with no treatment, which means that IM-9 cells show decreased g-H2A.X expression, but as a-tubulin expression was DNA damage response at the basal condition (Fig. 6A, 0 mmol/L in also reduced, we speculated that the decrease of g-H2A.X expres- the immunoblot for g-H2A.X). IBS003031 enhanced Bax expression might be due to the decrease of the total cell number. We also sion in a dose-dependent manner (Fig. 6A, the immunoblot for conﬁrmed in FACS analysis that IBS003031 increased sub-G1 Bax). The treatment with 6 and 9 mmol/L IBS003031 apparently population in a dose-dependent manner (Fig. 6B, top). YAP1

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Figure 4. The effect of IBS003031 on the phosphorylation at S909/S871 and T1079/T1041 of LATS1/2. A, HEK293FT cells were plated at 2 106 cells/6-cm dish and transfected with pCIneoMyc-LATS1 (5 mg) or pCIneoMyc-LATS2 (5 mg). The cells were cultured overnight, replated at 4 105 cells/well in a 12-well plate, and then treated with either DMSO or 10 mmol/L IBS003031 for 24 hours. The cell lysates were immunoblotted with anti-Myc antibody and the phospho-specific antibodies against phosphorylated S809/S871 and T1079/T1041 of LATS1/2. The immunoblotting with anti-a-tubulin antibody was shown as a loading control. IBS003031 increased Myc-LATS1 expression. B, HEK293FT cells were plated at 8 105 cells/well in a 6-well plate, transfected with pCIneoLuc-YAP1 (1 mg), pCIneoFLAG-LATS1 (1 mg), or pcDNA-LATS2-FLAG (1 mg) and cultured overnight. The cells were replated at 1.3 105 in a 24-well plate and culture for 24 hours with DMSO (the second column (Control)) or 10 mmol/L IBS003031. FLAG-LATS1 or LATS2-FLAG was immunoprecipitated. The luciferase activity coimmunoprecipitated was measured. The first column represents the luciferase activity in the immunoprecipitate from the cells, in which Luc-YAP1 was expressed with GFP instead of FLAG-LATS1 or LATS2-FLAG. The immunoblotting at the bottom shows that IBS003031 remarkably increased FLAG-LATS1 in the immunoprecipitate, which may reflect the enhanced expression of LATS1 in the presence of IBS003031 shown in Fig. 3A. LATS2-FLAG in the immunoprecipitate was also slightly increased by IBS003031. The data are mean SD. , P < 0.001.

depletion reduced the IBS003031-induced sub-G1 population cells than in YAP1-depleted IM-9 cells (Fig. 6C, left). In the (Fig. 6B, bottom). Trypan blue exclusion assay further supported immunoblotting, IBS003031-induced Bax expression was atten- that IBS003031 caused cell death more strongly in parent IM-9 uated by YAP1 depletion (Fig. 6C, right). Imatinib, ABL1

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Figure 5. The effect of IBS003031 on the dephosphorylation of YAP1 and the interaction between YAP1 and protein phosphatases. In A, ARPE-19 reporter cells were plated at 1.5 105 cells/well in a 6- well plate. In B,2 105 cells were plated in a 6-cm dish. The cells were treated with DMSO or 10 mmol/L IBS003031 for the indicated periods of time in A and for 24 hours in B.InA, the whole cell lysates were analyzed on Phos-tag gels and immunoblotted with anti-FLAG antibody. The samples were also run on the conventional SDS-PAGE and immunoblotted with anti-FLAG and anti- b-actin antibodies to demonstrate that all lanes contain almost the same amount of proteins. Under the treatment with IBS003031, the unphosphorylated YAP1 (an arrow) appeared at 3 hours and the phosphorylated YAP1 gradually decreased. In B, the subcellular fractionation was performed to separate the nuclear and the cytoplasmic fractions. IBS003031 increased the amount of unphosphorylated YAP1 in the cytoplasmic fraction. Unphosphorylated YAP1 (an arrow) in the nuclear fraction increased. a-Tubulin and Poly(ADP- ribose) polymerase (PARP) were used as the cytoplasmic and nuclear markers. C, Luciferase-fused protein phosphatase 1A and 2A (Luc-PP1A and Luc-PP2A) were coexpressed with GFP or FLAG-YAP1 in HEK293FT cells. The cells were treated with DMSO or 10 mmol/L IBS003031 for 24 hours and the immunoprecipitation was performed with anti-DYKDDDDK-tag antibody beads. The luciferase activity attached to the beads was measured. The first column represents the luciferase activity that nonspecifically bound to the beads. The value of the luciferase activity of the immunoprecipitate from the cells treated with DMSO is set as 1. The data are mean SD. n.s., not significant; , P < 0.05.

inhibitor, reduced the effect of IBS003031 in the MTT assay (Fig. The effect of IBS003031 on p73-dependent transcription 6D). All these ﬁndings support that IBS003031 induces apoptosis IBS003031 increased the amount of MDM2, FAS, and BAX in in IM-9 cells through ABL1 and YAP1. We also observed that the mRNA level, which are p73 target genes (Fig. 7A). Accordingly, IBS003031 suppressed cell viability and induced cell death in in HEK293FT cells, IBS003031 stimulated MDM2-promoter lucif- human myeloid leukemia K562 cells depending on YAP1 (Sup- erase reporter activity and YAP1 knockdown antagonized the plementary Fig. S6). effect of IBS003031 (Fig. 7B). Moreover, IB003031 enhanced the

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Figure 6. The effect of IBS003031 on human MM IM-9 cells. A, IM-9 cells were plated at 2 104 cells/well in a 96-well plate. The cells were cultured in the presence of various doses of IBS003031 for 48 hours. The cell viability was assessed by the MTT assay, and the viability of the cells with no treatment of IBS003031 was set as 1. For the immunoblotting, the cells were harvested at 24 hours and the cell lysates were immunoblotted with the indicated antibodies. B, Parent and YAP1- depleted IM-9 cells were plated at 2 105 cells in a 6-cm dish and treated with IBS003031 at various doses for 48 hours. The cells were ﬁxed in the ice-cold

70% (v/v) ethanol and incubated for 30 minutes with 1 mg/L propidium iodide and 10 mg/L RNaseA. The sub-G1 population was evaluated with FACS Calibur. The data were analyzed with BD CellQuest Pro Software. YAP1 depletion was conﬁrmed by qRT-PCR (the right histogram). C, Parent and YAP1-depleted IM-9 cells were plated at 1 105 cells/6-cm dish and treated with DMSO or 9 mmol/L IBS003031 for 48 hours. The viable cells were evaluated in the trypan blue exclusion assay (left). The ratio of trypan blue-positive cells for each condition is indicated. Parent IM-9 cells and YAP1-depleted IM-9 cells were treated with IBS003031 at various doses for 48 hours and the cell lysates were immunoblotted with anti-Bax antibody (right). , P < 0.001. D, IM-9 cells were plated at 1 106 cells/10-cm dish and cultured without or with 1 mmol/L imatinib overnight. The cells were replated at 2 104 cells/well in a 96-well plate and cultured with IBS003031 at various doses without or with 1 mmol/L imatinib. The cell viability was assessed by the MTT assay, and the viability of the cells with no treatment of IBS003031 was set as 1. , P < 0.01.

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control DMSO or IB003031 (a ﬁnal concentration of 5 mg/kg body weight) was injected intraperitoneally daily for 1 week (Fig. 8A). Tumor growth was evaluated in the bioluminescence imaging (Fig. 8B). The trend of the suppression of the growth by IBS003031 was observed (Fig. 8C). In the parallel experiment, we subcutaneously transplanted IM-9 cells and after tumor was formed, DMSO or IBS003031 was injected intraperitoneally (Supplementary Fig. S7A). One day later, tumor was isolated and endogenous YAP1 was immunostained. IBS003031 indeed increased nuclear YAP1 (Supplementary Fig. S7B).

Discussion In this study, we expressed YAP1 and TEAD-responsive fluorescent protein reporter in human retinal pigment ARPE-19 cells and used them to detect small chemical compounds that enhance the reporter activity (Fig. 1). ARPE-19 cells are immortalized cells, but show contact inhibition, suggesting that the Hippo pathway is intact. In the initial screening by using 18,606 compounds, we tested 80 compounds as one set in a 96-well plate and calculated Z-score in each set. The first screening gave 124 compounds with Z-score higher than 3.5 (Fig. 2). In the second screening, we directly compared them with the control and further selected 47 compounds that enhanced the reporter by more than 1.5. These compounds did not enhance the fluorescence reporter in ARPE-19 cells expressing only TEAD-responsive reporter, supporting that the compounds upregulated the reporter via exogenously expressed YAP1. In the conventional reporter assay using HEK293FT cells and TEAD-responsive luciferase reporter, 6 compounds attenuated the reporter activity (Fig. 2). ARPE-19 reporter cells were exposed to the compounds for 72 h, whereas HEK293FT cells were treated with the compounds for 18 h. The reason why 6 compounds show the unexpected effect may be the difference of the experimental condition. The phosphorylation by LATS kinases is best characterized and the most prominent regulatory mechanism of YAP1. According to the canonical Hippo pathway, YAP1 activators are predicted to decrease YAP1 phosphorylation and increase the amount of unphosphorylated YAP1. Among 6 compounds that remarkably enhanced the reporter activity in the conventional reporter assay, Figure 7. IBS003031 and IBS012851, faithfully to the canonical Hippo The effect of IBS003031 on p73-dependent transcription. A, IM-9 cells were pathway, increased the amount of unphosphorylated YAP1 and plated at 3 105 cells/well in a 12-well plate. Twenty-four hours later, decreased that of phosphorylated YAP1 (Fig. 4). Intriguingly, the IBS003031 was added to a final concentration of 9 mmol/L. For control cells, DMSO was added. The cells were cultured for 24 hours. Total RNA was isolated structures of both compounds and ethacridine that we previously and qRT-PCR was performed. The value for the control cells is set as 1. reported as a YAP1/TAZ activator are similar. We speculate that , P < 0.05; , P < 0.01; , P < 0.001. B, YAP1 was knocked down. Forty-eight these compounds target the same or the related molecule. hours later, HEK293FT cells were transfected with MDM2-promoter In this study, we wanted to conclude whether bona fide YAP1 luciferase reporter (0.3 mg), and pCMV alkaline phosphatase (0.3 mg). Five hours activators can be obtained through the screening with ARPE-19 later, the cells were replated at 3.2 105 cells/well in a 12-well plate and reporter cells. To facilitate the analysis, we focused on IBS003031 cultured with or without IBS003031 for 24 hours. The reporter assay was performed as described for Fig. 2A. YAP1 depletion was confirmed by the that remarkably increased the amount of unphosphorylated YAP1 immunoblotting (the bottom). , P < 0.001. C, GFP-YAP1 and FLAG-p73 were and decreased that of phosphorylated YAP1. IBS003031 expressed in HEK293FT cells. The cells were treated with 10 mmol/L enhanced the expression of LATS kinases. IBS003031 did not IBS003031. FLAG-p73 was immunoprecipitated. The coimmunoprecipitated inhibit the activities of LATS kinases and did not block the GFP-YAP1 was increased by IBS003031 treatment. interaction between YAP1 and LATS kinases (Fig. 3). We could conclude that the IBS003031-induced upregulation of unpho- coimmunoprecipitation of FLAG-p73 and GFP-YAP1 from sphorylated YAP1 is not caused by the inhibition of the phos- HEK293FT cells (Fig. 7C). phorylation. On the other hand, IBS003031 enhanced the interaction of YAP1 with protein phosphatases and induced the The effect of IBS003031 on MM cells in vivo dephosphorylation of YAP1 (Fig. 4 and Supplementary Fig. We transplanted luciferase-expressing IM-9 cells into the S5). The subcellular fractionation experiment revealed that immunocompromised mice. Twenty days after transplantation, unphosphorylated YAP1 increased not only in the nucleus but

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Figure 8. The effect of IBS003031 on the proliferation of IM- 9 cells in mice. A total of 2 106 IM-9 cells carrying luciferase were injected into C.B17/Icr-scidJcl mice intraperitoneally. Twenty days later, control DMSO or IBS003031 (5 mg/kg body weight) was injected intraperitoneally daily for 7 days. A, Design of the experiment. B, Representative images from bioluminescence imaging of mice carrying IM-9 cells. C, Distribution of the fold increase of the bioluminescence signal intensity at day 7.

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also in the cytoplasm under the treatment with IBS003031. This (19). We found that IBS003031 induced cell death in human finding suggests that the import of unphosphorylated YAP1 into myeloid K562 cells via YAP1. Therefore, YAP1 activation may be the nucleus is limited and that a slight increase in the nuclear useful to control not only MM cells but also other blood cancer unphosphorylated YAP1 may be sufficient to upregulate gene cells (Supplementary Fig. S6). The study including animal experi- transcription. The precise mechanism how IBS003031 enhances ments will be awaiting. the interaction between YAP1 and protein phosphatases is not In conclusion, the compound screening by use of ARPE-19 clear. Apoptosis-stimulating of p53 protein 2 functions as a reporter cells provided us with a novel YAP1 activator. We have scaffold for YAP1 and PP1A to promote the interaction of both also demonstrated the possibility that YAP1 activation can be a proteins (40). a-Catenin inhibits the interaction between YAP1 choice in the treatment of MM cells. and PP2A (41). Proteomic studies have revealed that the protein network underlying the Hippo pathway includes several interac- Disclosure of Potential Conflicts of Interest tions with serine and threonine protein phosphatases (44). No potential conflicts of interest were disclosed. IBS003031 may work through these interactions. YAP1 is primarily thought to be an oncoprotein (9). The Authors' Contributions expression of nuclear YAP1 correlates with poor prognosis in Conception and design: J. Maruyama, X. Jiang, M. Ishigami-Yuasa, Y. Hata cancer patients. Numerous reports demonstrate that YAP1 acti- Development of methodology: J. Maruyama, K. Inami, F. Michishita, vation leads to oncogenesis. Meanwhile, YAP1 is implicated in K. Nakagawa p73-dependent transcription of proapoptotic genes (17). YAP1 Acquisition of data (provided animals, acquired and managed patients, depletion suppresses cell death and promotes tumor formation in provided facilities, etc.): J. Maruyama, K. Inami, F. Michishita, H. Iwasa, K. Yamamoto, Y. Hata certain cancers. ABL1 is important for YAP1 to adopt the fate as a Analysis and interpretation of data (e.g., statistical analysis, biostatistics, tumor suppressor (18). DNA damage induces the nuclear local- computational analysis): J. Maruyama, K. Inami, F. Michishita, H. Kagechika ization of ABL1, which phosphorylates YAP1. Tyrosine-phos- Writing, review, and/or revision of the manuscript: J. Maruyama, X. Jiang, phorylated YAP1 interacts with p73 and upregulates p73-depen- H. Iwasa, M. Ishigami-Yuasa, H. Kagechika, Y. Hata dent transcription. A recent study has revealed the tumor sup- Administrative, technical, or material support (i.e., reporting or organizing pressive aspect of YAP1 in MM cells (19). MM cells, in which YAP1 data, constructing databases): K. Nakagawa, H. Kagechika, N. Miyamura, J. Hirayama, H. Nishina expression is low, do not undergo apoptosis in response to DNA damage, but begin to die when YAP1 is exogenously expressed or upregulated by the suppression of mammalian Ste20-like Acknowledgments kinase 1. Based on this report, we speculated that YAP1 activator This work was supported by research grants from the Japan Society for the could induce cell death in MM cells. As expected, IBS003031 Promotion of Science (JSPS; 26460359 to H. Iwasa and 26293061 to Y. Hata) and the Mitsubishi Foundation (26138). triggers cell death in human MM IM-9 cells (Fig. 5). YAP1 The authors acknowledge Hideyuki Saya, Tadashi Yamamoto, Sumiko depletion and ABL1 inhibitor blocked this effect. IBS003031 Watanabe, and Hiroshi Sasaki for materials. strengthened the interaction between YAP1 and p73 (Fig. 6). All these findings indicate that IBS003031 induces apoptosis in IM-9 cells via the ABL1–YAP1–p73 axis. We further confirmed that The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in IBS003031 suppressed the growth of IM-9 cells in vivo and accordance with 18 U.S.C. Section 1734 solely to indicate this fact. increased the nuclear YAP1 (Fig. 7 and Supplementary Fig. S7). The importance of the ABL1–YAP1–p73 axis as a barrier against Received July 18, 2017; revised September 20, 2017; accepted October 10, hematological malignancies was discussed in the previous paper 2017; published OnlineFirst October 23, 2017.

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YAP1 Activator Suppresses Multiple Myeloma Cell Viability

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www.aacrjournals.org Mol Cancer Res; 2017 OF15

Novel YAP1 Activator, Identified by Transcription-Based Functional Screen, Limits Multiple Myeloma Growth

Junichi Maruyama, Kazutoshi Inami, Fumiyoshi Michishita, et al.

Mol Cancer Res Published OnlineFirst October 23, 2017.

Updated version Access the most recent version of this article at: doi:10.1158/1541-7786.MCR-17-0382

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